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In 1872, menzal described a variant of ossifying fibroma, calling it a cemento - ossifying fibroma (cof). Cof is a well - demarcated and occasionally encapsulated neoplasm that contains fibrous tissue and varying amounts of calcified tissue resembling bone, cementum, or both . This neoplasm occurs in patients of a wide age range of third and fourth decades of life . Female - to - male ratio as high as 5:1, indicates a definite female proclivity . The world health organization (who) classified four types of cementum containing lesions: fibrous dysplasia, ossifying fibroma, cementifying fibroma and cemento - ossifying fibroma . The second who classification, divided benign fibro - osseous lesions in the oral and maxillofacial regions into two categories; osteogenic neoplasm and non - neoplastic bone lesions . Slow growth and lack of symptoms are the cardinal features; pain or paraesthesia may be elicited if pressure on an adjacent nerve ensues . Teeth in association with the lesion retain their vitality and as a rule, there is no associated root resorption . The term giant ossifying fibroma is used for large lesions increasing in size to over 80 mm in their greatest diameter . Although some cases of cof have been reported with massive expansile lesions, lesions measuring more than 10 cm are rare . This case report details a remarkable massive expansile lesion involving the mandible in a 34 year old female patient . A 34 year old female patient reported to department of oral medicine and radiology, with a chief complaint of swelling of the lower jaw since six years . She stated that a small swelling on the left side of jaw was noticed six years ago, measuring about 1 cm in diameter, which gradually increased to the present size, involving both sides of the face . Gross facial asymmetry was noticed due to a swelling involving the middle and lower one third region of the face [figure 1]. Bilaterally fullness of the cheeks and lower lip with obliteration of the mentolabial sulcus and nasolabial fold was noticed . A well demarcated swelling was noticed over the left cheek region and a diffuse swelling over the right side, which extended from the left ramus of the mandible to the right body of the mandible with obliteration of inferior border of the mandible . Swelling on the left side was roughly ovoid in shape, measuring about 8 6 5 cm . On palpation the swelling is hard in consistency, lobulated and nontender; with no accompanying cervical lymphadenopathy . Frontal view of the patient showing a well defined swelling on left side and diffuse swelling on the right side of the mandible on intraoral examination [figure 2], a diffuse swelling was seen extending from the left to right retromolar region . Bilaterally, vestibular obliteration was noticed due to buccal labial and lingual cortical plate expansion with normal appearing mucosa . Swelling was hard, lobulated and nontender . Correlating the history and clinical findings, a provisional diagnosis of benign fibro - osseous lesion of mandible was given and differential diagnosis of fibrous dysplasia, ossifying / cementifying fibroma and central giant cell granuloma (non- aggressive lesion) was given . Routine hematological examination, serum protein, calcium, phosphorus and alkaline phophatase levels were estimated and the values obtained were within the normal range . Intra - oral photograph shows an expansile swelling of the mandible with complete bucco - lingual cortical plate expansion and lingually displaced teeth panoramic radiograph [figure 3] revealed a well defined mixed radiopaque and radiolucent lesion, extending from left ramus region to the right angle of the mandible, measuring about 11 7 cms . Centrifugal growth fashion is appreciable, which causes a ball - like circular lesion and bowing of the inferior border of mandible . The inferior cortex is parallel to the tumor mass above with no evidence of erosion . Occlusal radiograph [figure 4], revealed irregular expansion of both bucco - lingual cortical plates and lingual displacement of involved teeth . Posterior - anterior (pa) mandible view [figure 5] revealed a mixed radiopaque, radiolucent lesion involving the left ramus region and extending up to the right angle of the mandible . Orthopantomogram reveals a massive high density, mixed radiopaque - radiolucent lesion exhibiting cotton wool appearance . Demarcation between the lesion and normal bone with bowing of the inferior border of mandible is appreciable lower occlusal radiograph shows irregular bucco- lingual cortical plate expansion posterior anterior mandible view reveals an expansile mixed radiolucent radiopaque lesion extending from the ramus of left side to the right angle of the mandible plain axial view and 3d - ct scan [figures 6 and 7] revealed a large expansile lesion within the mandible, which involves right and left body and left ramus of mandible, which exhibits mixed sclerotic and lucent areas measuring 11.8 7.0 7.3 cm . Axial ct scan view shows a large lobulated bone density mass involving both right and left body and left ramus of mandible, which exhibits mixed sclerotic and lucent areas . Central low - density areas (arrow) are seen image of 3d - ct scan shows a large expansile swelling within the mandible, which involves both right and left body and left ramus of mandible, which exhibits mixed sclerotic and lucent areas measuring 11.8 7.0 7.3 cm an incisional biopsy was performed and the tissue was sent for histopathological evaluation [figure 8] which irregularly shaped bony trabaculae interspersed in connective tissue stroma which is fibrocellular . Several spherical masses of a cellular calcified material resembling cementum were seen through the fibrous tissue . Fibroblasts in the stroma are numerous and spindle shaped with prominent nuclei which were suggestive of cemento- ossifying fibroma . Based on clinical, radiographic and histopathological findings a final diagnosis of giant cemento - ossifying fibroma of the mandible was made . Photomicrography shows several spherical masses of a cellular calcified material resembling cementum were seen throughout the fibrous tissue (h and e stain 250) the origin of cof is thought to be the periodontal membrane which harbors the potential for elaboration of both bone and cementum . Bernier and thompson speculated that infection with resulting inflammation and fibrosis of the periapical area might stimulate the periodontal membrane . After trauma, such as tooth extraction, the remaining periodontal tissue that is attached to the wall of the alveolus may serve as the origin of cof . Explained that ectopic periodontal membrane differentiated from primitive mesenchymal cells in the petrous bone may serve as a cause of development of cof in this area and that trauma such as severe whiplash may be a factor in the induction of proliferation of cof . His research showed g protein mutation; located in chromosome number 13 and was further investigated for three types of fibro- osseous lesions (fd, cof, and fcod) to see if this mutation has a diagnostic value . The pathologic nature of cof is not yet clearly understood . A close histogenetic relationship exists between the central cof and the central ossifying fibroma . The only difference between the two is that, in cof, there is cementum formation along with bony trabeculae; this cementum is not seen in ossifying fibroma . The late charles waldron wrote in absence of good clinical and radiologic information a pathologist can only state that a given biopsy is consistent with a fol . Therefore, the diagnosis of the majority of histopathologically proven fols affecting the jaws is made upon clinical and radiological features . Radiologically, depending on degree of mineralization these tumors may present a number of patterns . Two basic patterns are, one characterized by the presence of a unilocular or multilocular radio transparent image and another showing mixed density due to a variable internal amount of radiopaque material . The appearance of fibroma is concentric within the medullary zone of the bone and the cortical layers are preserved . Root reabsorption and displacement of the roots of the neighboring teeth is seen in some cases . Slootweg and muller described an objective of marginal definition; a lesion with zone of transition of less than 1 mm can be considered to be well defined . The first edition of the who classification was clear that fd and cof can be distinguished by radiology, in which the former has a poorly defined margin, whereas the latter has a well defined margin . The well - defined border of the cof helps differentiate it from the aggressive sarcomas and carcinomas . Conservative surgery is recommended even if the tumor is large with bowing and erosion of the inferior border of the mandible . En bloc resection of the tumor should only be considered as radical treatment, if there are recurrences due to its aggressive nature . Advantages for treating large cof conservatively are, there is minimal morbidity after surgery, good bone formation and consolidation, no loss of sensation and no bone graft required from a second surgical site . In long term follow - up cases where bowing or contours formed by these large lesions do not disappear completely with time, surgical intervention such as aesthetic re - contouring of the bone may then be taken into consideration . In the current case, total mandibulectomy without disarticulation was performed and reconstructed with the tibial graft from tibia was placed . Considering the patient's young age, aesthetic facial features and to improve the contour of the mandible, a further cosmetic operation was considered after resection of the mandible . A good result in regard to cosmetic and functional deformity was achieved by using this approach for a large ossifying fibroma, resulting in a fair prognosis . Cemento - ossifying fibroma of the jaw is a benign fibro - osseous lesion with a significant growth potential . Thus, it is not always easy to diagnose and manage these lesions because of their clinical, radiographic and histological criteria's, which often overlap causing confusion to clinicians, radiologists, pathologists and oral surgeons.
Realizar a traduo e a validao cultural para a lngua portuguesa falada no brasil e determinar a concordncia e a confiabilidade dos instrumentos perme intensive care unit mobility score (designado perme escore) e icu mobility scale (designada escala de mobilidade em uti, emu). Os processos de traduo e adaptao cultural seguiram as seguintes etapas: preparao, traduo, reconciliao, sntese, traduo reversa, reviso, aprovao e pr - teste . Aps esses processos, as verses em portugus dos dois instrumentos foram utilizadas por dois pesquisadores na avaliao de pacientes crticos em uti . O ndice kappa ponderado e a disposio grfica de bland - altman foram utilizados para verificar a concordncia entre os instrumentos . O coeficiente alfa de cronbach foi utilizado para verificar a confiabilidade entre as respostas dos avaliadores dentro de cada domnio dos instrumentos . A correlao entre os instrumentos foi verificada pelo teste de correlao de spearman a amostra foi composta por 103 pacientes, sendo a maioria homens (n = 56; 54%), com mdia de idade = 52 18 anos . O principal motivo de internao nas utis foi insuficincia respiratria (em 44%). Os dois instrumentos apresentaram excelente concordncia interobservador (> 0,90) e confiabilidade (> 0,90) em todos os domnios . Constatou - se um baixo vis interobservador na emu e no perme escore (0,048 0,350 e 0,06 0,73, respectivamente). Os ic95% para os mesmos instrumentos variaram, respectivamente, de 0,73 a 0,64 e de 1,50 a 1,36, respectivamente . Alm disso, verificou - se alta correlao positiva entre os dois instrumentos (r = 0,941; p <0,001). Early mobilization is part of the rehabilitation process for icu patients and is currently considered a way to prevent icu - acquired muscle weakness and worsening of physical function . Some studies have associated the practice of early mobilization with decreased duration of mechanical ventilation and reduced length of icu and hospital stays, as well as with the promotion of functional improvement in icu survivors . There are currently 26 published instruments that purport to assess function in icu patients . Of those, the functional independence measure and the barthel index have been used both in clinical practice and in research . However, few of these instruments were developed and validated to assess function and/or mobility in icu patients . In fact, only 6 were developed specifically for the icu setting and have published clinimetric data . These instruments are the physical function in intensive care test scored, the chelsea critical care physical assessment tool, the perme intensive care unit mobility score, the surgical intensive care unit optimal mobilization score, the icu mobility scale, and the functional status score for the icu . However, none of them are considered " gold standard " to assist multidisciplinary teams in quantifying the patient's degree of mobility in a rapid, easy, and objective way . In addition, there are conditions extrinsic to the patient that affect the patient's mobility in bed, such as the presence of access ports, lines, and chest tubes, which can be interpreted as a barrier to mobility, and this presence is not scored or considered in most instruments . Taking into account such limitations, perme et al . Developed a specific instrument for measuring improvement in mobility status, with a view to standardizing the evaluation of icu patients - the perme intensive care unit mobility score - hereafter referred to as the perme score, which is an instrument that objectively measures the mobility status of icu patients, starting with the ability to follow commands and culminating in the distance walked in two minutes . This mobility instrument is scored from 0 to 32 and comprises 15 items grouped into 7 categories: mental status; potential mobility barriers; functional strength; bed mobility; transfers; gait (with or without assistive devices); and endurance . In this instrument, in contrast, a low score indicates low mobility and an increased need for assistance . With similar objectives to those of the perme score, hodgson et al . Designated the icu mobility scale (ims), this single - domain instrument is scored from 0 to 10, with a score of 0 meaning low mobility (interpreted as a patient being capable of performing only passive exercises in bed) and a score of 10 meaning high mobility (interpreted as a patient being capable of independent ambulation, without aid). The clinical utility of a tool has to be determined on the basis of a logical assessment of its validation, reliability, and applicability . In order to choose the best tool that can effectively assess the functional changes that will occur in the patient during the icu stay, health care professionals and researchers should consider which tools have clinimetric data that are more robust and appropriate for the functional outcomes that they want to analyze . To date, neither the perme score nor the ims has been appropriately translated and validated for use in brazil, taking into account the language and cultural differences . Therefore, the objective of the present study was to translate these two icu mobility instruments into portuguese, creating versions that are cross - culturally adapted for use in brazil, and to determine inter - rater agreement and reliability for both versions . The present study was approved by the research ethics committee and the comisso de anlises de projetos de pesquisa (cappesq, committee for the analysis of research projects) of the university of so paulo school of medicine hospital das clnicas (ruling no . The translated instruments were evaluated at two clinical icus (10 beds) and one surgical icu (20 beds) of the university of so paulo school of medicine hospital das clnicas central institute, in the city of so paulo, brazil, between april and june of 2015 . In the phase of instrument testing, since physical therapy evaluation was part of the routine care at those icus, being performed several times a day, the health care professionals who participated in the study, scoring and comparing the instruments, gave written informed consent, rather than the patients . The methodology for translating and cross - cultural adapting and validating the instruments followed a rigorous process, in accordance with current guidelines for the translation and cross - cultural adaptation of instruments . The following steps were performed: 1) preparation: the author of the project contacted the authors of the original instruments and obtained the rights to use, translate, and cross - culturally validate the instruments; 2) translation from english into portuguese: the instruments were independently translated into the target language by two translators who were native speakers of portuguese and fluent in english, one of whom was familiar with the instruments and was aware of the objective of the present study and the other of whom was not familiar with the instruments; 3) reconciliation and synthesis: the two initial portuguese - language versions were compared, item by item, with the original english - language versions by two physical therapists who were familiar with the instruments . Any existing discrepancies were analyzed and discussed by three researchers, leading to the production of a second portuguese - language version for each of the two instruments; 4) back - translation: the second portuguese - language version of each of the two instruments was sent to two translators who were native speakers of english and fluent in portuguese, neither of whom had contact with the original english - language versions, for back - translation; 5) review and harmonization of the back - translation: the back - translated versions of the instruments were compared with their original english - language versions by a review committee comprising three researchers, in order to identify potential discrepancies and make the necessary adjustments, item by item, thereby producing the final back - translated version of each of the two instruments; 6) approval from the authors of the original instruments: the final back - translated versions were sent to the authors of the original instruments for evaluation and comments on their consistency . An expert committee comprising three physical therapists analyzed the evaluations and comments of the authors of the original instruments, incorporating their suggestions, and thereby produced the final portuguese - language version of each of the two instruments; and 7) pre - test: raters were trained on the administration and scoring of the final portuguese - language version of each of the two instruments . After training, a pilot study involving 40 patients was conducted in which two raters administered the two instruments following the methodology described in the original articles; during these evaluations, the raters could discuss the scores and the difficulties in administering each instrument . Data were collected by two raters, one of whom was a senior (> 5 years of experience) physical therapist (rater 1) and one of whom was a junior (<5 years of experience) physical therapist (rater 2). The two raters performed the scoring according to the rules of the two mobility instruments, which were administered after an initial evaluation made by the icu physical therapist . While one of the raters evaluated the patient, the other one only observed the procedure, without having any physical contact with the patient . Each rater was responsible for 50% of the evaluations, and the functions of rater and observer were swapped every two patients . Both raters completed the scoring sheet of the perme score and of the ims according to the highest activity level . In an attempt to avoid biases, the scoring sheets were completely separate and there was no communication between the raters . Data on age, gender, reason for icu admission, mechanical ventilation use, vasoactive drug use, and score on the simplified acute physiology score 3 were collected to determine the clinical characteristics of the population . The sample size was calculated with a level of significance of 5% and a power of 80%, taking into account that the instruments could be equal (50%) or not (50%). This is possible through the use of the bernoulli probability distribution; in addition, we considered a delta of 10%, that is, the probability of equality could range from 40% to 60%, and found that a sample size of 100 individuals was required . Statistical analysis was performed with the statistical package for the social sciences, version 17 (spss inc ., the clinical characteristics of the patients were descriptively expressed as mean and standard deviation, median and interquartile range, or proportion, depending on data type and normality of distribution . The level of inter - rater agreement in the scoring of each instrument was determined using weighted kappa statistics and 95% ci . Inter - rater reliability (internal consistency) in scoring was determined using cronbach's alpha coefficient . For the perme score, inter - rater agreement and reliability were assessed individually for each domain (items 1 to 15). In addition, bland - altman plots were used to determine inter - rater agreement in total score (sum of all domains) both for the perme score and the ims . The proportions of evaluations with minimum scores (floor effect) and maximum scores (ceiling effect) were also calculated . Finally, the kolmogorov - smirnov test and levene's test were used to determine normality and homoscedasticity, respectively . Since these principles were not met, spearman's correlation coefficient was used to test the correlation between the two instruments . For this correlation analysis table 1 shows the clinical characteristics of the patients evaluated in the present study . Slightly more than half (54%; n = 56) of our sample was male and 67% (n = 69) of the patients were admitted for clinical reasons, the most prevalent being respiratory disorders (n = 45). Mechanical ventilation was present in 36% (n = 37) of the cases, and vasoactive drug use occurred in 51% (n = 53). Appendices show the portuguese - language versions of the ims and the perme score, both of which were translated from the original instruments . They are available online at http://www.jornaldepneumologia.com.br/detalhe_anexo.asp?id=47 table 1characteristics of the patients (n = 103). Characteristicresultage, years 52 18male gender56 (54)saps3 66 reason for icu admission clinical69 (67)respiratory45 (44)renal9 (9)neurological8 (8)rheumatological5 (5)hepatic2 (2)surgical26 (25)gastroenterological11 (11)hepatic9 (9)cardiac3 (3)neurological2 (2)respiratory1 (1)trauma8 (8)vasoactive drug use53 (51)mechanical ventilation37 (36)duration of mechanical ventilation, days 4 tracheostomy9 (9)length of icu stay at the time of evaluation, days 5.5 saps3: simplified acute physiology score 3 . Table 2 shows the inter - rater agreement (kappa statistics and 95% ci) and reliability (internal consistency, cronbach's alpha coefficient) for the ims and for each domain of the perme score . In addition, the inter - rater agreement for each item of the perme score ranged from 78% to 100%, and the inter - rater reliability (cronbach's alpha coefficient) ranged from 88% to 100%, meaning that there was excellent inter - rater agreement and reliability for all items . Figure 1 presents the bland - altman plots for the ims and for the total score on the perme score . Inter - rater bias was low both for the ims (0.048 0.35) and the perme score (0.06 0.73). The 95% cis ranged from 0.73 to 0.64 for the ims and from 1.50 to 1.36 for the perme score . Table 2inter - rater reliability and agreement for the icu mobility scale (ims) and the perme icu mobility score.instrumentrater 1rater 2reliabilityagreement median [min - max]median [min - max](cronbach's alpha coefficient)(95% ci)ims1 [0 - 10]1 [0 - 10]0.990.99 (0.98 - 0.99)perme icu mobility score mental status: item 12 [0 - 2]2 [0 - 2]0.970.94 (0.92 - 0.96)mental status: item 21 [0 - 1]1 [0 - 1]1.001.00potential barriers: item 31 [0 - 1]1 [0 - 1]1.001.00potential barriers: item 40 [0 - 1]0 [0 - 1]0.960.92 (0.88 - 0.94)potential barriers: item 50 [0 - 1]0 [0 - 1]0.970.95 (0.93 - 0.96)potential barriers: item 60 [0 - 1]0 [0 - 1]0.880.78 (0.70 - 0.85)functional strength: item 7 (left leg)1 [0 - 1]1 [0 - 1]0.990.98 (0.97 - 0.98)functional strength: item 7 (right leg)1 [0 - 1]1 [0 - 1]1.001.00functional strength: item 8 (right arm)0 [0 - 1]0 [0 - 1]0.990.98 (0.97 - 0.98)functional strength: item 8 (left arm)1 [0 - 1]1 [0 - 1]0.990.98 (0.97 - 0.98)bed mobility: item 90 [0 - 3]0 [0 - 3]0.980.97 (0.96 - 0.98)bed mobility: item 100 [0 - 3]0 [0 - 3]0.990.99 (0.99 - 0.99)transfers: item 110 [0 - 3]0 [0 - 3]0.980.97 (0.95 - 0.98)transfers: item 120 [0 - 3]0 [0 - 3]0.990.99 (0.99 - 0.99)transfers: item 130 [0 - 3]0 [0 - 3]0.990.99 (0.99 - 0.99)gait item 140 [0 - 3]0 [0 - 3]1.001.00endurance: item 150 [0 - 3]0 [0 - 3]0.990.99 (0.98 - 0.99)perme icu mobility score (total) 8 [0 - 32]8 [0 - 32] figure 1bland - altman plots of inter - rater score differences and mean scores for the icu mobility scale (in a) and the perme icu mobility score (in b). Ula: upper 95% limit of agreement; and lla: lower 95% limit of agreement . The floor effect for the ims and the perme score was found to be 36% and 20%, respectively . The ceiling effect for the ims and the perme score was found to be 6% and 3%, respectively . The mean completion time for the scoring sheets was two minutes for the perme score and less than one minute for the ims . There was also a strong positive correlation between the use of the two instruments in the evaluation of the patients (r = 0.941; p <0.001). For the present study, two instruments for evaluating mobility in icu patients were carefully translated into portuguese and validated for use in brazil, with technical and semantic equivalence having been achieved between the original versions and the portuguese - language versions . Our results show that, in their versions adapted for use in brazil, both instruments showed high inter - rater agreement and reliability after a brief period of familiarization and training with them . In addition, there was a strong positive correlation between the two instruments . Performing physical therapy in critically ill patients is currently in the spotlight, with numerous publications commenting on its prevalence and benefits . In this context, some instruments for evaluating function and mobility have been developed specifically for this population in order to improve physical therapy care in terms of performing and progressing the exercises in icu patients according to the mobility milestones that each individual can reach . To date, as mentioned above, no instrument for evaluating mobility in icu patients has been cross - culturally adapted for use in brazil . The careful translation and cross - cultural validation of such an instrument makes it possible for health care professionals nationwide to have access to a tool that can improve the quality of care to critically ill patients in the icu, as well as allowing the comparison of results across studies conducted in different countries . In their versions adapted for use in brazil, both the ims and the perme score showed excellent inter - rater agreement and reliability (> 0.9 and> 0.9 for most domains). Although the group who developed the perme score reported moderate to high reliability, our study reported an even higher level of inter - rater reliability . One possible explanation for this finding it that, in our study, the sample size was larger than those of the two previous studies . In view of the ease of learning and ease of use of the perme score, any disagreements in score had a lesser impact in our study than in the validation studies for the original english - language version of the instrument . Our study also reported higher inter - rater reliability for the portuguese - language version of the ims than that reported in the validation study for the original english - language version of the instrument . In this case, the raters performed the scoring simultaneously but in an independent fashion, each being blinded to the scoring by the other rater, whereas in the study by hodgson et al . Interestingly, the ims showed excellent inter - rater reliability and agreement, although its single domain is scored from 0 to 10, an 11-point range . Although it is a greater range than that in each domain of the perme score, the ims comprises mobility milestones that are clear and can easily be evaluated by the rater . Item 6 in the perme score, " potential mobility barriers - continuous intravenous infusion ", was the one showing the lowest inter - rater reliability and agreement in our study . Although some patients had venous access for administration of saline or drugs, in some cases, the infusion was not being administered at the time of evaluation, which may have confused the raters in scoring this item . However, we emphasize that, even with this likely difference, the reported inter - rater reliability and agreement were higher than 75 - 80% . The bland - altman plots showed high inter - rater score agreement and low inter - rater score variability for the ims and for the sum of all domains of the perme score . Since the evaluation of each domain had shown excellent inter - rater agreement and reliability, the sum of all domains did not change this behavior . On the basis of the 95% cis, the maximum inter - rater difference was 2 points for the perme score and less than 1 point for the ims . It was expected that the scores on the two instruments would be highly correlated, given that both instruments measure the same property and therefore should show a similar behavior . Finally, instrument floor and ceiling effects of 15% or less are considered acceptable . In our study, floor effects were found to be higher for the two instruments (20% and 36% for the perme escore and the ims, respectively). Knowing that these instruments purport to assess functioning, floor effects were expected to be higher than normal, given the high incidence of sedated or unconscious patients in icus . Although data collection was performed at three different icus in order to try to minimize this drawback, in 35% of the evaluations, the patients were unconscious or had lethargic responses, which made it impossible to perform more functional tasks or mobilizations at the time of evaluation . The lower floor effect of the perme score as compared with that of the ims can be explained by the scoring of the different domains of the former, such as patient cooperation, presence of pain, and presence of barriers to mobilization . Although they are not mobilization aspects per se, they end up affecting the ease or difficulty of mobilization first, no clinimetric analyses were performed other than inter - rater reliability and agreement testing and inter - instrument correlation analysis . However, the primary objective of the present study was the cross - cultural validation of the instruments for use in brazil . We recognize that the applicability of the instruments and their predictive and concurrent validity have yet to be tested . Although the clinimetric properties of the ims were tested against those of the physical function in intensive care test scored, the same was not true for the perme score . Second, in our study, the two participating raters who were tested for inter - rater reliability and agreement were physical therapists . Knowing that such health care professionals are directly related to the process of functional evaluation and early mobilization of critically ill patients, analysis of inter - rater reliability and agreement involving such professionals was fundamental . However, we cannot state that the characteristics reported in the present study can be obtained by the other health care professionals who comprise multidisciplinary icu teams, such as nurses and physicians, and will eventually use the instruments . Finally, the evaluations took place concurrently . Therefore, aspects such as tone of voice, personal approach, and instruction of patients, all of which may differ from one health care professional to another, were not fully evaluated in our study . However, each rater was responsible for half of the evaluations, which to some extent minimized this effect . Therefore, we conclude that the brazilian portuguese - language versions of the ims and the perme score were appropriately translated and cross - culturally validated, following strict guidelines, and can be used in brazil.
Nasa's mercury surface, space environment, geochemistry, and ranging (messenger) spacecraft [solomon et al ., 2007] operated at close solar distances (0.3 to 0.4 au) between january 2008 and april 2015 . This close proximity to the sun, and the inclusion of a neutron spectrometer (ns) in the messenger payload, provided the possibility of detecting lowenergy (0.58 mev) solar neutrons . Solar neutrons at these energies cannot be detected at earth because free neutrons have an ~15 min mean life and therefore decay before reaching 1 au . Detecting and characterizing lowenergy neutrons can provide new insight regarding ion acceleration processes at the sun [dorman, 2010; vilmer et al ., 2011]. To date, two studies have reported evidence for the detection of solar neutrons from messenger ns observations [feldman et al ., 2010; lawrence et al ., a key challenge for identifying solar neutrons with messenger ns data is quantifying locally generated neutrons produced by the impact of energetic ions onto the spacecraft . Because the messenger payload does not include instrumentation that can unambiguously characterize the species and energy of the chargedparticle population for energies greater than a few mev, several techniques have been employed to constrain our knowledge of the flux of energetic ions and the neutrons such ions can produce . These techniques include using ns chargedparticle counters and independently measured gammaray data to constrain the maximum ion fluxes and energies and then using these ion constraints to model the maximum local neutron production on the spacecraft . With these techniques, lawrence et al . Showed that the measured neutron count rate during a neutron event on 4 june 2011 was 13 orders of magnitude larger than could be attributed to locally generated neutrons . On the basis, in part, of these results, lawrence et al . Concluded that there was strong evidence that the detected neutrons were solar in origin . In particular, share et al . Concluded that the excess neutrons detected on 4 june 2011 were generated locally at the spacecraft . We respond to the four principal arguments offered by share et al ., and we show that their claim is unwarranted . Share et al . Made four primary arguments to support their claim that the 4 june 2011 neutron event is the result of locally generated neutrons rather than solar neutrons . Before we respond to each of these arguments, we address an important misunderstanding that fundamentally affects the first three of their arguments . At the beginning of their discussion of the ns singles count rates, these (singles) rates also have a peak showing that there was a large flux of energetic ions at the time of the neutron transient . As argued in detail by feldman et al . And lawrence et al ., however, the presence of an enhanced singles count rate does not establish the presence of energetic ions . The ns singles counters can be triggered by any combination of energetic ions, electrons, neutrons, and photons . In fact, enhanced singles count rates are regularly triggered by bremsstrahlung photons created by energetic electron events in mercury's magnetosphere [ho et al . Because of this fundamental ambiguity, the ns singles counters cannot provide a reliable constraint on the presence of energetic ions . The assumption made by share et al . That the large singles count rates show that there was a large flux of energetic ions the first three arguments of share et al . Are undercut on this basis alone . The first argument made by share et al . Is that the energetic particle environment at messenger prior to the neutron event was similar to that at the solar terrestrial relations observatory (stereo) a spacecraft after accounting for the difference in solar distance between the two spacecraft . Because the stereo a spacecraft has instrumentation that can quantify energetic particle species and their energy for energies greater than a few mev, such a comparison provided share et al . Increased confidence in the messenger ns particle measurements, allowing them to demonstrate that the neutron transient at 16:00 utc was due to secondary neutrons produced by ion interactions in the spacecraft . To support this claim, share et al . Estimated that the singles count rate in the messenger ns lithium glass 2 (lg2) sensor expected for energetic protons should be approximately 2500 counts per second (cps), a factor of only 2.5 greater than the measured lg2 count rate of 1000 cps . First, the estimate was based on the assumption that lg2 singles are triggered entirely by protons, which as noted above is not a valid assumption . Second, share et al . Assumed a solid angle of 1 sr for the lg2 singles . However, this assumed solid angle was substantially underestimated, as the lg scintillators are planar detectors with a field of view of 2 sr . Third, on the basis of an analysis of two energetic electron events on 14 and 18 august 2010 by lario et al ., share et al . Assumed that the proton flux increases by a factor of 5 from 1 au to 0.32 au . (on the basis of a study by lario et al . Of energetic protons), a more appropriate distancescaling factor at 0.32 au is 1/0.32 10 . 's assumption that lg2 singles are due only to protons, the scaling given here shows that the lg2 count rate should be higher by a factor of 2 to 4. the correctly estimated lg2 count rate for such energetic protons would then 15,000 to 30,000 cps, values that are 15 to 30 times higher than the measured count rate of 1000 cps . Thus, the singles counters cannot be used to support share et al . 's claim that messenger and stereo a share a similar solar energetic proton environment . Moreover, the messenger ns double coincidence count rates, which are less ambiguous than the singles count rates in identifying energetic ions and electrons, show that messenger and stereo a were not in similar energetic particle environments [lawrence et al ., validated the absolute calibration of the double coincidence count rates using quiettime measurements of galactic cosmic rays . The energetic particle identification from the double coincidence count rates is thus well supported by independent calibration . The second argument of share et al . Was that the estimated number of locally produced neutrons as derived from the lg2 singles count rate was comparable to the measured neutron count rate enhancement . Specifically, share et al . Estimated that the number of locally produced neutrons was 9 counts per second (cps), which is 30% smaller than the measured count rate of 13 cps (share et al . Quoted a measured count rate of 15 cps, but that value includes a 2 cps cosmicray background) however, the 9 cps value is likely a significant overestimate, for several reasons . First, share et al . Assumed that the entire lg2 singles count rate was due to energetic ions, which is almost certainly not the case during the neutron enhancement, as energetic electrons and photons will also contribute to the total singles count rate . Second, share et al . Assumed a neutron production on the basis of 20 mev protons . However, if there was a fluence spectrum of 20 mev protons sufficient to produce the large neutron enhancement, an extended proton spectrum that is distributed in energy as is normally observed during solar energetic particle events should trigger the ns double coincidence counter, which has a threshold only 10 mev higher . As shown by lawrence et al . Is a steep function of energy, with lowerenergy protons and alpha particles producing substantially fewer neutrons than higherenergy protons and alpha particles, the use of the singular 20 mev value almost certainly yields a large overestimate . For these three reasons, we consider the neutron count rate of 9 cps for locally produced neutrons estimated by share et al . To be unreliable . Did not provide a good explanation for the presence of the large singles count rates during the neutron event . . Acknowledged this issue by stating that the large singles count rate during the neutron event is not fully understood . However, the presence of an enhanced singles count rate does not invalidate other evidence indicating an absence of an energeticproton enhancement and locally generated neutrons . First, some portion of the singles count rates is likely to be the result of energetic electrons and photons generated by the interaction of neutrons with spacecraft material as well as the neutrons themselves . These particles will be present regardless of the source of the neutrons and therefore must be taken into account when attempting to understand the total singles count rate . Second, although share et al . Rightly asked why mercuryoriginating neutrons did not generate an enhanced singles count rate similar to that seen during the neutron enhancement on 4 june 2011, one mitigating factor to note is that the energy spectra for neutrons from mercury and solar neutrons need not be the same, especially for neutrons with energies greater than 8 mev . Whereas the ns efficiently detects and positively identifies neutrons with energies less than 8 mev, a solar spectrum can extend higher in energy, and such neutrons will more effectively generate energetic electrons and photons . In addition, the presence of betadecay electrons and protons that accompany the neutrons cannot yet be ruled out as a contribution to the singles count rates . Finally, for their fourth argument, share et al . Misinterpreted the messenger gammaray data and therefore contended that these data do not prove that energetic protons were absent during the neutron enhancement . Specifically, share attributed all of the deexcitation lines to inelastic neutron interactions and state that the line at 1.635 mev can only be produced by proton interactions . Specifically, we should have clarified the point that the 1635kev line is not produced by (n, n) neutron inelastic reactions but rather from the decay of na, which is not present on the spacecraft at levels detectable by messenger's gammaray spectrometer (grs) [peplowski et al .,, this gamma ray is produced by the deexcitation of na produced by spallation processes on other spacecraft materials (e.g., mg and al) and therefore requires protons (and neutrons) of higher energies than those from (n, n) reactions ., as illustrated by the markedly higher threshold energies for 1635kev gammaray production relative to the other examples shown in that figure . It is the higherenergy thresholds for 1635kev gammaray production that make the 1635kev gamma ray well suited for constraining the particle environment at messenger . We showed that the 1635kev count rate did not increase during the neutron event, even though other lines with lower gamma ray production thresholds did show large enhancements (see figures 1a and 10a of lawrence et al . ). This observation severely restricts the flux of protons (and neutrons) having energies> ~20 mev to levels below that detectable by the grs via 1635kev gammaray emission . The ~20 mev cutoff is derived from the gamma ray production cross sections (figure 3 of share et al . ). Further presented a simulated gammaray spectrum that would be produced by spacecraftlike material in response to an increased flux of protons and alpha particles that follow a powerlaw energy spectrum . Stated that the calculated photon spectrum using protons and alpha particles reproduced most of the features in the observed energy loss spectrum . Have therefore illustrated our point that for a plausible energetic ion spectrum, the 1635kev line should show an enhancement . That no statistically significant 1635kev enhancement above the cosmicray background was observed during the 4 june 2011 neutron event, despite the large enhancements seen in other gammaray peaks (figure 1a), locally generated gammaray fluxes observed by the messenger grs during the (a) 4 june 2011 and (b) 22 september 2011 events . Data represent difference spectra (before and during the enhanced particle events) derived from measurements acquired far from mercury (altitudes> 3000 km). Data were smoothed over 1.8 kev prior to subtraction, an interval that is approximately onethird the width of the gammaray peaks in this energy region . The insets highlight the 1635kev region and show that this peak was enhanced by a value of 2.3 0.4 relative to background during the 22 september 2011 event, but there was no statistically significant enhancement for the 4 june 2011 event . To demonstrate that some events do show an enhancement in the 1635kev gammaray line, figure 1b shows a solar particle event observed on 22 september 2011 when an increase of 2.3 0.4 in the 1635kev line relative to the background count rate was observed . This measurement indicates the presence of protons with energies in excess of 20 mev . We note that the energetic particle environment for the 22 september 2011 event differs from that for the 4 june 2011 event (figure 2). The ns singles count rates (figure 2a) are more than an order of magnitude smaller than the count rates for the 4 june 2011 event, whereas the double and triplecoincidence counters show clear enhancements (figure 2b), indicating that the 22 september 2011 event had a much harder energeticparticle spectrum than the 4 june 2011 event . The fast neutrons for the former event show a slight enhancement over background that reached a maximum at 1 cps around 18:00 utc (figure 2c). If we assume that all the coincidence count rates from the 22 september 2011 event are due to protons, we can use the modeled proton response of the double and triplecoincidence counters [lawrence et al ., 2014] and the measured coincidence count rates to estimate a spectral powerlaw index for this event . This proton spectrum may then be used to estimate the ratio of measured to locally generated neutrons following the formulation given by lawrence et al . . We determine this ratio to be 1.5, which is significantly smaller than the values of 2.75.4 obtained for the 31 december 2007 event [feldman et al . 2014] and> 500 obtained for the 4 june 2011 event [lawrence et al ., thus, for 22 september 2011, there is a much higher likelihood that the detected neutrons were locally generated than for the other reported neutron enhancements . This event thus supports the idea that the presence or absence of the 1635kev gammaray line indicates the presence or absence of energetic protons and further validates our model estimates of local neutron production . Ns count rate data for (a) singles counters, (b) coincidence counters, and (c) fast neutrons during the 22 september 2011 neutron event submitted . For the singles and coincidence counters, lg1 data are shown in red traces and lg2 data are shown in black traces . Borated plastic singles count rates, shown as the blue trace in figure 2a, are multiplied by 0.2 to be seen on the same scale as the lg singles . Triple coincidence count rates, shown as the blue trace in figure 2b, are multiplied by 3.5 to be seen on the same scale as the double coincidence count rates . The three fastneutron count rate peaks at rates greater than 10 cps denote neutron detections from mercury periapsis passes and are not related to the solar particle event . The lack of an increase in the 1635kev count rate during the 4 june 2011 event places strong constraints on whether a population of energetic protons (or neutrons) having energies greater than 20 mev was present . This upper limit restricts the allowed population of protons capable of producing local neutrons via spallation processes [e.g., filges and goldenbaum, 2009] to those with energies of 5 mev to 20 mev . The lower limit is derived from the coulomb barrier for the types of light nuclei present in the messenger spacecraft, which ranges from ~5 mev (c, atomic number z = 6) to ~12 mev (al, z = 13). Heavier nuclei, such as fe and ti, have enhanced neutron production via spallation, but their coulomb barriers for protons are above the 20 mev threshold established by the 1635kev gamma ray . In either case, for protons <20 mev, neutron production via spallation occurs via single neutron emission during the evaporation of compound nuclei . Compound nucleus formation is inhibited for light nuclei [krane, 1998] for which <20 mev protons are above the coulomb barrier . Regardless, this neutron production mechanism is taken into account in our simulations of particle transport through the messenger spacecraft . For these energies, neutron production per incident particle is highly inefficient, with a value of ~0.01 neutrons per incident proton [mckinney et al ., direct reactions (e.g., protons or alpha particles on c) offer an alternative source of neutrons from compound nucleus evaporation . Local neutrons from these reactions were treated by lawrence et al . In response to a prior critique by share et al . . Our models, which included the specific cross sections [koning and rochman, 2012] recommended by share et al ., considered incident particle energies of 10 mev or greater . Our models show that neutron production for lower ion energies is negligible, consistent with the general behavior of total nonelastic cross sections [barashenkov, 1993; carlson, 1996]. These observations reinforce our conclusion that local neutrons cannot account for the observed neutron enhancement, and as a consequence, the best explanation for the neutrons detected on 4 june 2011 continues to be that they are nonlocal in origin and most likely solar . Specifically, share et al . Grounded multiple arguments on the invalid assumption that only energetic protons trigger ns singles count rates . 's estimate of the energetic proton population prior to the neutron enhancement is inaccurate, and their estimate of local neutron production from the lg2 singles count rate is unreliable . Finally, their own simulated gammaray spectrum supports the contention that energetic protons with energies> 20 mev were absent during the neutron enhancement . There is therefore strong evidence that the neutrons detected from 15:45 utc to 16:45 utc on 4 june 2011 were solar in origin . We acknowledge that not all data taken during the neutron enhancement, especially the ns singles count rates, are understood . Further modeling of the ns instrument response and data analysis for the 4 june 2011 event and other events is therefore needed . Concluded their analysis by describing a type of directional neutron measurement that would enable future measurements to make less ambiguous identifications of solar neutrons . A simpler approach that requires fewer spacecraft resources particularly important for resourceconstrained inner heliosphere missions would be to use a simple neutron detector coupled with the type of energetic particle detectors used on the stereo and solar probe plus (spp) missions [von rosenvinge et al . The capability to model energeticioninitiated neutron production and transport is sufficiently mature that as long as the spectrum of the incoming energetic ion flux is known, modeled absolute neutron count rate uncertainties should be less than 20% [mckinney et al ., 2006; lawrence et al ., 2006], and relative neutron count rate uncertainties should be less than 0.5% for a variety of spacecraft locations and orientations with respect to an incoming directional flux [lawrence et al ., 2013]. The messenger payload was constrained by mass and cost limits and was, of course, selected on the basis of planetary science objectives [solomon et al ., 2007]. Nonetheless, had messenger been equipped with the type of energetic particle detectors that are on stereo and will fly on spp, the ambiguities associated with the neutron measurements of feldman et al . And lawrence et al.
It has a horse shoe shaped body which lodges the teeth and a pair of rami which project upwards from the posterior ends of the body and provide attachment to muscles . Each half of the mandible ossifies from only one center which appears at about the 6 week of intrauterine life in the mesenchymal sheath of meckels cartilage near the future mental foramen . The mental foramen opens below the sockets for the two deciduous molar teeth near the lower border, this is because the bone is made up of only the alveolar part with sockets . The mental foramen opens mid way between the upper and lower borders because the alveolar and sub alveolar parts of the bone are equally developed . The alveolar border is resorbed, so that the height of the body is markedly reduced . The mental foramen is an important anatomical structure situated in antero lateral aspect of the body of the mandible which transmits mental nerve, artery and vein . Mental nerve is a terminal branch of the inferior alveolar nerve which supplies sensory innervations to lower lip, buccal vestibule and gingiva mesial to the first mandibular molar . Mental foramen serves as an important anatomical landmark, the orientation and position of which facilitate local anesthetic, surgical and other invasive procedures for oral and maxillofacial surgeries . Its location and the possibility that an anterior loop of the mental nerve may be present mesial to the mental foramen needs to be considered before any surgery in the foramina area in order to avoid injuring of the neurovascular bundles passing through these foramina and notches . The foramen may be occasionally misdiagnosed with a radiolucent lesion in the apical area of the mandibular premolar teeth . So identification of accurate anatomical position of mental foramen is very important in periodontal surgery especially during flap surgery in lower teeth, apical curettage of mandibular premolars, retrograde amalgam fillings and surgical orthodontics . Replacement of missing teeth by dental implant and the increasing frequency of orthognathic surgery have increased the possibility of surgical procedures near the mental foramen . Local anesthesia of the terminal incisive branches of the inferior alveolar and mental nerves can be obtained effectively if the mental foramen is correctly identified . Localization of mental foramen radiographically is difficult due to lack of consistent anatomical landmarks for reference and the foramen cannot be clinically visualized or palpated . Therefore the location of the mental foramen had been studied by means of direct measurement on dry mandibles or by using radiographs of dry mandibles in patients . As the bone density increases the mental foramen becomes more difficult to identify on radiographs . Knowledge of the most common position of the mental foramen in the population may give additional information in the mental nerve blocks and related mandibular surgeries . So the present study had been undertaken to determine the morphological features and morphometrics of mental foramen with reference to surrounding anatomical landmarks in both dentate and edentulous mandibles in coastal andhra population of andhra pradesh state . A total of 219 dry dentate and edentulous mandibles are examined in this study, out of these 127 were dentulous and 92 were edentulous . Various parameters were measured by using digital vernier caliper, metallic wire and metallic scale on both the right and left sides . Before measuring, the mandibles were placed on a standard horizontal plane to which the lower border of the mandible get its most contact when vertical pressure is applied to the molar region . [pr - mf] distance between posterior border of ramus [pr] of mandible and most anterior margin of mental foramen [mf] [figure 1][sm - mf] distance between symphysis menti [sm] and most anterior margin of mental foramen [figure 2][mf - ifb] distance between inferior margin of mental foramen and inferior border of mandible . [ifb] [figure 3][ac - mf] distance between alveolar crest and inferior margin of mental foramen [figure 4][a1] distance between alveolar crest to upper most point of mental foramen [figure 5][a2] distance between alveolar margins to the apex of second premolar socket [figure 6][a1-a2] distance between alveolar crest to upper most point of mental foramen - distance between alveolar margins to the apex of second premolar socket [figure 7]incidence of mental foramen in relation to first premolar, second premolar and first molar on right side in dentulous mandibles [figure 8]incidence of mental foramen in relation to first premolar, second premolar and first molar on left side in dentulous mandibles [figure 9]edentulous mandible showing accessory mental foramen [figure 10]. [pr - mf] distance between posterior border of ramus [pr] of mandible and most anterior margin of mental foramen [mf] [figure 1] [sm - mf] distance between symphysis menti [sm] and most anterior margin of mental foramen [figure 2] [mf - ifb] distance between inferior margin of mental foramen and inferior border of mandible . [ifb] [figure 3] [ac - mf] distance between alveolar crest and inferior margin of mental foramen [figure 4] [a1] distance between alveolar crest to upper most point of mental foramen [figure 5] [a2] distance between alveolar margins to the apex of second premolar socket [figure 6] [a1-a2] distance between alveolar crest to upper most point of mental foramen - distance between alveolar margins to the apex of second premolar socket [figure 7] incidence of mental foramen in relation to first premolar, second premolar and first molar on right side in dentulous mandibles [figure 8] incidence of mental foramen in relation to first premolar, second premolar and first molar on left side in dentulous mandibles [figure 9] edentulous mandible showing accessory mental foramen [figure 10]. [pr - mf] distance between posterior border of ramus of mandible and most anterior margin of mental foramen [sm - mf] distance between symphysis menti and most anterior margin of mental foramen [mf - ifb] distance between inferior margin of mental foramen and inferior border of mandible [ac - mf] distance between alveolar crest and inferior margin of mental foramen [a1] distance between alveolar crest to upper most point of mental foramen [a2] distance between alveolar margins to the apex of second premolar socket [a1-a2] distance between alveolar crest to upper most point of mental foramen - distance between alveolar margins to the apex of second premolar socket incidence of mental foramen in relation to first premolar, second premolar and first molar - right side of dentulous mandibles incidence of mental foramen in relation to first premolar, second premolar and first molar - left side of dentulous mandibles edentulous mandible showing accessory mental foramen the mean values are expressed as mean standard deviation with confidence interval of 95% . [pr - mf] distance between posterior border of ramus [pr] of mandible and most anterior margin of mental foramen [mf] [figure 1][sm - mf] distance between symphysis menti [sm] and most anterior margin of mental foramen [figure 2][mf - ifb] distance between inferior margin of mental foramen and inferior border of mandible . [ifb] [figure 3][ac - mf] distance between alveolar crest and inferior margin of mental foramen [figure 4][a1] distance between alveolar crest to upper most point of mental foramen [figure 5][a2] distance between alveolar margins to the apex of second premolar socket [figure 6][a1-a2] distance between alveolar crest to upper most point of mental foramen - distance between alveolar margins to the apex of second premolar socket [figure 7]incidence of mental foramen in relation to first premolar, second premolar and first molar on right side in dentulous mandibles [figure 8]incidence of mental foramen in relation to first premolar, second premolar and first molar on left side in dentulous mandibles [figure 9]edentulous mandible showing accessory mental foramen [figure 10]. [pr - mf] distance between posterior border of ramus [pr] of mandible and most anterior margin of mental foramen [mf] [figure 1] [sm - mf] distance between symphysis menti [sm] and most anterior margin of mental foramen [figure 2] [mf - ifb] distance between inferior margin of mental foramen and inferior border of mandible . [ifb] [figure 3] [ac - mf] distance between alveolar crest and inferior margin of mental foramen [figure 4] [a1] distance between alveolar crest to upper most point of mental foramen [figure 5] [a2] distance between alveolar margins to the apex of second premolar socket [figure 6] [a1-a2] distance between alveolar crest to upper most point of mental foramen - distance between alveolar margins to the apex of second premolar socket [figure 7] incidence of mental foramen in relation to first premolar, second premolar and first molar on right side in dentulous mandibles [figure 8] incidence of mental foramen in relation to first premolar, second premolar and first molar on left side in dentulous mandibles [figure 9] edentulous mandible showing accessory mental foramen [figure 10]. [pr - mf] distance between posterior border of ramus of mandible and most anterior margin of mental foramen [sm - mf] distance between symphysis menti and most anterior margin of mental foramen [mf - ifb] distance between inferior margin of mental foramen and inferior border of mandible [ac - mf] distance between alveolar crest and inferior margin of mental foramen [a1] distance between alveolar crest to upper most point of mental foramen [a2] distance between alveolar margins to the apex of second premolar socket [a1-a2] distance between alveolar crest to upper most point of mental foramen - distance between alveolar margins to the apex of second premolar socket incidence of mental foramen in relation to first premolar, second premolar and first molar - right side of dentulous mandibles incidence of mental foramen in relation to first premolar, second premolar and first molar - left side of dentulous mandibles edentulous mandible showing accessory mental foramen the mean values are expressed as mean standard deviation with confidence interval of 95% . Morphometric features of 219 mandibles revealed the following results [table 1]. Depicting the values of all parameters distance between anterior margin of mental foramen to the posterior border of ramus of mandible [mf - pr] in dentate mandibles is 69.61 6.02 mm on right side and 69.17 6 mm on left side, whereas for edentulous mandibles the values have slightly decreased and it is 68.39 6.4 mm on right side, 68.82 6.55 mm on left side . The distance between symphysis menti and most anterior margin of mental foramen [mf - sm], appears to be the same for both dentate and edentulous mandibles . Mf - sm in dentate mandibles on right side is 28.24 5.09 mm and on left side it is 27.45 3.7 mm whereas for edentulous mandibles it is 28.51 4.53 mm on right side and is 27.99 4.5 mm on left side . Distance between alveolar crest and inferior margin of mental foramen [ac - mf] in dentate mandibles is 15.29 3.71 mm on right side and is 14.38 3.75 mm on left side whereas for edentulous mandibles it is 15.50 3.71 mm on right side and on left side it is 14.70 4.43 mm which also appears to be the same for dentate and edentulous mandibles . Distance between inferior margin of mental foramen and inferior border of mandible [mf - ifb] in dentate mandibles on right side is 12 2.37 mm and on left side is 12.39 2.74 mm whereas for edentulous mandibles on right side it is 13.40 4 mm and on left side it is 13.56 4.04 mm which shows that the distance between these parameters is increased in edentulous mandibles compared to dentulous mandibles . Distance between alveolar crest to upper most point of mental foramen [a1] in dentate mandibles is 13.18 2.32 mm on right side and is 12.56 2.40 mm on left side, whereas for edentulous mandibles on right side it is 11.09 3.41 mm and on left side is 10.01 2.68 mm which shows the distance has significantly reduced in edentulous mandibles . Distance between alveolar margins to the apex of second premolar socket [a2] in dentate mandibles is 8.93 2.33 mm on right side and is 8.75 2.34 mm on left side whereas for edentulous mandibles it is 6.16 2.60 mm on right side and is 5.38 2.42 mm on left side which reflects a decrease of values for edentulous mandibles . Distance between alveolar crest to upper most point of mental foramen - distance between alveolar margins to the apex of second premolar socket [a1-a2], in dentate mandibles on right side is 4.18 1.71 mm and on left side it is 3.85 1.85 mm and for edentulous mandibles on right side it is 4.80 2.10 mm and on left side is 5.18 1.92 mm . The shape of mental foramen was visually observed and analyzed in all the 219 mandibles and it was found that in 87 cases it is round and 40 cases it is oval in dentate mandibles and in 56 cases it is round and in 36 cases it is oval in edentulous mandibles on right side . On left side, it is round in 99 cases and oval in 28 cases in dentate mandibles and in edentulous it is round in 57 cases and oval in 35 cases . The position of mental foramen on right side and left side in both dentate and edentulous mandibles in maximum cases is in between the first and second premolars [40 - 50%]. The mental foramen is usually a single opening on the antero lateral surface of the mandible, 13 - 15 mm superior to the inferior border of the body of mandible which is generally seen to be circular or oval in shape . Through this foramen, the mental neurovascular bundles exits . It remains incomplete till the 12 gestational week until the mental nerve separates into several fasciculi at that site . Identifying the precise location of mental nerve is critical while performing peri apical surgery, cyst enucleation, endosseous implant, periodontal surgery and surgical orthodontic treatments such as a mandibular body osteotomy, in order to prevent damage to the mental nerve . In addition, knowing the site of the mental foramen allows for the accurate delivery of local anesthesia during dental procedures . There can be neuro sensory disturbances encountered if this important landmark is ignored while doing any invasive treatment in this region . The interval between the two premolars is the most common site of the mental foramen and the apex of second premolar is the second most common site . The results of the present study conclude the same . In 42.5% mandibles on right side and 40.9% on left side of dentate mandibles the common site is the interval between the two premolars and in 18.1% of the mandibles it is at the apex of second premolar . Santini and land et al ., reported that in chinese the mental foramen was in line with second premolar and in british between first and second premolars . In edentulous mandibles, 50% of the mandibles on right side and 40.9% of the mandibles on left side, the mental foramen was located between first and second premolars . The second most common site is apex of second premolar which is seen in 25.2% of the mandibles, which is in accordance with the study conducted by shankland, wang, green . The position of the mental foramen varies depending on various factors like symmetry of mental triangle, microscopic and macroscopic morphology and maturity of the human mandible, bone remodeling activity and anthropologic features of the facial skeleton in different populations . According to the present study the distance between the apex of lower second premolar socket to superior border of mental foramen (a1-a2) is 3.8 mm in dentate mandibles and 4.5 mm in edentulous mandibles . Thus, when endodontic treatment is planned for these teeth, over obturation of root canals can lead to impingement and irritation to the mental nerve . Wang reported that the average distance between the apex of the lower second premolar socket and superior border of mental foramen is 2.5 mm . Such a short distance can harm the mental nerve if the root canal is overfilled during root canal treatment procedures . In the present study, the distance between inferior margin of mental foramen to the inferior border of mandible (mf - ifb) is 12.39 mm in dentate mandibles whereas 13.5 mm in edentulous mandibles which is in correlation with chung et al ., they reported that the average distance from the inferior border of the mandible to the center of mental foramen was 15.5 mm for korean males and 14.0 mm for korean females . Gershchenson et al ., reported that the location of the mental foramen in relation to the border of the mandible and teeth depended on age, tooth condition and the degree of resorption . In adults as age advances, mental foramen shifts toward the superior border of mandible . This can be attributed to tooth loss followed by bone resorption . With the loss of teeth, there is change in the relative position from midlevel toward the upper border of the mandible . In severe cases of alveolar resorption the bone loss is so much that the incisive and inferior alveolar nerves are exposed out of bone and lie just below the mucosa which may cause pain on denture wearing . Accordingly, the results of the present study also showed that the values decreased in edentulous mandibles . Soikkonen et al ., reported that the mental foramen moves toward the lower cortex of the mandible as a result of alveolar atrophy and was situated on average 3.8 mm lower in edentulous jaws than in dentate jaws . In our present study, the distance between alveolar crest and inferior margin of mental foramen [ac - mf] in dentate mandibles is around 3.71 mm and in edentulous mandibles it slightly increased to 4.43 mm . The distance between the posterior border of ramus of mandible and most anterior margin of mental foramen is very important, as nerve and vessels pass through it and care is taken to avoid complications during inferior alveolar nerve blocks, peri apical surgeries, apical curettage and implant placement and nerve repositioning . In the present study, in dentate mandible the distance between most anterior margin of mental foramen and posterior border of ramus of the mandible (mf - pr) on right side is 69.61 6.03 mm, left side is 69.17 6.0 mm and in edentulous mandibles (mf - pr) on right side is 68.39 6.4 mm, left side is 68.81 6.55 mm . The distance between symphysis menti and most anterior margin of mental foramen (mf - sm) in dentulous mandible on right side is 28.24 5.09 mm, and on left side is 27.45 3.7 mm and in edentulous mandible (mf - sm) on right side is 28.51 4.53 mm, and on left side are 27.99 4.50 mm . Clinically, there may be instances where the mental foramen cannot be localized without a fixed reference point . In such cases, it can be localized if the distance from the symphysis menti is known . Even so, in order to avoid neurovascular complications, particular attention should be paid to the possible occurrence of one or more accessory mental foramen during surgical procedures . Utmost care to the accessory mental foramen and nerve is essential during dental implant surgery and any surgical procedure involving the mandibular molar and premolar region . This care may reduce the rate of paralysis and hemorrhage in mental region, lower lip and gingiva from the mental foramen to the midline of the ipsilateral side . In the present study out of 127 dentulous mandibles 13.97% accessory mental foramens were found whereas out of 92 edentulous mandibles 2.76% of accessory mental foramen were found . Out of these 219 mandibles none of them presented with more than two accessory mental foraminae . Gershchenson et al ., reported 7.51% of double foramina in indian and israeli mandibles . And zografos and mutzuri studied the incidence of double foramina in 11.48% of north indians and 6.68% of greek population, respectively . In the present study, 68.5% of dentulous mandibles on right side and 78% on left side, the foramen is round in shape and oval in 31.5 and 22%, respectively . In edentulous mandibles, it is found that 60.9% is round on right side and 62% is round on left side, whereas, it is oval in 39.5 and 38%, respectively . These values are similar to study reported by mbajiorgu et al ., and junior et al . According to al - khateeb et al ., majority of foraminae were round in shape which is similar to the present study . The present analysis revealed variations in position, shape and size of the mental foramen . The knowledge of exact position and various distances of mental foramen are necessary for its anesthetic and surgical intervention . Thus, it is important to know the anatomical variations of mental foramina in order to avoid nerve damage in connection with surgical procedures . Since this study had been conducted in the local population, the ethnic and racial variations are not highlighted . Further studies focusing on racial and ethnic variations in different population groups need to be conducted on a large scale to substantiate this study . The knowledge of the position of mental foramen is important for day - to - day clinical practice . This variability of mental foramen should alert the dental surgeons while performing periodontal or endodontic surgery . The knowledge of the relationship of the foramen, in surgical procedures, in relation to anatomical landmarks can be of assistance in the localization of important maxillofacial neurological structures.
The incision extends along the anterior third of the iliac crest to the anterior superior spine, then curves slightly medially along the lateral border of the sartorius muscle . Immediately inferior to the anterior superior spine one finds the plane of division between the sartorius and the tensor fasciae femoris . The femoral fascia is incised along the lateral border of the sartorius, exposing the direct head of the rectus femoris . By sharp and blunt dissection the attachment of the direct head of the rectus femoris to the anterior inferior spine is defined.fig . 2the incision extends along the anterior third of the ilium and curves mesially along the lateral border of the sartorius . The incision extends along the anterior third of the ilium and curves mesially along the lateral border of the sartorius . 3) is to identify the plane of division between the iliopsoas muscle and the proximal portion of the reflected head of the rectus femoris; the latter takes origin from the anterior acetabular wall as well as from the anterior capsule . In order to obtain a better exposure of the lateral portion of the acetabulum, the tendon of the direct head of the rectus femoris is divided, leaving sufficient tendon attached to the anterior inferior spine to allow for suture . When the distal portion of this muscle is retracted, the reflected head of the rectus comes into view, partly fused with the anterior capsule, partly attached to the anterior acetabular margin . This is dissected free and retracted laterally with the direct head of the rectus femoris.fig . 3exposure of the anterior superior spine, of the lateral border of the sartorius, and of the mesial border of the tensor fasciae latae . Between these muscles is a compartment of fat with the rectus femoris as its floor.fig . 4reflection of the abdominal oblique, sartorius, and iliopsoas muscles mesially, exposing the anterior aspect of the hip joint . Exposure of the anterior superior spine, of the lateral border of the sartorius, and of the mesial border of the tensor fasciae latae . Between these muscles is a compartment of fat with the rectus femoris as its floor . Reflection of the abdominal oblique, sartorius, and iliopsoas muscles mesially, exposing the anterior aspect of the hip joint . By the procedures described sufficient exposure of the anterior acetabular wall is usually obtained, but in some cases the iliopsoas muscle remains too taut to be retracted . In these cases it is advisable to reflect subperiosteally the origin of the sartorius and the abdominal oblique muscle from the anterior crest of the ilium, exposing the anterior portion of the iliac fossa . The iliacus may then be reflected subperiosteally down to the upper margin of the anterior acetabular wall, but at this point the periosteum is divided along the line of the intended osteotomy . The above procedures allow for retraction of the iliopsoas muscle and of the sartorius mesially, and of both the direct and the reflected heads of the rectus femoris, and of the tensor fasciae femoris laterally . If the retraction mesially of the iliopsoas muscle is found difficult, the hip may be flexed, adducted, and externally rotated; by this procedure, the iliopsoas becomes relaxed and consequently may be retracted more easily . 5. )fig . 5showing the line of osteotomy of the anterior acetabular wall immediately below the attachment of the direct head of the rectus femoris . Showing the line of osteotomy of the anterior acetabular wall immediately below the attachment of the direct head of the rectus femoris . The next step is the osteotomy of the anterior acetabular wall; this may be done with a thin osteotome or gouge . The osteotomy starts just below the attachment of the direct head of the rectus femoris and is carried mesially a distance of approximately an inch and a half and is then curved inferiorly clown to the cotyloid notch . As soon as the fragment thus outlined becomes mobile the anterior superior capsule is incised down to the region of the distal neck . It is also incised inferiorly from the cotyloid notch along a line just proximal to the capsular attachment to the neck of the femur . When the anterior acetabular wall with its attached capsule is lifted out, there is obtained a beautiful exposure of the articular surface of the head of the femur and of the anterior aspect of the neck.fig . 6result of plastic procedure: exposure of the mesial portion of the femoral head and of the anterior femoral neck . Result of plastic procedure: exposure of the mesial portion of the femoral head and of the anterior femoral neck . By moving the hip in flexion, extension, abduction, and internal rotation if not, further excision of the superior acetabular margin and of the superior anterior capsule is done . The surgeon must be radical in performing this plastic procedure, but he should not remove so much of the superior aspect of the acetabulum as to allow the head of the femur to become displaced anteriorly . Of this there is relatively little danger, since hyperextension is rarely obtained and this is the motion which would allow the femoral head to dislocate . The two blood vessels encountered are the deep iliac circumflex and the ascending branch of the internal femoral circumflex . The former is easily eliminated; the latter sometimes gives rise to a little difficulty because of its extensive ramifications in the fat anterior and inferior to the hip joint . The closure of the wound is very simple indeed . When the retractors are removed the iliopsoas drops back into place over the anterior aspect of the head and neck of the femur . The direct apposition of the posterior surface of this muscle to the anterior aspect of the head and neck of the femur is of great advantage, two moving surfaces are very much less liable to form adhesions to one another than are two surfaces in more or less constant apposition, such as would be true of the capsule and of the underlying joint surface . The direct head of the rectus is sutured to its origin, and the superficial portion of the wound is closed in layers . Postoperative convalescence is remarkable because of the relative absence of pain . The patient complains of soreness, but rarely of acute pain . The extremity is suspended with five pounds of traction, simply to overcome muscle spasm and thereby to diminish pain . The position should be one of extension with maximum abduction and maximum internal rotation . At the end of two weeks the patient is allowed up on crutches and, as a rule, he leaves the hospital at the end of three weeks; the maximum period of hospitalization is four weeks . He is taught exercises which he thoroughly understands by the time he leaves the hospital and which he carries on at home . As already stated the main effect of this procedure is relief from pain: however, there is also considerable improvement in function . The increased function is the direct result of the removal of the acetabular wall, thus preventing impingement . If the anterior femoral head and neck are very prominent, it may at times be advisable to do a plastic operation on these as well as on the acetabulum . There is no danger of new bone formation or exostoses as a result of this procedure, since mechanical interference has been eliminated and since medullary bone is in apposition to the moving posterior surface of the iliopsoas muscle . The removal of the anterior capsule is probably an important factor in eliminating pain, because of its sensory innervation . It is important to remove sufficient capsule, including the y ligament of bigelow, to diminish the chances of adhesions and of scar formation . Another point to be emphasized in the technique is this: the acetabular wall should not be exposed subperiosteally, but its periosteum should be removed with it . The first case treated by this operative method was a case of bilateral intrapelvic protrusion of the acetabulum the patient was a woman, fifty - five years of age, who for months had been disabled for her occupation as a housekeeper . She was in pain lying in bed, sitting, standing, and walking . As a result of the operation she was able to return to work, free from pain and without even a limp . Figure 7 shows very definite protrusion of the acetabulum on the left and the same condition, but to a lesser extent, on the right . Ten months after the operation function was as follows: complete extension (fig . 8), flexion to 90 degrees (fig . 9), and a perfectly definite amount of abduction (fig . 10).fig . 7preoperative roentgenogram showing definite protrusion of the acetabula, more marked on the left than on the right.fig . 8 fig 10 preoperative roentgenogram showing definite protrusion of the acetabula, more marked on the left than on the right . As has been stated in the introduction, the success in the above case led us to apply it to cases of malum coxae senilis . The first case was a man of fifty - eight, whose occupation was that of a brakeman . He had symptoms from the right hip for a period of years and had been disabled for several months . . 11 and 12) showed changes in both hips, hypertrophic changes and thinning of the joint cartilage, more marked on the right than on the left . He was allowed up on crutches in seventeen days and was discharged on the twenty fifth day . At the time of discharge he got about very well indeed and was able to put on his right shoe for the first time in years . In five months he returned to his former occupation, which required jumping on and off trains he was allowed to do this by promising that he would not jump on his right foot . When last seen, eight months after the operation, he had been back at work for three months; he complained of no pain and walked with a scarcely noticeable limp . Figures 13, 14, and 15 show this patient s function at this time . There was permanent flexion of 20 degrees, motion in flexion to a good 90 degrees, and very little abduction (it was estimated as 10 to 15 degrees).fig . The line of osteotomy of the acetabulum is indicated by arrows.fig . 13 fig . This case is important because the patient had two more years to go before he could obtain a pension . If it is proved that this procedure does not give permanent relief, it may at least be useful in producing temporary relief and improved function over an important period of time . He had been in constant pain for several months before admission to the hospital . According to his story in the course of five months the patient returned to his blacksmith shop to do a couple of hours work a day . At the end of nine months he worked all day when he could find the work to do . Even at the time when this report was written, ten months after the operation, he walked with a very distinct limp, but this limp was unaccompanied by pain . Function at the end of nine months was as follows: 30 degrees of permanent flexion, motion in flexion to a good 90 degrees, abduction of approximately 10 degrees (figs . Eleven cases have been treated by this method, eight of malum coxae senilis, two of old slipped femoral epiphysis, and one of intrapelvic protrusion of the acetabulum . They have all done well; the relief from pain is the outstanding feature; gain in motion is definite but not marked . The outstanding feature of the postoperative period is the slight amount of pain complained of; some patients state that they have no pain at all, others say they have soreness, but none complain of acute pain . The affected extremity is suspended with five pounds of traction in the optimum position, maximum abduction and internal rotation, minimum flexion . Following two weeks of recumbency the period of hospitalization should be a minimum of three weeks, a maximum of four . When the patient leaves the hospital he should be thoroughly trained in exercises to maintain the corrected position . In the cases so far operated upon, the author s attention has been directed chiefly to the anterior acetabular wall and to the anterior capsule of the hip joint . (see fig . 21 .) In some cases a plastic procedure has been performed on the anterior femoral head and neck, when they have been the seat of advanced proliferative changes . The inferior portion of the head and neck has likewise been removed for the same reason; this has been done in two cases only . This may be one reason why a greater improvement in range of motion has not been obtained . 21characteristic appearance of specimen removed, consisting of: a, acetabular wall; b, attached capsule . 1 shows the specimen as viewed from the outside; 2 shows the intra - articular synovial surface . Characteristic appearance of specimen removed, consisting of: a, acetabular wall; b, attached capsule . 1 shows the specimen as viewed from the outside; 2 shows the intra - articular synovial surface . Gentle manipulation for the sake of increasing the range of motion has been carried out in some of the cases at the end of the operative procedure . It is the author s feeling that this is distinctly worth while and should be done routinely . A plastic procedure has been proposed for the relief of hip - joint conditions resulting from interference with the normal mechanics of the hip joint . Such conditions are malum coxae senilis, intrapelvic protrusion of the acetabulum, old slipped upper femoral epiphysis, fractures of the neck of the femur with malposition, legg - calv - perthes disease, and fractures of the acetabulum . Sufficient time has not elapsed to obtain true end results, but the author feels justified in rendering this preliminary report because the method is non - destructive and seems effective in relieving pain in conditions for which there is no other adequate treatment.
Giardia lamblia, probably the most frequent pathogenic intestinal protozoan infection in man, is an important cause of diarrhoeal disease in children and adults throughout the world . Although giardiasis the disease this protozoan causes is usually perceived as mild and self - limiting, symptoms generally subside within 2 - 3 weeks in otherwise healthy individuals . This infectious disease may have both immediate and long - term consequences including chronic diarrhoea with or without dehydration and intestinal malabsorption, recurrent abdominal pain, and weight loss . Additionally, it has been currently related to chronic fatigue, postinfectious irritable bowel syndrome [3, 4], and particularly, in early childhood, poor cognitive function and failure to thrive; all of these have attracted an increasing attention to this protozoan infection in the recent years . Despite its worldwide distribution, data from surveys, excluding documented outbreaks, indicate that in industrialized countries the prevalence rate is between 2% and 5% . Data on the prevalence of giardiasis in cuba is limited; in a national survey the overall prevalence of giardia infection was estimated to be 6.02% . However, higher prevalences have been found among young children attending day - care centres and primary schools [710]. Clinical giardiasis also gives the impression to be a common reason for hospitalization in paediatric hospitals in the capital of cuba [11, 12]; where, according to a study of risk factors for giardia infection among hospitalized children, it seems that, at least at the individual level, giardiasis - prevention activities in havana should be focused on health education to improve personal hygiene and food - related practices . Health education (he) continues to be one of the most important strategies in the fight against intestinal parasites . Appropriate management of giardiasis, including patient education to promote adherence to preventive measures that may protect against infection and reinfection, not only might reduce the frequency of outpatient clinic visits and hospitalizations for giardiasis, but also could reduce the costs associated with this disease . In childhood giardiasis, mothers and other caregivers, are therefore, of foremost importance in recognizing the disease and seeking treatment for their wards . Very little is known about the perceptions of parents and carers when dealing with giardiasis in the paediatric setting . Their ideas and behaviours concerning giardiasis might enhance or interfere with the effectiveness of giardiasis prevention and control . A qualitative study in cuba by escobedo et al ., in 2011, reported deficiencies in the children caregivers' knowledge and practices concerning giardiasis . It is important to assess the current knowledge about giardiasis before devising an intervention strategy for education campaigns among general population . The purpose of this study was to (1) explore the perceptions of a group of caregivers about medical aspects of giardiasis (clinical features, modes of transmission, prevention, and consequences), its prevention and treatment; (2) assess the sources and channels of information caregivers would prefer using to be informed about giardiasis . All of this might highlight areas where the support of health care professionals can be improved . This was a descriptive cross - sectional study conducted between january and march 2010, at academic paediatric hospital centro habana (aphch), havana, cuba . This 226-bed study hospital is a government - funded and public facility which provides secondary and tertiary medical care and active ambulatory and emergency services, within all clinical and surgical specialities, for children from all over cuba but mainly to the paediatric population from the municipalities of centro habana, cerro, habana vieja, and plaza municipalities . The study included respondents aged> 18 years who were willing to participate in the study and gave individual verbal consent . Only health professionals and students in the medical professions a structured questionnaire was designed on the basis of a qualitative research conducted in the same hospital by the authors . The questionnaire was peer - reviewed and discussed with health care professionals working within paediatric wards and also was pilot - tested on random patients' relatives at the hospital over a 1-week period to evaluate the language used, to verify the relevance of the questions, and for clarity and consistency . It focused on basic demographic data (age and gender), education, and questions about their knowledge regarding giardiasis, mode of transmission, organ affected, its suggestive signs and symptoms, prevention, most reliable method of diagnosis, and whether giardiasis was a curable disease . Additionally, caregivers were also assessed about from which sources and channels they would like to receive information on giardiasis . Answers were categorized into either correct (if matching the medically correct answer) or incorrect . All consecutive parents / carers who fulfilled the eligibility criteria for inclusion and who agreed to participate were recruited if they were either new attenders or reattending . Respondents were adult members of patients' families (parents / carers) who visited the outpatient clinic of aphch for gastrointestinal problems . After the interviewers, who were three of the research team authors (y. salazar, m. alfonso, and i. v. dawkins), explained the purpose of the study and obtained individual verbal consent from the respondents, they administered the questionnaire to parents / carers, while waiting for the consultation of their child . The interviews were conducted in private to maintain confidentiality and to reduce the influence of relations of peers . Verbal informed consent was obtained from the study participants before the research instruments were administered . Confidentiality of all information obtained from participants was maintained by not allowing information to be accessible to nonmembers of the research team . Database was prepared using epi info version 6.0, and accordingly data entry was done . Frequency distributions were obtained . In the exploration of possible associations between respondent characteristics and awareness about transmission and prevention, analysis was done taking into account education of respondents, which was categorized into two categories (less than 12 years of formal education or 12 years of formal education) and age of respondents (less than 40 year of age and 40 years of age). Crude odds ratio (or), their 95% confidence intervals (cis) and p values were calculated with set at 0.05 . Of the 202 caregivers interviewed, 177 (87.6%) were females and 25 (12.4%) were males . A substantial proportion of caregivers, 157 (77.7%), had received 12 years of formal education or more, and 128 (63.3%) were employed outside home . Of the 202 caregivers, more than half (72.7%) considered that giardiasis is a modern problem, and, although almost half (47.5%) stated that this disease could not lead to death, 39.1% did, and 13.4% did not know . Small intestine was correctly identified as the site of giardiasis by 125 (61.9%) of the caregivers . However, more than one organ was mentioned as a possible target of giardia infection including gallbladder (66 (32.7%)) and liver (54 (26.7%)). For 127 (62.9%) of respondents those who harbour giardia infection thirty - two (15.8%) of the respondents did not have knowledge about the signs and symptoms of giardiasis . Those having knowledge mentioned abdominal pain (77.6%), diarrhoea (70.6%), and loss of weight 59.4% as the symptoms of giardiasis . However, other clinical manifestations including hives, itching, spots in the skin, and tiredness were mentioned for more than 30% (table 1). The majority of caregivers, 171 (84.6%), considered the microscopy of duodenal aspirate as the most effective diagnostic tool for giardiasis . The microscopy of faecal smears was only cited by 18 (8.9%) respondents (table 1). One hundred and sixty - nine caregivers (83.6%) in this study knew that giardia could be transmitted through drinking unboiled water and unwashed vegetables and fruits intake . The other modes of transmission mentioned by interviewees were walking barefoot and using utensils previously used by patients with giardiasis (table 2). Those with an education of 12 years of formal education or more were four times more likely to be aware of prevention than those with less than 12 years of education (or = 4.5, 95% ci = 1.910.8). Interaction between knowledge of transmission and age was tested and found to have no significance (table 3). Nearly all caregivers (95.5%) almost all the respondents 175 (86.3%) considered giardiasis to be preventable, most of them (169 (83.6%)) believe that prevention could be achieved by drinking boiled water, washing hands, and washing vegetables and fruits; 86.3%, 89.6%, and 91.5%, respectively . Other actions including avoiding walking barefoot and avoiding utensils used by patients with giardiasis were also mentioned by a group of them (table 5). Those with an education of 12 years of formal education or more were nearly three times more likely to be aware of prevention than those with less than 12 years of education (or = 2.8, 95% ci = 1.35.9). No significant association was observed between knowledge of prevention and age (table 5). One hundred and fifty - nine of the caregivers stated that they did not receive information on giardiasis during the last year from any source or channel . Caregivers were asked which methods they would prefer to know or to improve their knowledge about giardiasis . Among the suggested sources and channels, television was mentioned by 130 (64.3%), nurses and medical doctors 107 (52.9%), printed materials leaflets, posters, and pamphlets94 (46.5%), and radio 35 (17.3%). The goals of treating symptomatic giardiasis properly are to minimize the duration of illness, ensure cure, stop transmission of infection, and could also be to prevent the emergence of drug resistance . Caregivers' input, as in other areas of medicine involving children, seems to be critical to clinical management in paediatric giardiasis . The importance of providing caregivers with appropriate information is to enable them to participate effectively in giardiasis recognition, and preventative measures should be recognized . Even when there were some important gaps in various issues, it was encouraging to observe that in most issues caregivers' perceptions on giardiasis closely paralleled that of the biomedical understanding . This coincidence should be used as an entry point for a broader he activity, which is then expanded to also accommodate other disease aspects, that is, its mode of transmission, signs and symptoms, and appropriate prevention methods, to provide accurate and complete knowledge on giardiasis . Internationally, this disease is gaining increasing attention, first as a reemerging infectious disease in industrialized countries and, second, as a part of the neglected disease initiative by the world health organization . Its diagnosis has been established progressively with improving sanitation and with fading of the role of fatal epidemic infections as the overwhelming everyday problem . These, together with the fact that in cuba high prevalences have been reported among young children attending day - care centres and primary schools [7, 8], might be justifying the perception held by most respondents that this disease is a modern problem . Additionally, giardia has also been frequently isolated from faeces of cuban children with diarrhoea, and it seems to be a common cause of children hospitalization [11, 12, 16]. These could have led some caregivers to conclude that this disease could cause death . Giardia organisms primarily affect the proximal small intestine; however, during the last decades, there have been various reports concerning other localizations of giardia trophozoites including gallbladder, urinary tract, gastric mucosa [1921], colonic and ileal mucosa [22, 23], and pancreas . The majority of caregivers were able to recognize that giardia infection is frequently asymptomatic, and diarrhoea and abdominal pain are the clinical manifestations of symptomatic giardiasis . This is probably due to the educational message in the mass media stating that two of the primary clinical features of symptomatic intestinal parasitic diseases are diarrhoea and abdominal pain . This could be of importance, taking into account that an early treatment in some aspects could depend upon prompt recognition of symptoms and signs of giardiasis in the household, mainly by caregivers . It was surprising because, although this sign has been reported associated with giardiasis in the scientific literature [2528], it is not so common . Considering the frequent asymptomatic nature of this infection and the high prevalence of this protozoan worldwide, urticaria most of the times indicates that it is necessary to look for an alternative diagnosis . Nevertheless, it should be stated that, coincidently, in a survey of knowledge, perceptions and practices about giardiasis in 63 gastroenterologist of havana, it was evidenced that 55.6% of these specialists in havana stated that this parasitic infection was a frequent cause of cutaneous manifestations . The intermittent excretion of giardia cysts might be affecting the perceptions of the microscopic study of faecal specimens which for most of the caregivers considered useless . At the same time two studies on knowledge and practices on giardiasis among gastroenterologist and dermatologists in havana showed that 52.4% of gastroenterologists and 64% of dermatologist considered that duodenal aspiration was the best way to diagnose this parasitic disease . Certainly, duodenal aspirate is a valuable diagnostic aid for avoiding delays in diagnosis and prolongation of patient morbidity . However, this situation could be counterproductive because other parasitologic diagnoses potentially causing disease . However, there were some important gaps concerning these two issues which indicate that people today tend to combine all of the general preventive measures against intestinal parasitic infections, maybe, as a reflection of a result of previous activities directed at intestinal parasites control . Additionally, there exists a link between hygiene and infection, which is a common theme in the lay literature, and caregivers could be able to make connections and draw inferences from previous similar experiences . G. lamblia remains the leading waterborne diarrhoea - causing disease [31, 32], either when people drink accidentally or swallow water from contaminated or untreated sources (no heat inactivation, filtration, or chemical disinfection). The correct treatment of water and food and hand hygiene (i.e., hand washing with soap and water or the use of a waterless, alcohol - based hand rub) are considered the most important measures in preventing infections transmitted via the faecal - oral route . It has been recently shown in a systematic review the impact of interventions to improve hand washing with soap in the community could reduce diarrhoea risk by 47% . In the usa, an outbreak of giardiasis, involving multiple modes of transmission, eventually stopped, possibly influenced by a vigorous hand washing campaign . In the present study, the higher educational level of the caregivers had a positive influence on the knowledge of transmission and prevention of giardiasis . The parents' educational level is one of the factors of health inequalities among children . Education is important for health because of the opportunities it creates to obtain better material living conditions; well - educated people are less likely to be unemployed, and more likely to have full - time jobs, fulfilling work, and high income . A good example could be a study carried out in peru where participants with greater wealth indexes were associated with protection against giardia and persistent giardia infections (> 14 days). In a study carried out in portugal, children with mothers without any education were more likely to be infected with giardia than those with educated mothers . Similarly, children living with fathers with no education had a 12.26 times higher risk of being infected with giardia than those with educated fathers . This highlights the need for giardiasis he programmes and material targeted at groups with special needs . Even in a country like cuba, where the educational level is very high, groups still exist where low education and thus higher risk of diseases, such as giardiasis, may interact to create an unfavourable situation . To target populations for prevention messages, it is important to know which sources and channels of information are most frequently reported as being used to keep informed about a specific topic . Consistent with a previous study, it was found that television was the preferred channel of information, followed closely behind by their medical doctors, and printed materials . He materials such as posters and brochures may be an effective way to disseminate health information, providing such information in the local language that could be displayed at prominent places, such as health facilities and community centres . At present, access to culturally appropriate and easy - to - understand educational materials on giardiasis is lacking . Based on this study, it may be recommended that he materials oriented towards increasing the knowledge in different issues of giardiasis should be developed . The case group and their mothers were separately covered by the he programmes that included washing hands with soap, cutting nails, washing vegetables, and making the families knowledgeable about giardiasis and its effects on their children . All the programmes were performed by he methods, that is, poster, video film, face - to - face meetings, and pamphlets . Three months later, a triple stool examination was done in both groups to check reinfection . After the implementation of the programmes, the mean of the mother's awareness about controlling of giardiasis increased from 6.54% to 27.16% (p = 0.00001), without any variation in the control group in this item . The mean of children's information about giardiasis and its methods of control in the case and control groups before implementation of the he programmes was 4.76% and 4.86%, respectively, but after the implementation of the programs, it increased to 16.3% in the case group but remained at 4.86% in the control group (p = 0.00001). After implementation of he programs, the rate was 23.3% in the case group but 86.7% in the control group, indicating a significant difference between the two groups . The interpersonal communication, which is the most effective method of communicating health information, that takes place in the doctor's offices and the waiting room may offer the opportunity for physicians and other health care professionals to display their potential to play a significant role in promoting individual patient action and contributing to the development of social norms concerning health behaviour . Health care workers (medical doctors and nurses) occupy a position of high credibility and trust which make their pronouncement well received; they have a well - recognized role encouraging prevention . However, as key communicators, they need the necessary skills to carry out activities that allow discussions, feedback, and explanation to the target audience . In the cuban context, some studies carried out among gastroenterologist, and dermatologists have evidenced the necessity of improving the giardiasis management and prevention information they provide to their patients [29, 30]. This might be a fruitful area for future health intervention campaigns . Study participants were restricted to caregivers who were recruited from only one health facility, and, therefore, their views may be biased by contact with the same paediatric gastroenterology team and their standard educational practices . Participants were recruited among parents / carers attending to the hospital (a majority resided in havana), and thus we likely failed to capture the views of people in other provinces . As parents were recruited from those taking their children to the outpatient clinic for gastroenterology and within population are likely to be a large group of children who have giardiasis, it is reasonable to assume that parents of these children are more likely to know what giardiasis is than parents from the general population . Additionally, it should be noted that people attending health facilities at a hospital are expected to have a relatively greater awareness about common health issues . There is not a previous study in this area, and hence this questionnaire survey was the first to explore some of the issues in detail . Face - to - face administration of the questionnaire might have led to some degree of bias in the interviewees' responses . The study has revealed reasonable knowledge of the symptoms of giardiasis; however, there is a need for educational intervention directed towards correcting misconceptions . There was a need for an effective education programme focusing on giardiasis transmission and prevention which should be emphasised in order to avoid reinfections . The central necessity of caregivers' involvement in developing educational strategies that reflect their understandings and interpretations of this disease should not be overlooked.
Bilateral diaphragmatic paralysis is a rare clinical condition due to different entities that can damage the phrenic nerves . Peripheral neuropathy is one of the most common neurological complications of human immunodeficiency virus / acquired immunodeficiency syndrome (hiv / aids) disease . The commonest forms of hiv - associated neuropathies are the distal sensory polyneuropathy and antiretroviral toxic neuropathy; disorders characterized mostly by sensory symptoms afflicting 40 - 50% of patients whose hiv disease is otherwise controlled by antiretroviral therapy . However, it is commonly caused by surgical or traumatic injuries, malignant neoplasm, and neurodegenerative disorders . The relationship of phrenic paralysis with viral infections is rare but also well recognized and exceptionally is a manifestation of a hiv - associated neuropathy, especially when it represents the only manifestation of peripheral neuropathy . A 42-year - old hiv positive male smoker on antiretroviral therapy (stavudine, efavirenz, and lamivudine) with very poor adherence to the medication regimen was admitted because of community acquired pneumonia . Since past one year chest examination revealed paradoxical movement of the diaphragm, during inhalation, when the patient was in the supine position and coarse crepitations near the base of the left lung . All deep tendon reflexes were normal and there were no other signs of peripheral neuropathy . Cd4 cell count was 170/l and the plasma viral load was 74,746 copies / ml . Serological tests for chagas disease, hepatitis b virus (hbv), and syphilis (venereal disease research laboratory (vrdl) and fluorescent treponemal antibody absorption (fta abs)) were negative whereas imunoglobulin g (igg) anti - toxoplasma antibodies were positive . Chest x - ray revealed the elevation of the right and left hemidiaphragms in a full inspired film [figure 1] and its immobility detected through a radioscopic examination . There were no pulmonary infiltrates, pleural effusion, or mediastinal tumor in the computed tomography (ct) scan of the chest . The forced vital capacity in the sitting position was 1.84 l (48%) and it decreased to 1.29 l (34%) when brought to the dorsal decubitus position (change of <30%). Oxygen saturation while breathing ambient air was 96% . Transdiaphragmatic pressure was 2.99 cm h2o and confirmed the diagnosis of bilateral phrenic paralysis . During the examination, a negative deflection of the gastric pressure was observed as a sign of paradoxical movement of the diaphragm . The electromyography test showed an axonal damage to both phrenic nerves, without compromise in the other territories examined in his four limbs . Patient was started on a new scheme of antiretroviral therapy, without stavudine, and pulmonary rehabilitation exercises . Ten days later, he was discharged from the hospital in a good clinical condition . The diaphragm is the chief muscle of respiration and its paralysis can lead to dyspnea and can affect ventilatory function . Diaphragmatic paralysis can be unilateral or bilateral; the clinical symptoms being more prominent in bilateral paralysis . The diagnoses is usually suspected on chest x - ray and clinical exam and confirmed with sniff test or phrenic nerve stimulation / diaphragm electromyography . Prognosis is usually poor in patients with advanced lung disease, bilateral paralysis, and chronic demyelinating conditions . It generally manifests as an accompaniment of a generalized polyneuropathy such as the guillain barr syndrome and isolated compromise is rare . It may be the initial manifestation of a multifocal motor neuropathy, in the course of an amyotrophic neuralgia of the shoulder, or in the cases of post extracorporeal circulation pump paralysis . Postviral neuropathy and phrenic nerve involvement have been described following several viral infections like herpes zoster, poliovirus, west nile virus and hiv infection . The absence of these causes suggest that the phrenic nerve can be a target in idiopathic neuritis . Hiv - associated neuropathies (hiv - n) have become the most frequent neurological disorder associated with hiv infection . The most common forms of hiv - n are the distal sensory polyneuropathy and antiretroviral toxic neuropathies . These disorders are characterized mostly by sensory symptoms that include spontaneous or evoked pain that follow a subacute or chronic course and afflicts 40 - 50% of patients whose hiv disease is otherwise controlled by antiretroviral therapy . Advanced hiv disease (cd4 lymphocyte count 200/l) predisposes the patient to different neuropathies . Hiv - associated sensory neuropathy is consistently associated with previous exposure to nucleoside reverse transcriptase inhibitors including stavudine (d4 t), which is widely used in resource - limited settings . Stavudine, a nucleoside reverse transcriptase inhibitor, was used as first - line antiretroviral therapy (art) in our patient and is said to exacerbate distal symmetrical polyneuropathy in hiv / aids patients . Although recent world health organization guidelines recommend withdrawing d4 t from art, it remains commonly used in resource - limited settings . Genetic variants in mitochondrial dna haplogroups have been associated with increased risk of developing peripheral neuropathy in european non - hispanic and black patients on stavudine . A medline, embase, and cochrane databases search revealed that one report of a case of a 46-year - old patient, with tcd4 lymphocyte count of 250/l and a plasma viral load of less than 50 copies / ml, who suffered a unilateral phrenic neuropathy that improved after the administration of intravenous igg . Authors suggest that the pathogenicity of phrenic damage is of immune mediated type . In conclusion, unilateral or bilateral paralysis of phrenic nerves can be an infrequent manifestation of peripheral neuropathy in patients with hiv infection . This entity should be considered in the differential diagnosis of patients with hiv infection and respiratory symptoms.
Dry eye syndrome is a multifactorial disease, which is caused by a vicious cycle: abnormalities of tear film and lacrimal hyposecretion induce the break - up of tear film, and the following inflammation of ocular surface deteriorates the secretion and the composition of tears . Hyperosmolarity is induced by lacrimal hyposecretion or the increase of evaporation evokes desquamation, decreased intercellular connections, blunting and loss of microplicae, cell membrane disruption, and cellular swelling with decreased cytoplasmic density in the corneal epithelium . Moreover, hyperosmolarity provokes squamous metaplasia, loss of goblet cells, and inflammation in the conjunctival epithelium [35]. These phenomena decrease the production of mucin for lubricating corneal epithelium, and the reduction of mucin aggravates dry eye . Histologic findings of dry eye in patients with sjgren syndrome and immunosuppressant patients, such as postbone marrow transplantation state, were reported as a reduction of goblet cells, increment of inflammatory cells in cornea and conjunctiva, and inflammation with fibrosis of the lacrimal gland [710]. Dry eye also induces the secretion of cytokines such as il6, il-1, tnf-, and ifn- . Cyclosporin, one of the drugs suppressing those cytokines, reduces the infiltration of conjunctival cells and il6, regulates the necrosis of conjunctival epithelial cells, and elevates the number of goblet cells by preventing the loss of the cells . Il6 has been known as a representative cytokine with increased expression in tears and the conjunctival epithelium of eyes with dry eye syndrome . As evidence of the proinflammatory effects, a report revealed that il6 treatment reduced the survival of liver cancer cells and tocilizumab, an il6 blocker, has been used as a therapeutic drug for autoimmune diseases in rheumatology . Anti - inflammatory effects of il6 were revealed by the following evidence: il6 treatment on cells increased migration, il6 induced cell migration and wound healing of mouse biliary epithelial cells, blocking of il6 reduced inflammatory - related molecules in mouse alkali burn model, and il6-deficient mice showed delayed wound healing after skin resection . Although il6 has been well known as an important cytokine on disease progression and severity associated with immune mechanisms, its role in dry eye was still vague except for increased expression on the ocular surface . Therefore, this research investigated the role of il6 on apoptosis of conjunctival epithelial cells after hyperosmolarity . The wong - kilbourne derivative of chang conjunctival cells (wkd, atcc ccl-20, manassas, va, usa) were cultured in dulbecco's modified eagle's medium f12 (1: 3) culture medium (invitrogen, waltham, ma, usa), supplemented with 1% penicillin and streptomycin (welgene, daegu, seoul) and 5% heat - inactivated fetal bovine serum (wisent, quebec, canada). When cells were approximately 8090% confluence, culture medium was replaced with fresh medium with added 1 m nacl to increase the osmolarity (corresponding to 290, 500 mosm) for 24 h. cells were incubated for 24 h before protein extraction and conditioned medium collection . The blockade of il6 was done 24 h before incubation using the neutralizing anti - human il6 antibody (anti - il6, clone 1936, r&d systems, minneapolis, mn, usa) for neutralizing the il6 . To determine whether il6 cytokine has protected effect in the nacl exposure or not, conjunctiva epithelial cells were incubated with il6 for 24 h and then exposed to 500 mosm nacl for 24 h. cell supernatant was collected 290 mosm, 500 mosm nacl incubation after 24 h, and then centrifuged 12,000 rpm for 3 min at 4c . Cytokine levels in supernatant were determined using the human il-1, il6, tnf-, and ifn- quantikine enzyme - linked immunosorbent assay kit (r&d systems, minneapolis, mn, usa) and we followed the manufacturer's protocol . Briefly, for il6 kit 100 l of standard and samples were incubated in antibody - coated plate at 2 hrs . After being washed four times, add 200 l conjugate solution being incubated at 2 hrs . After being washed four times, add 200 l substrate solution being incubated at 20 min . Add 50 l stop solution and read at 450 nm with reader within 30 min . Cell viability was determined using the cell counting kit-8 (dojindo, kumamoto, japan) according to the manufacturer's instructions . Cells (1 10 cells / ml, 100 l) were seeded in 96-well plate . When cells were confluent 80%90%, cells were treated with anti - il6 for 24 h. after treatment for 24 h, add 10 l of cck-8 solution and incubate the plate for 1 h and cover the plate to protect from light, and then measure the absorbance using a microplate reader at 450 nm . Cells were seeded at 1.5 10 cells in a well of 60 mm plates and cultured until confluent 8090% . Cells were exposed to 290 mosm, 500 mosm nacl with / without anti - il6 50 ng for 24 h. after 24 h, cells were collected and resuspended . For labeling the annexin v / propidium iodide (pi), we were using the annexin v / pi kit (invitrogen, eugene, oregon, usa). The suspension cells were labeled with annexin v / pi and analyzed by flow cytometry (bd, franklin, new jersey). Extracts were incubated for 2 h at 4c and obtained by centrifugation (13,000 rpm for 20 min at 4c). Protein concentrations were determined using the bca assay kit (thermostat, hercules, ca, usa), and whole - cell extracts were adjusted to same amount of total protein (20 g). Proteins were then transferred onto a pvdf membrane (millipore corporation, billerica, ma, usa) at 300 ma for 90 min at 4c, and the membranes were incubated with 3% bsa (sigma - aldrich, st . We washed five times with tbst (0.5% tween 20 in 1x tbs) the secondary antibodies conjugated horseradish peroxidase (hrp) (santa cruz, dallas, texas, usa) that was applied and incubated for 1 h at rt . After five times of washing with tbst (0.5% tween 20 in 1x tbs), the membrane followed by chemiluminescent detection using immobilon western substrate (millipore corporation, billerica, ma, usa) with the chemidoc mp imaging system (bio - rad laboratories inc ., hercules, california, usa). The antibodies diluents were shown at table 1 . The results were analyzed by kruskal - wallis analysis, followed by a mann - whitney analysis . Wkd cells were cultured under 290 mosm and 500 mosm for 24 h and the cell viability was analyzed . There were no definite apoptotic cell deaths in 290 mosm, while cell viability was significantly reduced after 12 h in 500 mosm (0.95 0.04 at 12 h, 0.69 0.012 at 24 h, and 0.47 0.12 at 48 h, resp ., p = 0.05 at 12 h, 24 h) (figure 1(a)). We assessed the levels of il-1, tnf-, ifn-, and il6 from medium of cells cultured under 290 mosm and 500 mosm to identify the inflammatory cytokines which are well known to increase in epithelial cells by hyperosmolarity stress . Quantitative analysis showed that, whereas il-1 (0.07 0.002 in 290 mosm, 0.06 0.002 in 500 mosm), tnf- (0.08 0.004 in 290 mosm, 0.08 0.0004 in 500 mosm), and ifn- (0.08 0.009 in 290 mosm, 0.08 0.003 in 500 mosm) showed no significant change, il6 (1.47 0.44 in 290 mosm, 3.23 0.12 in 500 mosm) were increased (p = 0.05, figure 1(b)). When measuring the time - dependent expression of il6 in 290 mosm and 500 mosm, the expression of il6 in 500 mosm increased from 0.012 pg / ml at 1 h to 2.1 pg / ml at 24 h, which was higher than that in 290 mosm (p = 0.05, figure 1(c)). To evaluate whether il6 was secreted to protect cells or induce cell death in apoptosis by hyperosmolarity, 50 ng of anti - il6 was used for blocking il6 . When investigating change of morphology and cell distributions in 290 mosm, 500 mosm, and 500 mosm with anti - il6, the cells in 500 mosm showed shrinkage and more apoptosis than in 290 mosm . Nevertheless, cells in 500 mosm with anti - il6 showed less apoptosis and shrinkage than those in 500 mosm without anti - il6 (figure 2(a)). We have given a 500 mosm on the conjunctival epithelial cells and the effect of il6 was examined using annexin v / pi . The amount of the cells was significantly increased in the cell pretreated with the anti - il6 . The apoptosis rates are increased from 10.2 2.44 in 290 mosm to 73.88 5.84 in 500 mosm and 45.58 2.89 in 500 mosm nacl with anti - il6 . Treated anti - il6 was very effectively decreased for the apoptosis rates in 500 mosm nacl treated (figure 2(b)). There was significant difference in the proportions of apoptotic cells among 290 mosm, 500 mosm, and 500 mosm with anti - il6 (figure 2(c)), which implies that il6 could promote apoptosis by hyperosmolarity . Evaluating jak - stat signaling and apoptosis markers by western blot, the expressions of p - stat3, p - erk1/2, and p - mtor were higher in 500 mosm (resp ., 1.01 1.16, 1.19 0.22, and 1.15 0.08) than 290 mosm (resp ., 0.43 0.15, 0.44 0.08, and 0.48 0.03); however, those were lower in 500 mosm with anti - il6 (resp ., 0.6 0.17, 0.72 0.17, and 0.9 0.12) than in 500 mosm ., 1.39 0.37, 1.08 0.02) than in 290 mosm (resp ., 0.37 0.17, 0.49 0.14), and those were also lower in 500 mosm with anti - il6 (resp . Bcl2 showed lowest expression in 500 mosm (0.3 0.14), followed by that in 500 mosm with anti - il6 (0.66 0.08) and 290 mosm (1.24 0.3) (figure 3(a)). The significant difference of expressions of p - stat3/total stat3, p - erk / total erk, and p - mtor / total mtor was revealed between those in 500 mosm and 500 mosm with anti - il6 (p = 0.05) apoptosis markers such as bax, bcl2, and cleaved caspase-3/caspase-3 showed significant discrepancy between those in 500 mosm and 500 mosm with anti - il6 (p = 0.05, figure 3(b)). It has been reported that hyperosmolarity stress could induce apoptosis and promote the secretion of diverse proinflammatory cytokines . The association between conjunctival epithelial cells and il6 has been described in many studies: increased expression of il-1, il8, il6, and tnf- in conjunctival epithelial cells and tear fluid in the patients with dry eye syndrome [19, 20] and increase of il-1, il8, and, especially, il6 in impression cytology [21, 22]. Those studies suggested conjunctiva epithelial cells could aggravate dry eye and il6 had an important role in pathomechanism of dry eye . A previous study showed that blocking il6 could increase goblet cells in conjunctival epithelium and reduce inflammatory cells, which could soothe the symptoms and prevent the chronic progression of dry eye . Flow cytometry analysis showed that the blocking of il6 significantly suppressed apoptosis observed at 500 mosm, which suggests blocking il6 could modify apoptosis following hyperosmolarity . Il6 can be activated though the membrane - bound il6 receptor (classical pathway) or the soluble type of il6 receptor (trans - signaling). The il6 attached with receptor could initiate cascade via jak activation, and activated jak kinase phosphorylate induces forming of dimer by phosphorylation of stat3 [24, 25]. Although the activation of the jak - stat signal by il6 was observed in cancer, there have been few studies for the association between conjunctival epithelial cells and il6, especially about the role of increased il6 in a hyperosmolar state of conjunctival epithelial cells . This research revealed that the il6/jak / stat signaling pathway was associated with apoptosis induced by hyperosmolarity . Western blot showed that 500 mosm promoted the expression of stat3 (direct signal), erk, and mtor (indirect signal), and il6 reduced the increased expressions . Bax and caspase-3 as apoptosis markers showed higher expression in hyperosmolar state (500 mosm), which was suppressed by blocking il6 . These suggested that jak - stat signaling pathway of apoptosis was associated with il6 . In this study, blocking il6 inhibited the apoptosis of conjunctival epithelial cells under hyperosmolar condition, and the process was associated with the jak - stat signal pathway (figure 4). This indicated that il6 could be one of the important cytokines affecting the pathogenesis of dry eye . Therefore, in dry eye patients, il6 could be a biologic marker, and regulating il6 though the il6/jak / stat3 signaling pathway could be an effective therapeutic target . Hyperosmolarity induced apoptosis in conjunctival epithelial cells was suppressed by blocking il6, which suggests that il6 may play an important role in the pathogenesis of dry eye disease.
Readers of this review should consider that many age - related processing speed findings reported in the literature are cross - sectional in nature, including the primary findings reviewed here . This is important because there are developmental (laasonen et al ., 2002; deary et al ., 2010) and sampling factors that could affect the outcome of cross - sectional studies on processing speed in addition, the neural systems that are critical for understanding individual variation in cognition may differ across the lifespan (zimmerman et al ., 2006). There may be qualitative neurobiological changes that distinguish older adults from the oldest adults . Our approach has been to include adults across the lifespan because cognitive aging appears to begin when people reach 2030 years of age (salthouse, 2009). For this reason, we consider many of the findings summarized in this review to reflect effects of chronological age . Importantly, however, longitudinal findings of processing speed change have supported the robust cross - sectional findings of processing speed declines on cognitive function (finkel et al ., 2009). We anticipate that neural systems exhibiting cross - sectional processing speed changes will also exhibit robust within subject changes with processing speed . One early neurobiological explanation for age - related changes in processing speed focused on cortical declines in neuropil (morris and mcmanus, 1991). Age - related differences in total gray matter volume have been related to differences in processing speed (chee et al ., 2009), which theoretically could result in degraded or noisy representations that would slow the recognition of stimuli and decision making . For example, primary auditory cortex neurons in the aged rat exhibit elevated excitation that has been related to a loss of gabaergic function (caspary et al ., 2008). These changes likely stem from reduced inhibitory interneuron input onto dendritic arbors that are diminished in the aged rat (vaughan, 1977) and in older adult human auditory association cortex (jacobs and scheibel, 1993). Similar changes could occur across perceptual, planning, and motor systems that support speeded task performance . Thus, age - related changes in cognition are likely to be driven by changes in the connectivity of neural systems (dickstein et al ., 2007). Another prominent explanation for processing speed declines is that a loss of myelination could slow conduction rates and therefore slow processing speed (morris and mcmanus, 1991; fjell and walhovd, 2010). Age - related differences in whole brain white matter fractional anisotropy have been associated with processing speed (coffey et al ., 2001; charlton et al ., 2006; there is a myelin - specific explanation that could account for age - related changes in processing speed . The number of oligodendrocytes does not decline with age in the rhesus monkey and have been observed to re - myelinate axons whose oligodendrocytes have died (peters, 2009). The consequence, however, is (1) a shortening in the lengths of the new myelin sheaths, (2) additional nodes of ranvier that could alter conductance speed, and (3) the potential for altered neural firing patterns that could further diminish the quality of neural signals affected by axonal and neuropil loss (peters, 2009). The early hypotheses for processing speed decline focused on global changes in gray and white matter, due in part to the available evidence . Over the last 20 years there has been mounting evidence for regional specificity in the severity of age - related change in gray matter volume . 2009), anterior insula, inferior frontal, superior frontal, medial frontal, and cerebellar regions exhibit some of the most pronounced changes with age when measured with automated (good et al ., 2001;; tisserand et al ., 2004; lemaitre et al ., 2005; chee et al ., 2009) and manual measures (raz et al ., 1997, 2005, 2010;, 2005; raz and rodrigue, 2006). Given the substantial changes in frontal white matter that also occur with age (bendlin et al . 2010), these results suggest that frontal cortex is undergoing de - afferentation and subsequent decline with increasing age . We return to this premise below with respect to vascular explanations for changes in processing speed . One important point is that these findings could explain why frontal cortex exhibits fewer functional connections with other cortical and sub - cortical regions in older compared to younger adults (meunier et al ., 2009), with reduced control of sensory and perceptual processing . Similarly, the cerebellar hemispheres could be considered as having domain general roles in the support of cognitive function, where the substantial aging effects on morphology (raz et al ., because of their widespread influence on behavior, frontal and cerebellar regions are consistently related to a variety of speeded or timed cognitive tasks . Frontal cortex has been a primary target for the study of age - related processing speed changes because of the relatively pronounced aging effects on frontal cortex morphology and interest in prominent hypotheses for cognitive aging such as the frontal aging hypothesis (west, 1996) and the inhibition deficit hypothesis (hasher and zacks, 1988; hasher et al ., 1999). Volumetric, voxel - based, and sulcal morphometry examinations of frontal gray matter have demonstrated associations with performance across a wide variety of cognitive tasks . Importantly, the frontal gray matter findings appear to be dependent on the susceptibility of frontal white matter to age - related decline . Regions of frontal gray matter that exhibit cross - sectional change with age and predict processing speed are presented in figure 1 from a sample of 42 adults that ranged in age from 19 to 79 years (eckert et al . Younger adults with elevated medial frontal and lateral frontal gray matter volume demonstrated faster processing speed compared to older adults with lower gray matter volume in these regions . This pattern of frontal results is consistent with volumetric and voxel - based gray matter associations with processing speed that were observed in a sample of 248 subjects ranging in age from 55 to 86 years (chee et al ., 2009). Similarly, the volume of prefrontal cortex has been observed to predict a drawing speed (kennedy and raz, 2005), an estimate of perceptual comparison speed (schretlen et al ., 2000), and a measure of fluid intelligence in older adults (raz et al ., 2008; kennedy et al ., 2009) frontal sulcal span, an indirect measure of gray matter declines in frontal cortex, has also been associated with processing speed (kochunov et al ., 2010). Voxel - based gray matter variation (modulated) predicts connections simple processing speed performance (a). These results are not significant at the p <0.001 (uncorrected) level after controlling for age, thereby demonstrating the dependence of the results on age . Posterior cingulate, cuneus, cerebellar, and medial temporal regions exhibited weak associations with processing speed (p <0.05, uncorrected) after controlling for age, suggesting that age may exaggerate normal variation in brain morphology and processing speed . Sbm (please see figure 3 for an sbm summary) demonstrates unique patterns of frontal and cerebellar gray matter variance (b, c) that each accounted for different distributions of the processing speed results [(d): p <0.001 after controlling for variance representing either the frontal or the cerebellar independent components]. Importantly, by controlling for either gray matter component we can see that there are at least two sources that contribute to the association between gray matter and processing speed in this sample . In a separate analysis, these component specific effects were not significant using a p <0.001 threshold when age was covaried . There were weak p <0.05 associations with processing speed in cingulate and cerebellar regions after controlling for either component and age, again suggesting the influence of additive developmental factors (deary et al ., 2010). Individual variation in prefrontal gray matter volume among older adults is strongly related to frontal white matter volume (raz et al ., 2003). Perhaps then it is not surprising that multiple measures of frontal white matter morphology or integrity have been associated with processing speed, including transverse relaxation rate (bartzokis et al ., 2010), midline genu area of the corpus callosum (jokinen et al ., 2007), and fractional anisotropy and/or mean diffusivity (deary et al, 2007; bucur et al ., 2008; kennedy and raz, 2009a; schiavone et al ., 2009; bendlin et al ., 2010; an example of the robust association between fractional anisotropy in frontal lobe white matter and processing speed is presented in figure 2 . Individual variation in frontal lobe fractional anisotropy has been related to processing speed in healthy young adults (turken et al ., 2008), suggesting the interesting possibility that aging exaggerates normal structure function associations (harris et al ., 2009). Voxel - based analysis of fractional anisotropy data demonstrates a right frontal association with connections simple processing speed (p <0.01, family - wise discovery rate corrected) in 36 of the 42 subjects whose data were included in the figure 1 analyses . The graph shows the association between frontal fractional anisotropy and the frontal gray matter component from figure 1, the presence of white matter hyperintensities among people with low fractional anisotropy and low frontal gray matter (circle color: no hyperintensities blue; some hyperintensities orange; pronounced hyperintensities green), and that ps is related to each of these variables (larger circle size reflects faster processing speed). (the tspoon approach designed to control for smoothing kernel effects, lee et al ., 2009, was used to process the 2 mm 2 mm in plane resolution images that were collected on a philips 3 t with 32 directions using an 8-channel phased array head coil . The b0 and fa data were co - registered to each subject's t1 image and then normalized into study - specific space using the dartel parameters obtained for the t1 images .) A large body of literature implicates white matter pathology in the cognitive slowing of older adults (gunning - dixon and raz, 2000; kennedy and raz, 2009b; vernooij et al ., 2009; kochunov et al ., 2010; vidal et al ., 2010; please see gunning - dixon et al . White matter hyperintensities, a common age - related finding in periventricular regions (almkvist et al ., 1992; soderlund et al ., 2003; cook et al ., 2004), have a gradual impact on cognition over time (silbert et al ., 2009) and appear to affect regional gray matter morphology even before overt cognitive changes are observable (ota et al ., 2010). They are generally considered to be a sign of cerebral small vessel disease and the severity of the hyperintensities has been related to hypertension (brickman et al ., 2010), blood pressure (kochunov et al ., 2010; kuo et al ., 2010), coronary artery calcification (vidal et al ., 2010), and endothelial dependent vasodilation (hoth et al ., 2007). White matter pathology has been observed independently of effects related to systolic blood pressure and cholesterol (chowdhury et al . Age - related changes in pericytes, cells that contribute to the blood brain barrier, may provide an explanation for both hypoxia related damage and neurotoxic damage stemming from microvessel leakage . Pericyte deficient mice exhibit progressive loss of cerebral perfusion and a loss of dendritic spines that has been attributed to the leakage of damaging proteins through the blood brain barrier and subsequent neuronal loss (bell et al ., 2010). Both conditions would produce an inflammatory response and could explain why variation in interleukin-6 has been associated with changes in macaque frontal white matter (willette et al ., 2010). Regardless of the mechanism, damage to periventricular white matter appears to disrupt pathways projecting to and from frontal lobe regions that support performance on processing speed tasks . Cerebellar morphology also undergoes robust age - related structural change but has not received the same experimental attention as frontal cortex with respect to age - related changes in processing speed . This is due, in part, to image processing choices that exclude the cerebellum and because the prominent theories of cognitive aging (the frontal aging hypothesis and the inhibition deficit hypothesis) focus on frontal cortex . While there may be a publication bias related to negative findings, relations between cerebellar morphology and processing speed have been observed and provide support for a fronto - cerebellar aging hypothesis (hogan, 2004). Hogan (2004) proposed that changes in feedback and feedforward cerebro - cerebellar interaction result in greater within subject variation in task performance, reduced behavioral automaticity, and increased demands on cognitive control, as well as reduced working memory capacity . This hypothesis is based on normative and patient evidence linking the cerebellum to cognitive and perceptual functions (e.g., eckert et al ., 2003; chen and desmond, 2005; desmond et al ., 2005; steinlin, 2007) and its distinct patterns of structural and functional connectivity with motor, sensory, and attention - related cortical regions (e.g., kelly and strick, 2003; habas et al ., 2009; krienen and buckner, 2009; schmahmann, 2010). In particular, limits on the cerebellum's role in modifying motor function in response to sensory and perceptual feedback (ivry, 1997) could affect performance on perceptual and motor processing speed tasks . Cross - sectional age - related changes in cerebellar morphology have been associated with processing speed (maclullich et al ., 2004; paul et al ., 2009; eckert et al ., 2010) and a measure of general intelligence among older adults (hogan et al ., 2011). In particular, we observed that processing speed was predicted by regional cerebellar gray matter volume (figure 1). This voxel - based effect was present throughout the vermis and cerebellar hemispheres (superior to the horizontal fissure). The vermis finding is spatially consistent with a finding that the midline surface area of vermis (declive, folium, tuber region) predicted age - related digit - symbol substitution test performance, which was significant even after controlling for an estimate of intracranial volume (maclullich et al ., 2004). In addition, a volumetric measure of the same vermis region and total cerebellar gray matter volume were significantly related to age - related changes in trail making test (alternating condition) performance (paul et al ., 2009). In the paul et al . (2009) study, however, the vermis measure did not significantly predict trail making task performance after accounting for a measure of prefrontal volume (brodmann areas 9 and 46). The specificity of these frontal and cerebellar findings is addressed below . As suggested by hogan (2004), the processing speed and cerebellar findings could be explained by problems with sensory motor integration . For example, a history of falling and slow processing speed has been associated with delayed horizontal saccades during the initiation of a footlift for a stepping task (greany and di fabio, 2008). In addition, left cerebellar volume has been related to gait speed in older adults (rosano et al ., 2007; although see rosano et al ., 2008) and right cerebellar hemisphere volume has been associated with balance difficulty among older adults (rosano et al ., 2007). A sensory motor integration explanation could also explain why cerebellar volumes have been associated with perceptual motor skill as measured by total time on target for a pursuit rotor task (raz et al ., 2000), although not for a timed mirror tracing task (kennedy and raz, 2005). Age - related differences in cerebellar volume has also been significantly correlated with associative learning as measured by eye - blink conditioning (woodruff - pak et al ., 2000, 2001). Together, these findings suggest that the behavioral effects of cerebellar aging are variable across subjects, perhaps because of individual variability in developmental factors (deary et al ., 2010), and may have widespread effects on a variety of cerebellar functions . Based on the perceptual motor skill associations with cerebellar morphology, we asked whether performance on the connections processing speed test could be influenced by increasing performance across the two trials of each sub - test [numbers only, letters only, numbers letters (starting with numbers), letters numbers (starting with numbers)]. There was no evidence indicating that people with elevated cerebellar gray matter exhibited the greatest improvement in performance across the trials . People with low cerebellar gray matter volume were more likely to improve across the first to second trials of the alternating letters numbers condition compared to people with elevated cerebellar gray matter volume (p <0.05 uncorrected; no significant relations were seen for the other connections conditions). This finding suggests that the processing speed and cerebellar relation was not due to a specific learning effect, however it is possible that we could not detect evidence of procedural learning because of (1) the balanced order in which the sub - tests are administered or (2) ceiling effects on the first trial of the connections sub - tests in people with the greatest cerebellar gray matter volume . Importantly, however, these results are consistent with evidence that variation in cerebellar morphology is related to the initial stages of skill acquisition (raz, 2000). Returning to the hogan (2004) cerebellar hypothesis for cognitive aging, we also wondered whether variation in performance across the connections tests could be related to age and cerebellar morphology . A measure of each subject's variance in processing speed was obtained from the sd of subject z scores from the connections sub - tests, where the z score was based on the mean performance of the sample for each sub - test . Rather than greater variability among the oldest subjects, an inverted u function was observed where by middle - aged subjects exhibited the most variable connections performance (age connections variance; quadratic r = 0.41, p <0.05; linear r = 0.05, ns). There were not clear linear or non - linear relations with cerebellar morphology and the connections variance measure, but it is intriguing to consider that elevated variance in performance among middle - aged subjects may be an early behavioral marker for neurobiological declines that slow processing speed and cognition . Finally, there are questions about the etiological factors that drive age - related cerebellar changes and that would contribute to slowed processing speed . We observed that cerebellar associations with processing speed occurred independently of periventricular white matter hyperintensities, suggesting that cerebral small vessel disease was not a primary factor for changes in cerebellar morphology in our sample . Similarly, white matter hyperintensities did not account for the association between cerebellar volume and gait speed observed in the rosano et al . The results of other studies, however, have indicated that primary declines in prefrontal cortex contribute to cerebellar changes in morphology (bugalho et al ., 2007) there is the strong likelihood that cerebellar and frontal regions vary in susceptibility to different age - related risk factors . The high metabolic demands of cerebellar purkinje cells, which demonstrate a substantial loss in number with age (hall et al ., 1975; andersen et al ., 2003), may increase the risk for cell death due to oxidative stress . Indeed, oxidative stress is one factor contributing to cerebellar aging in mice (lee et al ., 2000). The results of human imaging studies indicate that leptin may serve to protect against oxidative stress based on evidence that plasma concentration of leptin was associated with elevated cerebellar and hippocampal gray matter in middle - aged and older adults (narita et al ., 2009) and evidence that leptin treatment increases cerebellar gray matter in leptin deficient patients (matochik et al ., 2005). These findings, together with developmental findings in c57 mice showing that leptin promotes cerebellar purkinje cell survival and neurite outgrowth (oldreive et al ., 2008), suggest the interesting possibility that leptin limits the impact of oxidative stress on cerebellar purkinje cells whose high metabolic rate places them at risk for age - related declines as it does for in vitro hippocampal purkinje cells (guo et al ., 2008). This leptin explanation for cerebellar health is just one example for what is likely a multifactorial aging process in the cerebellum . One early neurobiological explanation for age - related changes in processing speed focused on cortical declines in neuropil (morris and mcmanus, 1991). Age - related differences in total gray matter volume have been related to differences in processing speed (chee et al ., 2009), which theoretically could result in degraded or noisy representations that would slow the recognition of stimuli and decision making . For example, primary auditory cortex neurons in the aged rat exhibit elevated excitation that has been related to a loss of gabaergic function (caspary et al ., 2008). These changes likely stem from reduced inhibitory interneuron input onto dendritic arbors that are diminished in the aged rat (vaughan, 1977) and in older adult human auditory association cortex (jacobs and scheibel, 1993). Similar changes could occur across perceptual, planning, and motor systems that support speeded task performance . Thus, age - related changes in cognition are likely to be driven by changes in the connectivity of neural systems (dickstein et al ., 2007). Another prominent explanation for processing speed declines is that a loss of myelination could slow conduction rates and therefore slow processing speed (morris and mcmanus, 1991; fjell and walhovd, 2010). Age - related differences in whole brain white matter fractional anisotropy have been associated with processing speed (coffey et al ., 2001; charlton et al ., 2006; vernooij et al ., 2008; schiavone et al ., 2009), although this variation likely reflect a loss of myelinated axons . There is a myelin - specific explanation that could account for age - related changes in processing speed . The number of oligodendrocytes does not decline with age in the rhesus monkey and have been observed to re - myelinate axons whose oligodendrocytes have died (peters, 2009). The consequence, however, is (1) a shortening in the lengths of the new myelin sheaths, (2) additional nodes of ranvier that could alter conductance speed, and (3) the potential for altered neural firing patterns that could further diminish the quality of neural signals affected by axonal and neuropil loss (peters, 2009). The early hypotheses for processing speed decline focused on global changes in gray and white matter, due in part to the available evidence . Over the last 20 years there has been mounting evidence for regional specificity in the severity of age - related change in gray matter volume . 2009), anterior insula, inferior frontal, superior frontal, medial frontal, and cerebellar regions exhibit some of the most pronounced changes with age when measured with automated (good et al ., 2001;, 2004; tisserand et al ., 2004; lemaitre et al ., 2005; chee et al ., 2009) and manual measures (raz et al ., 1997, 2005, 2010; jernigan et al ., 2001; tisserand et al ., 2002; decarli et al ., 2005; raz and rodrigue, 2006). Given the substantial changes in frontal white matter that also occur with age (bendlin et al . 2010), these results suggest that frontal cortex is undergoing de - afferentation and subsequent decline with increasing age . We return to this premise below with respect to vascular explanations for changes in processing speed . One important point is that these findings could explain why frontal cortex exhibits fewer functional connections with other cortical and sub - cortical regions in older compared to younger adults (meunier et al ., 2009), with reduced control of sensory and perceptual processing . Similarly, the cerebellar hemispheres could be considered as having domain general roles in the support of cognitive function, where the substantial aging effects on morphology (raz et al ., 2010) could affect perceptual and cognitive task performance . Perhaps because of their widespread influence on behavior, frontal and cerebellar regions are consistently related to a variety of speeded or timed cognitive tasks . Frontal cortex has been a primary target for the study of age - related processing speed changes because of the relatively pronounced aging effects on frontal cortex morphology and interest in prominent hypotheses for cognitive aging such as the frontal aging hypothesis (west, 1996) and the inhibition deficit hypothesis (hasher and zacks, 1988; hasher et al ., 1999). Volumetric, voxel - based, and sulcal morphometry examinations of frontal gray matter have demonstrated associations with performance across a wide variety of cognitive tasks . Importantly, the frontal gray matter findings appear to be dependent on the susceptibility of frontal white matter to age - related decline . Regions of frontal gray matter that exhibit cross - sectional change with age and predict processing speed are presented in figure 1 from a sample of 42 adults that ranged in age from 19 to 79 years (eckert et al ., younger adults with elevated medial frontal and lateral frontal gray matter volume demonstrated faster processing speed compared to older adults with lower gray matter volume in these regions . This pattern of frontal results is consistent with volumetric and voxel - based gray matter associations with processing speed that were observed in a sample of 248 subjects ranging in age from 55 to 86 years (chee et al ., 2009). Similarly, the volume of prefrontal cortex has been observed to predict a drawing speed (kennedy and raz, 2005), an estimate of perceptual comparison speed (schretlen et al ., 2000), and a measure of fluid intelligence in older adults (raz et al ., 2008; kennedy et al ., frontal sulcal span, an indirect measure of gray matter declines in frontal cortex, has also been associated with processing speed (kochunov et al ., 2010). Voxel - based gray matter variation (modulated) predicts connections simple processing speed performance (a). These results are not significant at the p <0.001 (uncorrected) level after controlling for age, thereby demonstrating the dependence of the results on age . Posterior cingulate, cuneus, cerebellar, and medial temporal regions exhibited weak associations with processing speed (p <0.05, uncorrected) after controlling for age, suggesting that age may exaggerate normal variation in brain morphology and processing speed . Sbm (please see figure 3 for an sbm summary) demonstrates unique patterns of frontal and cerebellar gray matter variance (b, c) that each accounted for different distributions of the processing speed results [(d): p <0.001 after controlling for variance representing either the frontal or the cerebellar independent components]. Importantly, by controlling for either gray matter component we can see that there are at least two sources that contribute to the association between gray matter and processing speed in this sample . In a separate analysis, these component specific effects were not significant using a p <0.001 threshold when age was covaried . There were weak p <0.05 associations with processing speed in cingulate and cerebellar regions after controlling for either component and age, again suggesting the influence of additive developmental factors (deary et al ., 2010). Individual variation in prefrontal gray matter volume among older adults is strongly related to frontal white matter volume (raz et al ., 2003). Perhaps then it is not surprising that multiple measures of frontal white matter morphology or integrity have been associated with processing speed, including transverse relaxation rate (bartzokis et al ., 2010), midline genu area of the corpus callosum (jokinen et al ., 2007), and fractional anisotropy and/or mean diffusivity (deary et al kennedy and raz, 2009a; schiavone et al ., 2009; bendlin et al . An example of the robust association between fractional anisotropy in frontal lobe white matter and processing speed is presented in figure 2 . Individual variation in frontal lobe fractional anisotropy has been related to processing speed in healthy young adults (turken et al ., 2008), suggesting the interesting possibility that aging exaggerates normal structure function associations (harris et al ., 2009). Voxel - based analysis of fractional anisotropy data demonstrates a right frontal association with connections simple processing speed (p <0.01, family - wise discovery rate corrected) in 36 of the 42 subjects whose data were included in the figure 1 analyses . The graph shows the association between frontal fractional anisotropy and the frontal gray matter component from figure 1, the presence of white matter hyperintensities among people with low fractional anisotropy and low frontal gray matter (circle color: no hyperintensities blue; some hyperintensities orange; pronounced hyperintensities green), and that ps is related to each of these variables (larger circle size reflects faster processing speed). (the tspoon approach designed to control for smoothing kernel effects, lee et al ., 2009, was used to process the 2 mm 2 mm in plane resolution images that were collected on a philips 3 t with 32 directions using an 8-channel phased array head coil . The b0 and fa data were co - registered to each subject's t1 image and then normalized into study - specific space using the dartel parameters obtained for the t1 images .) A large body of literature implicates white matter pathology in the cognitive slowing of older adults (gunning - dixon and raz, 2000; kennedy and raz, 2009b; vernooij et al ., 2009; kochunov et al ., 2010; vidal et al ., 2010; please see gunning - dixon et al ., 2009 for additional review). White matter hyperintensities, a common age - related finding in periventricular regions (almkvist et al ., 1992; soderlund et al ., 2003; cook et al ., 2004), have a gradual impact on cognition over time (silbert et al ., 2009) and appear to affect regional gray matter morphology even before overt cognitive changes are observable (ota et al ., 2010). They are generally considered to be a sign of cerebral small vessel disease and the severity of the hyperintensities has been related to hypertension (brickman et al ., 2010), blood pressure (kochunov et al ., 2010; kuo et al ., 2010), coronary artery calcification (vidal et al ., 2010), and endothelial dependent vasodilation (hoth et al ., 2007). White matter pathology has been observed independently of effects related to systolic blood pressure and cholesterol (chowdhury et al . Age - related changes in pericytes, cells that contribute to the blood brain barrier, may provide an explanation for both hypoxia related damage and neurotoxic damage stemming from microvessel leakage . Pericyte deficient mice exhibit progressive loss of cerebral perfusion and a loss of dendritic spines that has been attributed to the leakage of damaging proteins through the blood brain barrier and subsequent neuronal loss (bell et al ., 2010). Both conditions would produce an inflammatory response and could explain why variation in interleukin-6 has been associated with changes in macaque frontal white matter (willette et al ., 2010). Regardless of the mechanism, damage to periventricular white matter appears to disrupt pathways projecting to and from frontal lobe regions that support performance on processing speed tasks . Cerebellar morphology also undergoes robust age - related structural change but has not received the same experimental attention as frontal cortex with respect to age - related changes in processing speed . This is due, in part, to image processing choices that exclude the cerebellum and because the prominent theories of cognitive aging (the frontal aging hypothesis and the inhibition deficit hypothesis) focus on frontal cortex . While there may be a publication bias related to negative findings, relations between cerebellar morphology and processing speed have been observed and provide support for a fronto - cerebellar aging hypothesis (hogan, 2004). Hogan (2004) proposed that changes in feedback and feedforward cerebro - cerebellar interaction result in greater within subject variation in task performance, reduced behavioral automaticity, and increased demands on cognitive control, as well as reduced working memory capacity . This hypothesis is based on normative and patient evidence linking the cerebellum to cognitive and perceptual functions (e.g., eckert et al ., 2003; chen and desmond, 2005; desmond et al ., 2005; steinlin, 2007) and its distinct patterns of structural and functional connectivity with motor, sensory, and attention - related cortical regions (e.g., kelly and strick, 2003; habas et al ., 2009; krienen and buckner, 2009; schmahmann, 2010). In particular, limits on the cerebellum's role in modifying motor function in response to sensory and perceptual feedback (ivry, 1997) could affect performance on perceptual and motor processing speed tasks . Cross - sectional age - related changes in cerebellar morphology have been associated with processing speed (maclullich et al ., 2004; paul et al ., 2009; eckert et al ., 2010) and a measure of general intelligence among older adults (hogan et al ., 2011). In particular, we observed that processing speed was predicted by regional cerebellar gray matter volume (figure 1). This voxel - based effect was present throughout the vermis and cerebellar hemispheres (superior to the horizontal fissure). The vermis finding is spatially consistent with a finding that the midline surface area of vermis (declive, folium, tuber region) predicted age - related digit - symbol substitution test performance, which was significant even after controlling for an estimate of intracranial volume (maclullich et al ., 2004). In addition, a volumetric measure of the same vermis region and total cerebellar gray matter volume were significantly related to age - related changes in trail making test (alternating condition) performance (paul et al ., 2009). (2009) study, however, the vermis measure did not significantly predict trail making task performance after accounting for a measure of prefrontal volume (brodmann areas 9 and 46). The specificity of these frontal and cerebellar findings is addressed below . As suggested by hogan (2004), the processing speed and cerebellar findings could be explained by problems with sensory motor integration . For example, a history of falling and slow processing speed has been associated with delayed horizontal saccades during the initiation of a footlift for a stepping task (greany and di fabio, 2008). In addition, left cerebellar volume has been related to gait speed in older adults (rosano et al ., 2007; although see rosano et al ., 2008) and right cerebellar hemisphere volume has been associated with balance difficulty among older adults (rosano et al ., 2007). A sensory motor integration explanation could also explain why cerebellar volumes have been associated with perceptual motor skill as measured by total time on target for a pursuit rotor task (raz et al ., 2000), although not for a timed mirror tracing task (kennedy and raz, 2005). Age - related differences in cerebellar volume has also been significantly correlated with associative learning as measured by eye - blink conditioning (woodruff - pak et al . Together, these findings suggest that the behavioral effects of cerebellar aging are variable across subjects, perhaps because of individual variability in developmental factors (deary et al ., 2010), and may have widespread effects on a variety of cerebellar functions . Based on the perceptual motor skill associations with cerebellar morphology, we asked whether performance on the connections processing speed test could be influenced by increasing performance across the two trials of each sub - test [numbers only, letters only, numbers letters (starting with numbers), letters numbers (starting with numbers)]. There was no evidence indicating that people with elevated cerebellar gray matter exhibited the greatest improvement in performance across the trials . People with low cerebellar gray matter volume were more likely to improve across the first to second trials of the alternating letters numbers condition compared to people with elevated cerebellar gray matter volume (p <0.05 uncorrected; no significant relations were seen for the other connections conditions). This finding suggests that the processing speed and cerebellar relation was not due to a specific learning effect, however it is possible that we could not detect evidence of procedural learning because of (1) the balanced order in which the sub - tests are administered or (2) ceiling effects on the first trial of the connections sub - tests in people with the greatest cerebellar gray matter volume . Importantly, however, these results are consistent with evidence that variation in cerebellar morphology is related to the initial stages of skill acquisition (raz, 2000). Returning to the hogan (2004) cerebellar hypothesis for cognitive aging, we also wondered whether variation in performance across the connections tests could be related to age and cerebellar morphology . A measure of each subject's variance in processing speed was obtained from the sd of subject z scores from the connections sub - tests, where the z score was based on the mean performance of the sample for each sub - test . Rather than greater variability among the oldest subjects, an inverted u function was observed where by middle - aged subjects exhibited the most variable connections performance (age connections variance; quadratic r = 0.41, p <0.05; linear r = 0.05, ns). There were not clear linear or non - linear relations with cerebellar morphology and the connections variance measure, but it is intriguing to consider that elevated variance in performance among middle - aged subjects may be an early behavioral marker for neurobiological declines that slow processing speed and cognition . Finally, there are questions about the etiological factors that drive age - related cerebellar changes and that would contribute to slowed processing speed . We observed that cerebellar associations with processing speed occurred independently of periventricular white matter hyperintensities, suggesting that cerebral small vessel disease was not a primary factor for changes in cerebellar morphology in our sample . Similarly, white matter hyperintensities did not account for the association between cerebellar volume and gait speed observed in the rosano et al . The results of other studies, however, have indicated that primary declines in prefrontal cortex contribute to cerebellar changes in morphology (bugalho et al ., 2007) there is the strong likelihood that cerebellar and frontal regions vary in susceptibility to different age - related risk factors . The high metabolic demands of cerebellar purkinje cells, which demonstrate a substantial loss in number with age (hall et al ., 1975; andersen et al ., 2003), may increase the risk for cell death due to oxidative stress . Indeed, oxidative stress is one factor contributing to cerebellar aging in mice (lee et al ., 2000). The results of human imaging studies indicate that leptin may serve to protect against oxidative stress based on evidence that plasma concentration of leptin was associated with elevated cerebellar and hippocampal gray matter in middle - aged and older adults (narita et al ., 2009) and evidence that leptin treatment increases cerebellar gray matter in leptin deficient patients (matochik et al ., 2005). These findings, together with developmental findings in c57 mice showing that leptin promotes cerebellar purkinje cell survival and neurite outgrowth (oldreive et al ., 2008), suggest the interesting possibility that leptin limits the impact of oxidative stress on cerebellar purkinje cells whose high metabolic rate places them at risk for age - related declines as it does for in vitro hippocampal purkinje cells (guo et al ., this leptin explanation for cerebellar health is just one example for what is likely a multifactorial aging process in the cerebellum . The review above suggests that age - related changes across multiple systems could affect processing speed . The frontal findings, in particular, could explain why changes in auditory gap detection (harris et al ., 2010) and tactile temporal order thresholds (craig et al ., 2010) it is difficult to determine, however, from the results presented in figure 1a whether widespread anatomical effects stem from a common mechanism or whether unique neural systems are affected . This is a particularly significant problem for cross - sectional chronological aging studies where disentangling neurobiological patterns of aging can be difficult . Our approach to addressing this challenge has been to use ica or source based morphometry to identify unique patterns of structural covariance in the data that uniquely predict age - related differences in processing speed and that might provide insight into the affected neural systems . Independent component analysis of functional imaging data is helping to identify consistent patterns of neural activity across experiments that are thought to reflect distinct neural systems for focused and scanning attention, motor function, or auditory and visual processing (beckmann et al ., 2005; eckert et al ., 2008a). Indeed, this type of analysis may help to identify atypical coherence in neural systems that underlie changes in processing speed . For example, the ability to switch between functional networks that are thought to represent focused attention to a task (dorsal attention network; sridharan et al ., 2007) and self - referential thinking (default mode network; andrews - hanna et al ., 2010) has been related to within subject variability in children's task performance (kelly et al ., 2008), appears to be impaired in older adults (grady et al ., 2006), and may also relate to variable processing speed performance in middle - aged adults . Independent component analysis of structural mri data has been described as source based morphometry because unique patterns of variation are thought to have common underlying influences or sources (xu et al ., 2009; specific regions of gray matter may covary across subjects because of variation in fiber pathways connecting distant areas, the amount of neuropil, vascular support, or image artifact . For example, this technique identified periventricular white matter regions where white matter hyperintensities were observed (figure 6 in eckert et al ., 2010). In addition, the component included regions where there was less gray matter in frontal cortex (ic7, figure 3). This result suggested that a common source, presumably cerebral small vessel disease, was affecting gray and white matter segmentation in people with reduced frontal gray matter and lower fractional anisotropy in frontal white matter . Source based morphometry or ica of gray matter probability images across 42 subjects . Each subject's t1-weighted image is segmented to generate a gray matter probability image that is normalized to a common coordinate space and smoothed . The degree to which each independent component (ic) or unique pattern of variance can be compared to the other ics with a variety of metrics, including an estimate of similarity space . The brain regions that contribute most to each component can be identified by displaying each component with scaled intensity values (z score = 13 above). Each ic also has an inverse component or regions that are negatively correlated with regions in the ic . An example is presented for ic7 where white matter hyperintensity related segmentation error (yellow arrow) is identified by ica and is inversely correlated with decreased frontal gray matter (e.g., anterior cingulate, anterior insula, and superior frontal sulcus regions represented by hot signal intensities above). This is important because these results suggest there are independent age - based sources that affect gray matter variation in cerebellar (ic4) and frontal (ic7) regions that are associated with processing speed . The source based morphometry analysis also identified six other independent spatial patterns of gray matter covariance (figure 3). We identified multiple patterns of gray matter variation that were related to age but only some were related to processing speed . One component coincided spatially with the default mode network (ic1), which was nearly perfectly correlated with total brain volume (r = 0.90, p <0.001), related to age (r = 0.34, p <0.05), but not uniquely predictive of processing speed . The frontal component (ic7) related to vessel disease and a cerebellar component (ic4) were each associated with age and uniquely predicted processing speed (figures 1 and 3). Importantly, unique frontal - cerebellar predictors of processing speed were identified with this analysis . A nice feature of the source based morphometry approach is that each component can be covaried in a whole brain analysis to identify regions uniquely associated a variable of interest . This type of analysis is presented in figure 1 and demonstrates: (1) that the frontal and cerebellar regions were uniquely predictive of processing speed; (2) the spatial extent of these effects; and (3) that some brain regions were significantly correlated with processing speed after controlling for either component . The latter is a particularly exciting demonstration of the potential for source based morphometry as an analytical tool because some of these regions, the cingulate for example, had voxels that were influenced by both the source of the frontal component and the source of the cerebellar component . In other words, this is like height being influenced by iodine deficiency and parental height, but in this case the gray matter volume of a specific brain region appears to be influenced by multiple additive aging risk factors . Figure 4 provides a summary of these results, presented in the context of the multifactorial nature cognitive aging . Summary of potential factors affecting frontal and cerebellar morphology and processing speed . On the left side of the figure, aging affects brain morphology through the damaging effects of oxidative stress and inflammation (small vessel disease is in red because of consistent evidence linking cerebral small vessel disease to structural declines and slowed processing speed). These detrimental effects are likely buffered by neuroprotective factors such as (1) leptin and growth factor levels that limit the impact of oxidative stress, (2) norepinephrine or blueberry limits on inflammatory responses in animal models (heneka et al ., 2010; willis et al ., 2009), and (4) positive lifestyle behaviors such as aerobic exercise (rosano et al ., 2010; voss et al ., 2010). The gray arrow indicates that oxidative stress and inflammation likely affects cerebellar morphology, in comparison to the black arrows for which there is empirical evidence of significant associations . The right side of the figure is designed to emphasize that early development has a significant influence on the degree to which older adults will demonstrate atypical morphology and impaired processing speed (deary et al ., 2006, 2010), but could also influence the expression of neuroprotective factors and the development of lifestyle patterns of behavior and modulate risk for age - related neural declines . While this summary figure is general in nature, it is designed to emphasize the multifactorial and interactive effects of aging neural systems on processing speed . The structural components that we identified with source based morphometry included patterns of variation that have been observed in functional imaging studies using ica (beckmann et al ., 2005; damoiseaux et al ., 2006; de luca et al ., 2006; habas et al ., these results suggested that source based morphometry could be used to identify structural variation that corresponds to functionally defined neural systems . Although source based morphometry results will be dependent on variation within a sample, further support for the potential of the source based morphometry approach to identify neural systems was the observation that individual variation in a cerebellar component was related to individual variation in a white matter component that included the middle cerebellar peduncles, cerebral peduncles, and posterior limb of the internal capsule (figure 5 in eckert et al ., 2010). The possibility that source based morphometry captures variance reflecting neural systems may therefore provide some insight into the neural systems that influence processing speed . Our results and the existing literature suggest that a dorsal attention (sweeney et al ., 2001) or prefrontal - mediated system for maintaining focused attention, inhibitory control, and working memory is significantly affected by microvascular damage . This interpretation is consistent with evidence that prefrontal gray matter volume has been related to perseveration on the wisconsin card sorting task (raz et al ., 2003, 2008). It is important, however, to note that our processing speed results were most pronounced for the connections simple test compared to the connections complex task that required inhibiting a previous response . Older adults may become particularly dependent on prefrontal cortex to maintain normal performance when age - related declines in sensory systems make relatively easy tasks more challenging because of degraded stimulus representations (eckert et al . Indeed, elevated frontal activity is a common finding in functional imaging studies of aging (cabeza et al ., 2002; grady et al ., 2005) and appears to reflect compensatory engagement of frontal cortex in some studies (townsend et al ., 2006). Performance across a variety of tasks may slow or exhibit impairment with subsequent declines in a dorsal attention system, particularly for tasks that require sustained attention or the inhibition of distracting information . For this reason, changes in a dorsal attention system could account for slowed processing speed across sensory domains . Interestingly, the dorsal attention system appears to include regions of the cerebellar hemispheres (krienen and buckner, 2009). (2010) did exhibit some spatial overlap even though they each predicted additive variance in those overlapping regions . It is possible that primary aging effects are occurring across the dorsal attention system, but are differentially concentrated across people . This could explain why cerebellar measures might be uniquely predictive of processing speed in some studies and not others . The same neural system may be affected, but there may be regional specificity in the degree to which it is affected . Changes in a dorsal attention or executive attention system could also explain why older adults exhibit difficulty inhibiting attention, as measured by eye - movements, to distracting or irrelevant visual targets (kramer et al . This would have the downstream effect of slowing performance because of difficulty making a saccade to a new target, a function that is critical for many processing speed tasks . It is also possible that direct effects on an eye - movement system could influence processing speed, which is suggested by our findings that processing speed covaries with gray matter variation in the frontal eye - fields, cingulate, dorsolateral prefrontal cortex, and vermis (fujikado and noda, 1987; hayakawa et al . Older adults have been observed to exhibit impaired horizontal saccades (carter et al ., 1983; sharpe and zackon, 1987), but not in every study (pratt et al ., 2006). Understanding the degree to which frontal and cerebellar declines relate to horizontal saccade performance may provide insight into the statistical linkage between neuroanatomical variation in these regions and processing speed . Cerebellar morphology associations with processing speed most likely reflect a change in the ability of the cerebellum to integrate sensory and motor information to help guide smooth and quick movements . Impaired integration of visual and proprioceptive information because of granule cell loss and/or impaired output of this information because of purkinje cell loss could slow behavior and increase movement errors . This prediction is supported by evidence that older adults have difficulty stopping arm movements smoothly and make more corrective movements during reaching than younger adults (brown, 1996). It is unclear, however, whether specific cerebellar zones and related function are affected because of the widespread cerebellar vermis and hemisphere effects observed in mri studies (figure 1). In summary, the existing literature and our sbm findings strongly implicate a dorsal attention system in the processing speed declines of older adults . Impaired function of a domain general system would account for slowed task performance across such a wide variety of behavioral tasks . Questions remain how best to enhance healthy aging of this system, although exercise appears to have positive benefits (colcombe et al . In addition, the degree to which cerebellar declines reflect impairments in specific cerebellar systems and/or reflect changes in a dorsal attention system is unclear . Addressing these questions and identifying the mechanisms for declines in neural systems that support processing speed has the potential to enhance healthy cognitive aging . The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Clinicians and microbiologic laboratories report diagnosed cases to the swedish institute for infectious disease control ., we included all 5,282 ne cases reported to the swedish institute for infectious disease control and registered in the database during january 1, 1997december 31, 2007 . Openepi (www.openepi.com) was used to calculate the standardized mortality ratio (smr). Because ne is an acute viral infection, and as a comparison for the numbers of deaths during the different phases of ne, we used the number of deaths during the second year after diagnosis in the studied population . Incidence was highest in the group 5559 years of age for men and women (figure 1, panel a), and the mean sd age at diagnosis was 49.3 16.7 years for men and 50.7 16.5 years for women . Sex hormones can play major roles in susceptibility to infectious diseases (5,7). To test whether increased incidence of puuv infection in men was dependent on sex hormones, we compared sex ratios in ne cases between children (persons <10 years of age and presumably prepubertal) and adults (> 10 years of age). The male: female ratio for these children was 1.58:1, which was similar to the sex ratio for adult ne patients (1.52:1). This finding suggested that circulating sex hormones may not play a major role in the observed overrepresentation of ne in men . Incidence of acute nephropathia epidemica (ne) and case - fatality rates, sweden, 19972007 . A) age distribution for male and female patients with acute ne . B) age distribution of case - fatality rates for all patients with acute ne . The swedish death register was used to identify all deceased persons with a diagnosis of ne . Numbers of deaths during different periods after diagnoses were 21 (13 male patients and 8 female patients) during the acute phase (<3 months after diagnoses), 7 (5 male patients and 2 female patients) after the acute phase (> 3 months after diagnoses) but <1 year later, and 24 (19 male patients and 5 female patients) during the second year after diagnoses . Of persons with a diagnosis of ne, 21 (0.4%) the smr was 3.5 (95% confidence interval [ci] 2.225.26) for all patients and 6.4 (95% ci 2.9712.15) for female patients and 2.7 (95% ci 1.524.56) for male patients during the acute phase of ne . No persons <50 years of age died during the acute phase (figure 1, panel b). However, the case - fatality rate increased with age (figure 1, panel b), and the case - fatality rate was 6.5% for patients> 80 years of age, which showed age - dependent differences in mortality rates for ne . The mean sd age at time of death for patients with acute ne was 73.7 10.4 years (range 50.988.5 years). Women died at a slightly, but not significantly, younger mean sd age (71.5 12.4 years, range 50.985.7 years, n = 8) than men (75.1 9.2 years, range 57.888.5 years, n = 13) (p = 0.347, by t test). Mean age at time of death for patients with ne was 10 years less than the expected life span for women and 3 years less than the expected life span for men (mean age at time of death in sweden in 2003 was 78 years for men and 82 years for women; www.scb.se/statistik/_publikationer/be0701_1986i03_br_be51st0404.pdf). Because no patient <50 years of age died of ne during the acute phase and the mean age at time of death was relatively high, we suggest that ne is rarely life threatening . To assess this possibility, we analyzed mortality rate patterns early after acute ne . The smr decreased for the 9 months after acute ne in female (smr 0.67, 95% ci 0.112.20) and male (smr 0.36, 95% ci 0.130.79) patients (figure 2). Overall standardized mortality ratios (smrs) for male and female patients with a diagnosis of acute nephropathia epidemica (ne) and smrs 312 mo after diagnosis, sweden, 19972007 . We also analyzed smrs for the first year (including the acute phase of ne [<3 months after diagnosis] and early phase [312 months] after ne diagnosis). For 52 persons who died <2 years after ne diagnosis, the case - fatality rate was only slightly higher in the first year (n = 28) than in the second year (n = 24) after diagnosis (smr 1.17, 95% ci 0.791.66). Deaths did not increase in men between the first (n = 18) and second (n = 19) years after diagnosis (smr 0.95, 95% ci 0.581.49). However, we observed a 2 difference in deaths for women between the first (n = 10) and second (n = 5) years after diagnosis (smr 2.0, 95% ci 1.023.57). We report an age - dependent case - fatality rate for the hantavirus disease ne; most deaths occurred in older persons . There is a report of patients <50 years of age dying of ne (12), but this finding is rare . Increased case - fatality rates in older persons have been described for other infectious diseases . For example, during a typical influenza season, 90% of deaths caused by influenza occur among persons> 65 years of age (13). Whether this finding is unique to relatively mild infection with puuv or is a conserved feature of all hantaviruses causing hfrs / hcps is unknown . We previously showed that there are sex differences in cytokine responses during acute ne (11), which suggested that there might be sex differences in severity of infection . Women showed a 2 difference in number of deaths between the first year and second year after ne diagnosis . However, men showed no difference in number of deaths between the first year and second year after diagnosis . These results suggest that there are sex differences in mortality rates after infection with puuv . Whether ne is a more lethal disease in women than men or causes increased mortality rates in men up to 2 years after diagnoses is unknown . The finding that the case - fatality rate for ne is associated with the age and sex of patients might have practical implications on healthcare issues . It may also indicate that age and sex should be considered predictive variables in clinical studies of hantavirus infections.
Moderate to severe hyponatremia (serum sodium <125 mmol / l), when it occurs, is often an iatrogenic occurrence during hospitalization with significant associated morbidity . Seizures, cerebral edema and death resulting from cerebral edema have been described in some cases of water intoxication . Hyponatremia occurs from a variety of pathogenic processes, including the use of medications which affect water metabolism . One such medication with reported hyponatremia is duloxetine, a selective serotonin reuptake inhibitor (ssri) and selective norepinephrine reuptake inhibitor (snri) approved in the usa for treatment of major depressive disorder, diabetic neuropathic pain, stress urinary incontinence and fibromyalgia . We report here an instance of asymptomatic hyponatremia occurring during duloxetine treatment that became symptomatic during the course of a glomerular filtration rate (gfr) test that involved water loading the subject . A 48-year - old caucasian woman with past medical history of systemic lupus erythematosus, type 2 diabetes mellitus, hypertension, migraine, gastroparesis, fibromyalgia, mitral valve prolapse, hypothyroidism and hyperprolactinemia was scheduled for an i - iothalamate gfr testing as part of a research study . The participant consumed ~ 2 l of fluids at home following instructions from the staff recommending hydration before the test . She received five drops of super - saturated potassium iodide (sski) in 2 oz of water to block the thyroid uptake . At baseline, blood blood and urine were collected to serve as background values to compare with the serial blood and urine tests during the iothalamate gfr testing . The participant was asked to drink additional water per the protocol based on the background urine specific gravity . The gfr test was uneventful for the first 2 h. then, the subject complained of acute onset of generalized headache described as 56 out of 10 with nausea and vomiting . Her heart rate was 5060/min, blood pressure rose to 160170 mmhg systolic and 90108 mmhg diastolic, oxygen saturation registered 100% on room air and finger - stick blood glucose values were 99102 mg / dl . An intravenous solution of 0.9% saline was initiated, and the subject was transferred to the emergency department for further management . During the gfr testing, an intake of 3300 ml of water and a urine volume of 1325 ml were recorded . Her home medications included naratriptan, amitriptyline, cabergoline, propranolol, sitagliptin, meloxicam, hydroxychloroquine, pravastatin, lansoprazole, levothyroxine, vitamin d, azelastine, polyethylene glycol, aspirin and duloxetine . The serum sodium measured 3 months after the increase in the dose of duloxetine was 137 mmol / l . In the emergency department, she had normal pupil reactivity, a supple neck, and normal pulmonary, cardiovascular and abdominal examinations . Neurological examination showed the patient to be responsive but confused with intact deep tendon reflexes . A computerized tomography (ct) scan of the brain showed diffuse cerebral edema with basal cistern effacement . Her intravenous solution was altered to 3% hypertonic saline (212 ml of 3% saline = 109 mmol of sodium chloride), and she was administered furosemide 40 mg intravenously (iv), zofran 4 mg iv and lorazepam 1 mg iv . Serial serum sodium levels were checked, and the rate of hypertonic saline was adjusted accordingly . Symptoms and mental status improved with gradual increase in serum sodium (figure 1). She was discharged to home after 4 days with advice to taper the dose and then discontinue duloxetine . Gfr is considered the best measure of kidney function in health and disease . In large clinical studies, i - iothalamate the test requires water loading, multiple blood draws and 68 h of time . Despite the significant water loading recommended to achieve the most accurate gfr results, hyponatremia has not been reported previously during gfr testing . Renal capacity for free water excretion is up to 28 l / day . As a result, under normal physiological conditions,, our subject had undergone uneventful i - iothalamate testing in the past without complications prior to receiving duloxetine . In previously reported cases of duloxetine - related hyponatremia, the time course of developing hyponatremia was acute as compared with our subject whose serum sodium was normal even after 6 months of duloxetine usage . It is likely that the pre - admission water load specified in the gfr protocol provoked hyponatremia in our subject, which was worsened by the added water ingestion during the gfr procedure . Our patient illustrates the need for vigilance during water loading, even when well tolerated previously, especially when medications such as antidepressant and antipsychotic are in use . A stat serum sodium blood test could be performed prior to exposing the subject to water loading when such a situation like ours occurs in a clinical research setting . Our case illustrates the value of a detailed medication history when performing water loads and supports the need for vigilance when studying subjects who take an antidepressant or antipsychotic medication despite a documented history of eunatremia on such therapies.
Aggression is defined as behaviors intended to hurt, harm, or injure another person (1). It is rooted in an overall structure which could appear in the forms of anger, violence, physical, verbal, and relational aggression (2, 3). Aggression could be a product of our interactions with individuals in our environment (4) which, its severity greatly differs across countries and cultures (5). There are various studies indicating relations between aggressive behaviors and sex (6 - 11), household income (8 - 11), marital status (12), and age (7, 10, 13). Aggression is by no means a new concern in human society, especially in youth . Aggressive behaviors in the young people are complex, heterogeneous with diverse etiologies and consequences . So, no single term is adequate to capture all variegated and divers presentations of such behaviors in youth (14). Moreover, universities are among the institutions in which most of the members are young people (14) and because of facing with various personal and social stressors, the students usually experience high level of stress (15). They are a particular group of people in a critical transitional period (17). Many reasons such as academic stress, new community relation, and changing in life conditions could increase possibility of the students aggressive behaviors (18). It is also indicated that student aggression would be related with type of university (19) and level of education (20). This study aimed to evaluate potential associations between students aggression and their personal, family, and social characteristics . This study aimed to determine aggression among university students and its association with their personal, family, and social characteristics . This analytic, cross - sectional study was conducted in gonabad, a collegiate city in eastern iran in 2012 . There are 3 universities in the city with more than 10000 students, as the research population . The inclusion criteria were the students willingness to participate in the study, having no obvious mental and physical disorders, and studying in the first semester of 2011 - 2012 academic year . We excluded any participants who failed to complete the questionnaire as well as those who were not willing to continue the study . Using the following formula, a minimum size of 784 the participants was determined however, to gain a higher validity, in case of potential problems during the study process, the required sample size increased to 860 . Where, p = 0.5, q = 0.5, z (1 - /2) = 1.96, z (1 -) = 0.84, and d = 0.05 . The relevant official permits were issued to perform the sampling and accomplish the research ., the sampling frame was used . Using probability proportional to size stratified sampling, we selected 860 students of gonabad university of medical sciences (gmu) (n = 120), islamic azad university (gonabad branch) (n = 380), and payame noor university (gonabad branch) (360), based on their sex and marital status in each stratum . Later, 51 participants were removed due to filling incomplete questionnaire we applied bass - parry aggression questionnaire (21, 22) concentrating on physical aggression (9 items), verbal aggression (5 items), anger (7 items), and hostility (8 items). Buss and perry reported a cronbach coefficient at 0.89 and test - retest reliability at 0.80 (21). This is a bias free (23), self - report questionnaire, which is used in most countries (24) and consisting of 29 sentences valued at a likert scale from 1 (extremely uncharacteristic of me) to 5 (extremely characteristic of me). This questionnaire is validated in iran (2) via cronbach coefficient (0.89). To collect the data, we first distributed the questionnaires among the participants we continued the process until collecting the required questionnaires . Using spss (version 18), we analyzed the gathered data via t test, anova, and linear regression model . Regarding the regression assumption, the distribution of aggression scores was independent . Besides, the distribution of errors was normal and using durbin - watson, errors independency was acceptable . In this study, p this study was approved by student research committee of gonabad university of medical sciences . Before starting the study furthermore, the participants were informed about the goals of the study . After receiving the participants verbal consent, this analytic, cross - sectional study was conducted in gonabad, a collegiate city in eastern iran in 2012 . There are 3 universities in the city with more than 10000 students, as the research population . The inclusion criteria were the students willingness to participate in the study, having no obvious mental and physical disorders, and studying in the first semester of 2011 - 2012 academic year . We excluded any participants who failed to complete the questionnaire as well as those who were not willing to continue the study . Using the following formula, a minimum size of 784 the participants was determined . However, to gain a higher validity, in case of potential problems during the study process, the required sample size increased to 860 . Where, p = 0.5, q = 0.5, z (1 - /2) = 1.96, z (1 -) = 0.84, and d = 0.05 . The relevant official permits were issued to perform the sampling and accomplish the research . According to the performed coordination, the sampling frame was used . Using probability proportional to size stratified sampling, we selected 860 students of gonabad university of medical sciences (gmu) (n = 120), islamic azad university (gonabad branch) (n = 380), and payame noor university (gonabad branch) (360), based on their sex and marital status in each stratum . We applied bass - parry aggression questionnaire (21, 22) concentrating on physical aggression (9 items), verbal aggression (5 items), anger (7 items), and hostility (8 items). Buss and perry reported a cronbach coefficient at 0.89 and test - retest reliability at 0.80 (21). This is a bias free (23), self - report questionnaire, which is used in most countries (24) and consisting of 29 sentences valued at a likert scale from 1 (extremely uncharacteristic of me) to 5 (extremely characteristic of me). This questionnaire is validated in iran (2) via cronbach coefficient (0.89). To collect the data using spss (version 18), we analyzed the gathered data via t test, anova, and linear regression model . Regarding the regression assumption, the distribution of aggression scores was independent . Besides, the distribution of errors was normal and using durbin - watson, errors independency was acceptable . In this study, p this study was approved by student research committee of gonabad university of medical sciences . Before starting the study, furthermore, the participants were informed about the goals of the study . After receiving the participants verbal consent, we analyzed the data of 809 fulfilled questionnaires out of 860 (response rate: 94%). Relations of the respondents aggression score and variables under study are seen in table 2 . According to the results, the mean score of aggression among students who lived in dormitory (in - dorm) was significantly higher than those students who lived out of dorm . Since p value for age and sex was less than 0.200, we entered these variables with residency status in a linear regression model, simultaneously . The results showed that, after controlling age and sex, there was no difference between mean aggression score of the students in terms of residency status . Then, we evaluated mean score of aggression of the students in terms of residency status . The mean aggression score in male students who lived in dorm was significantly higher than female students residing in dorm . Meanwhile, the mean score of aggression in gonabad medical university students who lived in dorm was significantly lower than in dorm students of other universities . There were no significant differences between aggression scores and the independent factors among out - of - dorm students . We can see in table 5 that a significant, inverse association exists between age of the students living in dorm and their aggression scores . According to the results, residency status can be considered as a confounder in this research . So, we should analyze the data in two separate levels in terms of residency status . As it is seen, there was no association between the mean aggression score of the respondents who lived out of the dorm and their sex, marital status, residency situation, university, and age . In contrast, there were significant association between aggression scores of the students lived in dorm and their sex, university, and age . This study aimed to evaluate the students aggression and its association with their personal and social characteristic . Based on the study results, paying attention to aggressive behaviors among the students as well as recognizing some potential associations would be useful to control and diminish aggression among them . According to the results, there were significant relations between students aggression and their sex and residency status, while there were no such associations with the respondents sex, level of education, marital status, and residency condition . After controlling the potential confounding effect of residency status (in - dorm, out - of - dorm), aggression score of those students who lived out of dorm had no significant relation with their characteristics . Among in - dorm students, however, there were significant associations between their aggression score and sex, age, and university . Our results indicated that mean score of aggression for in - dorm students was higher than those students who lived out of dorm . This finding is justifiable as these students usually live in a stressful condition and far from their home . So, they may have low tolerance threshold against stressors and could not mange well such conditions . Besides, aggression would be a product of interactions with individuals in an environment (4). Therefore, living in dorm would increase aggressive behaviors among students because of personal contacts as well as maladaptive behaviors . There are various studies concentrating on aggressive behaviors and some of the causes among students who lived in dorm (25). Our results showed the aggression mean score of the male students was significantly higher than the female students who lived in dormitory which was comparable with many studies (26 - 28), except anderson et al . Results (29). Differences between the students age groups as well as using different tools to evaluate their aggression could probably be potential reasons for discrepancy of the results . We found an association between aggression and the students university; mean score of students aggression who lived in dorms of gonabad medical university which is a state university, was significantly lower than those in - dorm students of the private universities (i.e. Islamic azad and payame noor universities). This may be because of controlling residency status, as a confounder, in our study . According to our results, there was a significant and inverse association between the in - dorm students age and their aggression score which was similar to swanson (12) findings . Our result was not comparable with hess and hagen (30) as well as mousavi et al . We did not find any relation between the respondents education term and their aggression score . Contrary to our finding, sharma (20) indicated that mean scores of master students in anger scale were significantly lower than those of bachelor students . One of the potential causes to this discrepancy would be the use different tools for evaluating aggression . Swanson et al . (12), however, showed that aggression score of married persons was significantly more than that of singles . Surprisingly, grassi et al . (32) found that single individuals had more aggressive behaviors than married persons . Although use of different tools to evaluate aggression as well as research on different study groups would be potential causes of these discrepancies, more studies to discover the potential associations are recommended . One of the limitations of this study is the method of gathering data which was self - report . The researchers, albeit, offered required comments when the questionnaires delivered to the respondents . Besides, this study was conducted on university students; so, our results could not be generalized to all young people . Research on relevant sample size and sampling method as well as a separate analysis among in - dorm and out of dorm students are strong points of our study . Indeed, one of the important points of our study was considering the living condition of students (in - dorm, out of dorm) as a confounding variable . According to our results, we recommend holding educational programs related to stress management for students as well as providing more facilities with respect to cultural and exercise activities for refreshing students, especially for in - dorm students . These activities would fill both their leisure time and reduce their stress affecting aggressive behaviors . Increasing the number of day shift university students implicitly indicates the necessity of recognizing students characteristics and their relational problems as well as finding methods to diminish these difficulties . Therefore, our results could be beneficial for policy makers as well as deans of the iran universities . In conclusion, this study showed that mean aggression score of in - dorm students was significantly higher than those students who lived out of dorm . Besides, aggression score of the male students was higher than the female students . So, regarding the control of aggressive behaviors, paying attention to male, in - dorm students, especially younger ones has more priority . Besides, relation between the participants aggression score and type of the university in terms of state and private would be a proxy of family, social, and economical discrepancies . One of the limitations of this study is the method of gathering data which was self - report . The researchers, albeit, offered required comments when the questionnaires delivered to the respondents . Besides, this study was conducted on university students; so, our results could not be generalized to all young people . To determine aggression condition between student and nonstudent youth, research on relevant sample size and sampling method as well as a separate analysis among in - dorm and out of dorm students are strong points of our study . Indeed, one of the important points of our study was considering the living condition of students (in - dorm, out of dorm) as a confounding variable . According to our results, we recommend holding educational programs related to stress management for students as well as providing more facilities with respect to cultural and exercise activities for refreshing students, especially for in - dorm students . These activities would fill both their leisure time and reduce their stress affecting aggressive behaviors . Increasing the number of day shift university students implicitly indicates the necessity of recognizing students characteristics and their relational problems as well as finding methods to diminish these difficulties . Therefore, our results could be beneficial for policy makers as well as deans of the iran universities . In conclusion, this study showed that mean aggression score of in - dorm students was significantly higher than those students who lived out of dorm . Besides, aggression score of the male students was higher than the female students . So, regarding the control of aggressive behaviors, paying attention to male, in - dorm students, especially younger ones has more priority . Besides, relation between the participants aggression score and type of the university in terms of state and private would be a proxy of family, social, and economical discrepancies.
Children in developing countries bear a heavy burden of respiratory tract diseases and diarrhoeal diseases . Hand washing with soap is one of the principal means of preventing transmission of certain diseases . It is generally acknowledged that using latrines can also interrupt the transmission of diarrhoeal disease . Measures such as hand washing with soap, water supply, construction of latrines, and promoting general hygiene provide opportunities for improving children's health . In 2006, six major french actors on the international solidarity scene came together to form an innovative public - private partnership, the alliance for development . Members of the alliance include institutions from both the public sector (ministry of foreign affairs; ministry of economy, finance, and industry; and agence franaise de dveloppement) and the private sector (sanofi - aventis, institut pasteur, and veolia environnement). The aim is to take advantage of their complementary competences and networks in undertaking concrete action to contribute to improving local environmental and health conditions in developing countries . In order to have a positive impact on prevention of communicable diseases and thus on the health of the population in the long term the agence franaise de dveloppement (afd) was the lead partner for the alliance's project in niger . The agency is in charge of ensuring the smooth running of the project on water, environment, and health in schools, known as esamis . The maradi region of niger was identified as the main intervention area for this pilot phase . The goal of the esamis project is to improve access to clean drinking water, sanitation, and health education for school children and their teachers . The project focused on the following hygiene measures: water supply, with the construction of hydraulic infrastructure; sanitation, with the construction of latrines; proper management of this equipment through the education programmes and rules of hygiene . The esamis project lasted 18 months, from january 2007 to june 2008 (table 1). The latrines were built, clean water was provided, school children in maradi schools were educated on health and hygiene, and local management committees (comprising teachers and parents) were set up to ensure sustained use of the infrastructure . There has been very little research on the hygiene and living conditions of communities in niger and the positive effects that interventions produce for beneficiary populations . The centre de recherche mdicale et sanitaire (cermes) was therefore requested to evaluate the epidemiological impact of the esamis project . It is expected that putting in place hygiene and sanitation infrastructure and providing health education will lead to a reduction in symptoms of infection, absenteeism, and intestinal parasitic diseases carriage . Most of the existing research on the health impact of clean water supply and hygiene infrastructure projects focuses on the incidence of diarrhoeal diseases, malnutrition, and infant mortality [59]. Very little research has been carried out on the impact of hygiene and sanitation measures on intestinal parasitic diseases in sub - saharan africa . A study was carried out in cte d'ivoire to assess the impact of latrine construction, water supply, and health education on the incidence of parasitic diseases in children aged 2 to 4 years . It is generally accepted in theory that latrines do serve to reduce the incidence of parasitic disease . Also, when family heads are asked about household health problems and health problems in the community in general, they rarely ever mention intestinal worms or schistosomiasis as a major health problem . It is therefore clear that more studies must be undertaken to document the epidemiology of parasitic infections and estimate their impact on the affected populations . It is meant to consolidate the existing documentation on the impact of improved hygiene on intestinal parasitic diseases in sub - saharan africa . The aim is to assess how clean water supply, construction of latrines, establishment of hand washing stations, and health and hygiene education affected the health of school children in the maradi region . This was a before and after intervention study on a representative sample of school children from schools in the maradi region . Study since some schools were beneficiaries of the project (programme group) while others were not (control group). The population covered by the study was made up of school children from 20 schools in the aguie, mayahi, and tessaoua districts of the maradi region (eastern niger). Three primary schools (programme group) were first selected randomly, out of the 20 schools . For each school selected, another nonbeneficiary school (control group) was randomly selected from among all the schools within a 5 km radius of the programme group school . 120 pupils were then selected randomly from each of the 6 schools . For the two surveys, before and after the project, the same sampling plan was used for school children in both the programme group and the control group . The number of subjects needed to compare parasite carriage in the two groups of schools and demonstrate any difference in the programme group before and after the project was 720 in the six schools, that is, 120 per school . The final sample was made up of school boys and girls aged between 7 and 12 years from public primary schools in the awache and magaria villages in the tessaoua district, bougouzawa and el gueza in the aguie district, and zongon oumara and bassare in the mayahi district . Before the start of the project, only two schools (one from each group) were equipped with two old latrines each . The project set up clean water outlets, three latrines, and three hand washing stations in each of the programme group schools . The assigned trainer in each of the programme schools held 30-minute daily discussion sessions with the school children and teachers . The lessons focused on drinking water hygiene, use of latrines, and hand washing with soap . Four posters describing the use of latrines and hand washing techniques were distributed to each class . Teachers were given a package containing a guide describing the 5 steps in hand washing and including some basic concepts on the transmission of infectious diseases . The project provided drinking water in covered buckets equipped with a cup for each classroom in the programme schools . The school children were responsible for washing the equipment and changing the water each day . Two plastic kettles were placed in each latrine for washing the anus . For hand washing, a cake of soap was placed on each washing bowl and the trainers demonstrated correct hand washing methods . This learning programme was based on the theory that these measures would inculcate the habit of hand washing with soap and using latrines . They are therefore expected to reduce the symptoms of infection, as well as children's absence from school . The teachers then continued to reinforce the educational message in the school programmes by repeating what they had learned in the training phase and by using the guide . Each week, the teachers would do a 10- to 15- minute revision of the messages in the posters on the wall . This entailed a quick review of the hygiene rules and the symptoms caused by lack of hygiene . The first field survey was carried out between november 7, 2007, and november 19, 2007, prior to the start of the programme, and the second between 20 and may 31, 2008 . An individual questionnaire was administered to all the school children to gather demographic data; information about symptoms such as diarrhoea, abdominal pains, and vomiting; water consumption habits; sources of drinking water at school; and latrine usage . The interviews were carried out in standard format in individual conversations between the school children and four trained interviewers who were well aware of how to conduct the interview, as well as the possible biases that could occur . The teachers had to answer a group questionnaire to provide data on pupils' absence from school during the 5 days prior to the survey, as well as on the source, storage, and replenishment of drinking water at school . Two kato - katz slides were prepared and examined according to a previously described method [13, 14]. The aim was to find the eggs of schistosoma mansoni, hookworm, pinworms, and other parasites (entamoeba coli, entamoeba histolytica, taenia saginata, hymenolepis nana, and trichomonas intestinalis). Data were entered into the database using epi info (cdc, atlanta, ga, usa). The proportions were then compared using pearson's chi - squared test and fisher's exact test . Means were compared using student's t - test . The threshold for significance was set at p <0.05 . The factors studied were intestinal parasitic disease, symptoms caused by lack of hygiene, water consumption habits, absence from school, sources of clean drinking water at school, and latrine use . The study was approved by the national ethics committee of niger before the data were collected . Only children whose parents had given their approval were allowed to participate in the study . The primary school inspectorates of the schools concerned were also informed about the study, for each of the cermes studies . The school director was first briefed and subsequently accompanied a delegation including members of the mission to meet the village chief and inform him of the aims of the study . Information was then passed on to parents through the parents' association, of which most of them were members . Children who were infected by intestinal helminths were given the appropriate treatment by the study team . For schistosoma mansoni infection they were treated with praziquantel, and with albendazole for the other helminths . Two samples of children aged between 7 and 12 years were selected . During the preproject study, 665 pupils were selected, with a predominance of boys (sex ratio = 1.8). The average ages of the children were 9.1 and 9 years, respectively, before and after the project . A statistically significant reduction in cases of diarrhoea and abdominal pains was noted after the project; from 3.9% before to 2.7% after, p = 0.04, and from 4.7% to 3.2%, p = 0.02, this reduction was noted in both the programme group schools and those in the control group . There was no statistically significant difference in the prevalence of vomiting before and after the project (from 1.7% to 1.3%, p = 0.08). The number of children absent from school in the 5 days preceding the study increased significantly from 2.6% to 7.3% (p <0.001). The increase in absence from school was statistically significant in both the programme group schools and those in the control group . The rate went up from 4.4% before the project to 8.6% (p = 0.01) after the intervention in the schools in the programme group and from 0.6% to 25.4% (p <0.0001) in schools in the control group (table 2). According to 19.7% of respondents, the water consumed in schools on the eve of the study came mostly from the water tap (61.1%). In schools in the programme group, consumption of tap water increased from 87.0% to 99.6% (p <0.001), to the detriment of the two other sources of supply . Use of the water tap was clearly much higher in the programme group schools (87.0%). In schools in the control group, water mainly came from the well (89.7% of the 12.1% respondents). Use of the borehole was significantly higher in schools in the control group, from 10.3% to 42.1%, p <0.001 (table 3). Following the project activities, schools in the programme group totally abandoned the use of the well as a source of water supply (from 10.9% to 0%). There was no difference in the frequency of latrine usage at home in the programme group: from 24.8% to 29.8%, p = 0.20 . This frequency however fell significantly in schools in the control group, from 6.8% to 3.3% (p = 0.01) table 2 . At the time that stool samples were taken for the preproject study, 7.5% of the 664 school children who provided such samples were carriers of at least one intestinal parasite . Prior to the intervention, the intestinal parasites carriage rate was significantly higher for the programme group schools than for those in the control group (9.6% versus 5.3%, p = 0.02). Among those who had worms overall, carriage of at least one parasite increased from 7.5% before the project to 10.2% after (p = 0.04) table 4 . It went from 9.6% before the project to 8.1% after the project in the programme group, although this was not statistically significant (p = 0.24). There was however a significant increase in the control group, from 5.3% before the project to 12.5% after it (p <0.001). After the project, parasite carriage was higher in the control group schools, compared to the programme schools, although the results barely reached the significance threshold (8.1% versus 12.5%, p = 0.05). Only carriage of parasites other than pinworm, ancylostoma, and ascaris was significantly higher in the control schools (p = 0.03). Group schools, there was a statistically significant increase in the prevalence of hymenolepis nana, from 0 to 1.9 (p = 0.02). This is the first ever study in niger to describe the health impact of sanitation and hygiene for school children . It demonstrated a significant increase in the use of tap water systems as a source of water supply and in the adoption of hygiene measures . Intestinal parasitic diseases remained stable in the programme group but increased significantly in schools in the control group . This shows that construction of latrines, followed by a programme of health education, produces the required effects . Vomiting is not a pathognomonic sign for lack of hygiene and may occur in other infections such as malaria . It is worth noting that the study took place in may, at the start of the high malaria transmission period . Under such conditions, and in the absence of a differential diagnosis, it is difficult to observe the real positive effects of latrines and water supply systems on the occurrence of vomiting . The number of children absent from school in the five days preceding the survey increased in both the control schools and those in the programme group . A chinese study covering a period of 5 months showed that children who had been taught about hand washing with soap missed school significantly less frequently that those who had not had such lessons . It is of course difficult to link these observations directly with the project because the reasons for the children's absence are not always indicated . There may be several different reasons: truancy, farm work, and social events (births, weddings, deaths, etc . ). In this specific situation, indeed, the teachers often told us that the pupils had left the village in the company of their parents . The schools in the study are located in rural areas where families travel from the village with their children at this time, to work on their farms . There was also an increase in reported use of the water supply system, latrines, and hand washing after using the toilet in all the schools, after the health education campaign . These complementary hygiene practices are known to be effective and they must be promoted in school . Pupils in the programme group schools made more frequent use of drinking water from the tap . The school drinking water was stored in buckets that were washed every two to seven days, depending on the school . The longer period between washes nevertheless, this method is still safer than the wells, which are sometimes not covered . This study showed a significant increase in reported hand washing in schools in the programme group and to a lesser degree in schools in the control group . Reported latrine usage at home also increased slightly among pupils in the programme group schools . On the other hand the fact that children had been taught about promoting hygiene had a positive effect on the use of latrines at home . These results may be due to the fact that very little time had elapsed between the project and the evaluation . Some of the reasons that may explain the low impact of hygiene interventions on the population include the fact that most studies last only for a short time and that interventions are not flexible enough in the way they are set up to include research methodologies . Hand washing after using the toilet also increased in the programme group schools, which goes to show that the awareness - raising had been effective . The campaign, which was carried out in the schools, also focused on some material aspects such as providing water and availability of soap and latrines . Availability of soap close to the latrines and the time required to reach the water outlet are two of the constraints on the practice of hand washing after defecation . In order to encourage people to wash their hands, constraining factors such as an appropriate source of water and soap availability have to be resolved . Our study did not find any significant difference in parasite carriage prevalence in the programme group school . It was therefore not possible to demonstrate a significant reduction in parasitic diseases prevalence . In this, the study differed from what is sometimes reported in the literature . It is however important to note that unlike the case of the control group, there was no increase in the prevalence of parasitic diseases in the programme group . The study carried out in cte d'ivoire demonstrated that improvements in hygiene conditions reduced the incidence of ascariasis by 75% and ancylostomiasis by 82% . The population included in that study was however younger than ours (2 to 4 years), and the study was carried out two to four years after the initial preintervention study . In our study, overall carriage of intestinal parasites was low (7.5%) even before the project was implemented . Intestinal parasitic diseases are generally quite rare in niger, with prevalence rates below 10% . This low prevalence rate is also found in chad and mali, where the climate is similar to that of niger . The low prevalence of parasite carriage may also be due to the fact that deworming exercises using albendazole are carried out regularly in the maradi region as part of school health programmes and also as part of neglected tropical diseases control activities . Indeed a mass treatment campaign had taken place in the region in april 2008 and had covered both the schools in the programme group and those in the control group . For such a study, this represents a potential confounding factor that it was difficult to take into account . The programme schools and the control group schools were close to each other (within a radius of 5 km). Children from the control group may therefore have heard about the project, and, as such, its real effects may actually be underestimated . In the duration of the study, children from the programme group may have disseminated the educational messages about hand washing, especially after defecation . Because of the methodological limitations, we were not able to assess which external factors may have influenced the hygiene practices of pupils (especially latrine usage). Pupils' self - reported hand washing is not strongly correlated to observed behaviour, which requires immense resources . As a result although we were not able to assess the changes in pupils' hygiene behaviour or the impact on the hygiene practices and health of family members, this model of hygiene promotion is quite easy to replicate and should be useful for improving hygiene habits . Furthermore, interventions that focus on hygiene have been associated with a reduction in the number of visits to health centres . Programmes such as this, which promote better hygiene, should therefore be encouraged in low income countries . Our assessment of the health impact of building latrines and implementing an educational programme confirms the findings of corrales et al . On the fact that transmission of infections is not only linked to latrines . Putting health infrastructure in place in schools obviously had an impact on hygiene - related habits in the beneficiary schools and communities . This impact is not generally demonstrated in health (parasitic diseases, and symptoms related to lack of hygiene). Such activities nevertheless have at least short term beneficial effects since they increase access to clean drinking water and teach children hygienic habits . Monitoring should continue over a longer period, in order to consolidate the effects of such interventions . Awareness - raising on standard hygiene measures, especially hand washing and use of latrines, should be carried out regularly.
Increasing age and cancer are well - established risk factors for venous thromboembolism (vte).16 given that older persons bear most of the cancer burden, as the population ages the incidence of cancer - associated vtes will increase as well.7,8 however, the epidemiology of vte in older cancer patients has not been well described . Prior efforts have largely focused on either hospitalized patients or were not able to collect longitudinal data on a sufficient number of patients.1,9,10 as a result, we lack information about the degree to which vte increases the risk of death or anticoagulation - associated complications such as hemorrhage at the population level . Prior work has suggested that cancer patients with a vte have a two- or threefold greater risk of death.3,11 if a vte diagnosis truly does independently increase the risk of death threefold, it would have profound implications for prognostication, management, and prophylaxis of vte in cancer patients . It is also possible that the impact of vte on mortality may be less pronounced than prior studies have suggested because vtes are associated with more aggressive tumor characteristics and later stage at diagnosis.10 prior studies have not fully accounted for patient and tumor factors and it is unclear to what degree vtes are truly a risk factor for mortality or if they are simply a marker of more aggressive tumors.11 furthermore, given that cancers vary in the degree to which they are associated with thrombosis, it could be expected that the impact of vte on subsequent survival could vary across cancer types.12 information about whether vte is associated with increased risk of death for different types of cancer could inform decision - makers about the intensity and duration of vte prophylaxis and treatment . Although some work has suggested that patients with a vte could benefit from prolonged or even indefinite anticoagulation, the hemorrhage risks for patients with vte are uncertain.13,14 while the frequency of major hemorrhage reported in clinical trials was quite low, recent analyses of anticoagulation treatment of both cancer and noncancer patients in the community setting have suggested that major bleeding rates were up to tenfold higher.4,15,16 moreover, subgroup analyses suggested that patients with vte in the setting of cancer may have a higher hemorrhage risk than patients with vte alone.4,15,16 in addition, patients with some types of malignancies may be more prone to major hemorrhage than others because of anatomic location or disordered hemostasis associated with the underlying malignancy.17 as a result of these areas of uncertainty, there has been a call for population - based data documenting the rate of hemorrhage among cancer patients with vte.18 to address these knowledge gaps, we conducted a longitudinal population - based study of older cancer patients to determine how frequently vte is diagnosed concomitantly with cancer and the degree to which vtes increase the risk of death or major hemorrhage for different types of cancer . We obtained data on patients diagnosed with cancer during 1995 through 1999 from the linked national cancer institute (nci) surveillance, epidemiology, and end results (seer)medicare database . All incident cancer patients who reported to the seer registries are cross - matched with a master file of medicare enrollment.19,20 patient level information available includes sociodemographic characteristics, cancer type, grade, site, and stage.21 we identified all patients who were diagnosed with 1 of 10 common cancer types, selected on the basis of their high overall incidence rates as well as prior studies demonstrating a relationship with vte.11,12 to allow for a 2-year ascertainment period for previous vte as well as other comorbid conditions before their cancer diagnosis (see below), we only included patients who were 67 years of age and older at the time of their cancer diagnosis . Table 1 demonstrates the construction of the study sample . Of a total of 410,315 patients in the database, we included only the 247,785 individuals who were 67 years of age or older at the time of diagnosis, had a malignant primary lesion diagnosed during 1995 through 1999, and had a known cancer type and month of diagnosis . Exclusions included date of death before cancer diagnosis (23 patients), ineligible for medicare part a or part b (17,477), and enrollment in a managed care plan during a 2-year period before cancer diagnosis (60,771). These latter groups of patients were excluded because claims from these beneficiaries were not included in medicare claim files . Patients who dropped coverage and/or enrolled in a managed care plan after their cancer diagnosis were censored at the month the fee - for - service coverage terminated . Finally, we restricted our study sample to patients who had no diagnosis of vte (defined as the absence of any icd-9 codes) between 6 and 24 months before their cancer diagnosis to increase the likelihood that patients defined as having a concomitant vte did not have a prior vte . Table 1construction of study sample number of patientspercentagetotal patients410,315100.0exclusions not malignant primary30,4507.6 first cancer diagnosis outside of 1995199940,20710.0 age <67111,14227.7 cancer type not of interest12,2413.1 unknown month of cancer diagnosis2,0510.5eligible247,78561.7additional exclusions date of death before cancer diagnosis230.0 not eligible for part a or part b during 2-year period before cancer diagnosis17,4777.1 hmo enrollment during 2-year period before cancer diagnosis60,77124.5 prior claim for vte3,6320.9study sample167,38540.8vte = venous thromboembolism . A vte was defined as the diagnosis of either deep venous thrombosis (dvt) or pulmonary embolism (pe). Cases were ascertained through hospital admissions associated with specific international classification of diseases, 9th revision, clinical modification (icd-9-cm) codes . The relevant icd-9-cm codes used for the diagnosis of dvt or pe were 451.1, 451.11, 451.19, 453.2, 451.81, 453.2, 453.3, 453.8, 453.9, 415.1, 415.11, and 415.19 . We defined a vte as presenting concomitantly with a cancer diagnosis if the vte diagnosis was made between 6 months prior and 1 month after the initial cancer diagnosis . The 6-month prediagnosis window was selected because many cancer - related vtes may be diagnosed before recognition of the underlying malignancy.3,22,23 we defined major hemorrhage as an intracranial or gastrointestinal hemorrhage requiring hospital admission . Codes 430, 431, or 432were derived from validated work using administrative claims data.24 similarly, icd-9-cm codes for gastrointestinal hemorrhage (456.0x, 530.7, 530.82, 5315, 537.83, 562.0203, 562.1213, 569.3, 569.85, and 578.xx) were also derived from previously published approaches.25 cancer stage was determined using seer american joint committee on cancer (ajcc) stage or historical stage (coded as local, regional, or distant) for cancer types in which ajcc was not available (lymphoma, pancreatic, renal, and prostate cancers). We used median income according to patient zip code of residence as a proxy for socioeconomic status (ses). Dichotomous cancer treatment variables were created using seer data and medicare claims to identify patients who had received cancer - specific surgery, chemotherapy, and radiation therapy.26,27 pertinent comorbidity diagnosis codes during the period before each patient s cancer diagnosis were obtained from the inpatient and outpatient claims to identify conditions that comprise the charlson comorbidity index.28 we only included conditions that appeared on either one inpatient or two outpatient claims.28 to enable us to capture additional conditions that could alter the risk of developing a vte, we also identified patients with a diagnosis code for atrial fibrillation (icd-9 code 427.3) prosthetic heart valve (icd-9 code 35.20, 35.22, 35.24, 35.26, or 3528), obesity (278.x), hip fracture (icd-9 code 352.0, 352.2, 352.4, 352.6, or 352.8), or insertion of a central venous catheter (cpt code 36533) during the 2-year period before their cancer diagnosis . To understand the degree to which the relation between vte and mortality is mediated by cancer - related and other factors the initial cox proportional hazards model incorporated only age (by 5-year age group because of the nonlinear relation between age and mortality), race, and gender as covariates, with vte as the independent variable and death as the outcome . We then added cancer characteristics and therapy to the model, including stage at diagnosis, histological grade, chemotherapy, radiation, and cancer - specific surgery . Because comorbid conditions could affect the probability of receiving treatment or of developing a vte, comorbidity was then added to the model . While ses would not be expected to alter the physiologic impact of a vte, we considered lower ses as a potential mediator of stage and comorbidity . We calculated the proportion of patients who had a major hemorrhage during the first year after cancer diagnosis for each cancer type according to vte status . The excess hemorrhage risk in vte patients was estimated by deriving the absolute difference in hemorrhage rates between the vte and non - vte groups . Finally, we explored the potential role of hemorrhage in mediating the relationship between vte and mortality by adding hemorrhage as a covariate to the final multivariate model . We obtained data on patients diagnosed with cancer during 1995 through 1999 from the linked national cancer institute (nci) surveillance, epidemiology, and end results (seer)medicare database . All incident cancer patients who reported to the seer registries are cross - matched with a master file of medicare enrollment.19,20 patient level information available includes sociodemographic characteristics, cancer type, grade, site, and stage.21 we identified all patients who were diagnosed with 1 of 10 common cancer types, selected on the basis of their high overall incidence rates as well as prior studies demonstrating a relationship with vte.11,12 to allow for a 2-year ascertainment period for previous vte as well as other comorbid conditions before their cancer diagnosis (see below), we only included patients who were 67 years of age and older at the time of their cancer diagnosis . Table 1 demonstrates the construction of the study sample . Of a total of 410,315 patients in the database, we included only the 247,785 individuals who were 67 years of age or older at the time of diagnosis, had a malignant primary lesion diagnosed during 1995 through 1999, and had a known cancer type and month of diagnosis . Exclusions included date of death before cancer diagnosis (23 patients), ineligible for medicare part a or part b (17,477), and enrollment in a managed care plan during a 2-year period before cancer diagnosis (60,771). These latter groups of patients were excluded because claims from these beneficiaries were not included in medicare claim files . Patients who dropped coverage and/or enrolled in a managed care plan after their cancer diagnosis were censored at the month the fee - for - service coverage terminated . Finally, we restricted our study sample to patients who had no diagnosis of vte (defined as the absence of any icd-9 codes) between 6 and 24 months before their cancer diagnosis to increase the likelihood that patients defined as having a concomitant vte did not have a prior vte . Table 1construction of study sample number of patientspercentagetotal patients410,315100.0exclusions not malignant primary30,4507.6 first cancer diagnosis outside of 1995199940,20710.0 age <67111,14227.7 cancer type not of interest12,2413.1 unknown month of cancer diagnosis2,0510.5eligible247,78561.7additional exclusions date of death before cancer diagnosis230.0 not eligible for part a or part b during 2-year period before cancer diagnosis17,4777.1 hmo enrollment during 2-year period before cancer diagnosis60,77124.5 prior claim for vte3,6320.9study sample167,38540.8vte = venous thromboembolism . A vte was defined as the diagnosis of either deep venous thrombosis (dvt) or pulmonary embolism (pe). Cases were ascertained through hospital admissions associated with specific international classification of diseases, 9th revision, clinical modification (icd-9-cm) codes . The relevant icd-9-cm codes used for the diagnosis of dvt or pe were 451.1, 451.11, 451.19, 453.2, 451.81, 453.2, 453.3, 453.8, 453.9, 415.1, 415.11, and 415.19 . We defined a vte as presenting concomitantly with a cancer diagnosis if the vte diagnosis was made between 6 months prior and 1 month after the initial cancer diagnosis . The 6-month prediagnosis window was selected because many cancer - related vtes may be diagnosed before recognition of the underlying malignancy.3,22,23 we defined major hemorrhage as an intracranial or gastrointestinal hemorrhage requiring hospital admission . The icd-9-cm codes for intracranial hemorrhage codes 430, 431, or 432were derived from validated work using administrative claims data.24 similarly, icd-9-cm codes for gastrointestinal hemorrhage (456.0x, 530.7, 530.82, 5315, 537.83, 562.0203, 562.1213, 569.3, 569.85, and 578.xx) were also derived from previously published approaches.25 cancer stage was determined using seer american joint committee on cancer (ajcc) stage or historical stage (coded as local, regional, or distant) for cancer types in which ajcc was not available (lymphoma, pancreatic, renal, and prostate cancers). We used median income according to patient zip code of residence as a proxy for socioeconomic status (ses). Dichotomous cancer treatment variables were created using seer data and medicare claims to identify patients who had received cancer - specific surgery, chemotherapy, and radiation therapy.26,27 pertinent comorbidity diagnosis codes during the period before each patient s cancer diagnosis were obtained from the inpatient and outpatient claims to identify conditions that comprise the charlson comorbidity index.28 we only included conditions that appeared on either one inpatient or two outpatient claims.28 to enable us to capture additional conditions that could alter the risk of developing a vte, we also identified patients with a diagnosis code for atrial fibrillation (icd-9 code 427.3) prosthetic heart valve (icd-9 code 35.20, 35.22, 35.24, 35.26, or 3528), obesity (278.x), hip fracture (icd-9 code 352.0, 352.2, 352.4, 352.6, or 352.8), or insertion of a central venous catheter (cpt code 36533) during the 2-year period before their cancer diagnosis . To understand the degree to which the relation between vte and mortality is mediated by cancer - related and other factors, we constructed a series of sequential models for each cancer type . The initial cox proportional hazards model incorporated only age (by 5-year age group because of the nonlinear relation between age and mortality), race, and gender as covariates, with vte as the independent variable and death as the outcome . We then added cancer characteristics and therapy to the model, including stage at diagnosis, histological grade, chemotherapy, radiation, and cancer - specific surgery . Because comorbid conditions could affect the probability of receiving treatment or of developing a vte, comorbidity was then added to the model . While ses would not be expected to alter the physiologic impact of a vte, we considered lower ses as a potential mediator of stage and comorbidity . We calculated the proportion of patients who had a major hemorrhage during the first year after cancer diagnosis for each cancer type according to vte status . The excess hemorrhage risk in vte patients was estimated by deriving the absolute difference in hemorrhage rates between the vte and non - vte groups . Finally, we explored the potential role of hemorrhage in mediating the relationship between vte and mortality by adding hemorrhage as a covariate to the final multivariate model . The median age of patients in the study sample was 75 years (interquartile range 71, 81 years). Overall, about 1% of the patients was diagnosed with a vte concomitantly with their cancer diagnosis (table 2). The cancer types that were most frequently associated with vte were ovarian, kidney, and pancreatic (2.7%, 2.5%, and 2.2%, respectively). Conversely, breast (0.4%) and prostate (0.4%) cancers were rarely associated with concomitant vte . In the bivariate (unadjusted) analysis, concomitant vte was strongly associated with mortality for patients with all types of cancer (table 3). The hazard ratio (hr) for the risk of mortality in patients with a concomitant vte compared with those without a vte ranged from 1.30 (95% confidence interval [ci] 1.181.44) among patients with lung cancer to a high of 3.06 for patients with cancer of the uterus (95% ci 2.324.04). Table 2concomitant venous thromboembolism (vte) according to cancer typecancer typenumber of patientsconcomitant vte (occurring 6 months before to 1 month after cancer diagnosis)n%prostate40,7101520.37breast26,5631020.38bladder11,063790.71uterus5,685711.25lung32,3484491.39colorectal29,1014531.56lymphoma8,0221451.81pancreas6,3931392.17kidney4,1411032.49ovary3,359922.74total167,3851,7851.07table 3concomitant venous thromboembolism (vte) and risk of death according to cancer typecancer typehazard of death associated with vte (vs no vte)model a: unadjustedmodel b: adjusted for age, sex, racemodel c: adjusted for factors in model b + cancer characteristics and treatmentmodel d: adjusted for factors in model c + comorbiditymodel e: adjusted for factors in model d + socioeconomic statushazard ratio95% cihazard ratio95% cihazard ratio95% cihazard ratio95% cihazard ratio95% ciprostate2.241.782.811.811.442.271.441.141.811.200.951.511.210.961.52breast2.301.763.011.951.482.551.110.841.451.010.771.321.010.771.33bladder2.802.173.622.842.203.661.571.212.031.541.192.001.431.131.80uterus3.062.324.043.472.634.592.081.572.751.981.482.641.961.472.62lung1.301.181.441.321.201.451.201.0911.11.151.041.271.161.051.27colorectal1.331.181.491.321.171.481.241.101.391.191.061.331.191.061.34lymphoma1.951.622.341.821.522.191.781.482.141.621.351.961.631.351.97pancreas1.311.101.551.351.141.601.281.081.521.261.061.491.261.061.49kidney1.491.191.881.611.282.021.491.181.881.411.121.781.431.131.80ovary1.271.001.611.160.911.471.351.071.721.321.041.681.321.031.68each row represents a unique model, as patients with each type of cancer were analyzed separately . Hazard ratio represents the hazard of death for patients with a concomitant vte compared to patients with the same cancer type, but without a concomitant vte . Concomitant venous thromboembolism (vte) according to cancer type concomitant venous thromboembolism (vte) and risk of death according to cancer type each row represents a unique model, as patients with each type of cancer were analyzed separately . Hazard ratio represents the hazard of death for patients with a concomitant vte compared to patients with the same cancer type, but without a concomitant vte . Mortality after accounting for demographic factors, cancer characteristics, and comorbidity, the relation between concomitant vte and mortality was attenuated substantially for patients with cancer of the breast or prostate . For example, among patients with breast cancer, the unadjusted hr associated with vte was 2.30 (95% ci 1.763.01). After adjusting for age and gender, the hr decreased to 1.95 (95% ci 1.482.55) (table 3). After accounting for cancer characteristics and anticancer treatment received, the hr decreased to 1.11 and was no longer statistically significant (95% ci 0.841.45). Finally, after accounting for ses and comorbidity, the hr was 1.01 (95% ci 0.771.33). A similar trend was noted among patients with prostate cancer, in that in the unadjusted hr associated with vte was 2.24 (95% ci 1.782.81); the hr decreased sequentially after adjusting for demographics (hr 1.81, 95% ci 1.442.27), cancer characteristics and treatment (hr 1.44, 95% ci 1.141.81), and finally ses and comorbidity (hr 1.21, 95% ci 0.961.52).unlike breast and prostate cancers, concomitant vte was significantly associated with mortality for patients with the other eight cancer types, even after adjusting for patient and cancer factors (table 3). The hrs associated with vte ranged from 1.16 (95% ci 1.051.27) for patients with lung cancer to 1.96 (95% ci 1.472.62) for patients with cancer of the uterus . When we added hemorrhage as a covariate to the analysis, there was little change in the hrs associated with vte for any of the cancer types (data not shown). Hemorrhage approximately 16.8% (95% ci 14.918.8) of patients with a concomitant vte were admitted to the hospital with a major hemorrhage during the year after their cancer diagnosis . In contrast, 7.9% (95% ci 7.78.0) of patients without a vte experienced a hemorrhage - related admission . The excess risk of hemorrhage in vte patients was 8.9% (95% ci 7.0, 10.8; p value for difference <.001). A concomitant vte was associated with a significantly higher hemorrhage risk for patients with 8 of the 10 cancer types studied (table 4). There was substantial variation across cancer types with regard to both baseline hemorrhage risk (i.e., proportion of patients with no vte who had a hemorrhage) and the excess hemorrhage rate associated with vte . Table 4concomitant venous thromboembolism (vte) and major hemorrhage during the first year after cancer diagnosis according to cancer typecancer typeconcomitant vteno concomitant vteexcess hemorrhage rate in vte patients (%) p valuen% hemorrhagen% hemorrhageprostate1298.538,3713.64.90.007breast8010.024,8223.16.90.0035bladder6813.210,3275.37.90.001uterus541.95,2484.42.50.36lung38212.626,8978.14.50.003colorectal38825.825,83617.97.9<0.001lymphoma13122.16,93610.611.5<0.001pancreas11524.34,83616.47.90.020kidney9510.53,6128.42.10.45ovary8214.62,7738.06.60.033total1,52416.8149,6587.98.9<0.001concomitant vte: vte diagnosed between 6 months before and 1 month after cancer diagnosis . Major hemorrhage defined as intracranial or gastrointestinal bleeding requiring hospitalization. p value with two - sided fisher s exact test for difference in hemorrhage rate between patients with vte versus without vte for each cancer type.the excess risk of hemorrhage associated with concomitant vte (and the related anticoagulation therapy) was approximately 68% for most cancer types . Among patients with bladder cancer, for instance, approximately 13.2% of those who had a concomitant vte suffered a major hemorrhage, in comparison to only 5.3% of patients without a concomitant vte, yielding an excess hemorrhage rate of 7.9% . The attributable risk for hemorrhage associated with vte ranged from no significant excess (kidney and uterine cancer) hemorrhage risk to 11.5% (lymphoma). Concomitant venous thromboembolism (vte) and major hemorrhage during the first year after cancer diagnosis according to cancer type concomitant vte: vte diagnosed between 6 months before and 1 month after cancer diagnosis . Major hemorrhage defined as intracranial or gastrointestinal bleeding requiring hospitalization . * p value with two - sided fisher s exact test for difference in hemorrhage rate between patients with vte versus without vte for each cancer type . In a population - based cohort of older patients with cancer, we found concomitant vtes were associated with increased risk of death as well as major hemorrhage for patients with most types of cancer . The 9% absolute increase in hemorrhage rate associated with vte suggests that treatment of vte is associated with substantial risk . The risk of death associated with vte did not change substantively after adding hemorrhage to the multivariate model, suggesting that hemorrhage is not the mechanism through which vte increases the risk of death . A major hemorrhage leading to hospitalization is costly, frightening, and can be associated with substantial morbidity among patients with a relatively short life expectancy . Our analysis builds upon these studies by providing quantitative, population - based estimates of hemorrhage risk in the older cancer population . In one analysis of 181 cancer patients with vte, the 12-month cumulative incidence of major bleeding was 12.4% (95% ci 6.518.2).4 of note, the same analysis also investigated the hemorrhage rate in vte patients without cancer and found that it was only 4.9% (95% ci 2.57.4).4 similarly, a review of more than 2,000 patients with vte, with and without cancer, demonstrated a bleeding - related hospitalization rate of 11.4 to 14.9 per 100 patient - years, and that patients with cancer had a higher hemorrhage risk.15 these hemorrhage rates are substantially higher than the rate (1.1 events/100 person - years) reported in a recent meta - analysis of anticoagulation trials, which included patients with and without cancer, across a spectrum of age groups.16 this discrepancy between trial and community outcomes is likely because of differences in study populations, monitoring of therapy, and the fact that many of the trials have included patients with and without cancer.16,29 a previously validated tool for assessing hemorrhage risk is the outpatient bleeding risk index, which includes risk factors such as age greater than 65 years, prior gastrointestinal hemorrhage, and specific comorbid conditions including atrial fibrillation stroke, anemia, prior myocardial infarction, or renal insufficiency.18,30 in the context of prior work showing that cancer increases the risk of hemorrhage, and our finding that hemorrhage risk can vary substantially across cancer types, future work should seek to identify bleeding prediction tools for validation in cancer patients . Our findings suggest that prior estimates of the relation between vte and mortality may have been inaccurate because of confounding by tumor characteristics.1 in one case control study of cancer patients that accounted for age, gender, and cancer type, patients with a vte had a hr for mortality of 2.20 (95% ci 2.052.45).3 although patients with a vte were significantly more likely to have metastatic disease than patients without a vte, cancer stage was not accounted for in this analysis . In contrast, we found that the impact of vte on mortality was attenuated after accounting for these factors in patients with breast or prostate cancer . This suggests that a substantial portion of the increased mortality risk previously attributed to vte for patients with these cancer types may actually be attributed to underlying tumor characteristics and stage at presentation . Because they are common malignancies and are frequently diagnosed at an early stage, inadequate adjustment for cancer type and stage can lead to biased assessments of vte outcomes . Although other authors have used icd-9 codes to identify patients with diagnosed vte, and some studies have reported that this is a relatively accurate approach, many cases of vte may not be clinically recognized, and some clinically recognized cases may not be appropriately coded.1,23,3133 we focused on vtes that were diagnosed with an associated hospital admission to increase the likelihood that the vtes were acute . However, because it is unclear whether administrative data can reliably capture the clinical diagnosis of vte, future work should use alternate data sources to assess outcomes associated with vte . Similarly, while there has been a trend toward increased treatment of vtes in the outpatient setting, low molecular weight heparin was not approved for outpatient vte treatment until december 31, 1998near the end of our study period.34 our study included only older persons, so it is unclear whether our findings would generalize to a younger cancer patient population . Finally, because pharmacologic and laboratory data were not available, we were unable to identify anticoagulation management patterns for patients with vte . Further work should identify the degree to which the substantial hemorrhage rate noted in our sample is attributable to appropriateness of therapeutic monitoring, or the interaction of anticoagulation risks with specific cancer characteristics or other comorbid illnesses . Finally, we repeated the analysis after excluding patients who were diagnosed in 1999, as low molecular weight heparin was available during this time and outpatient treatment would have affected the analysis . We found no substantive change in the adjusted hazard of death associated with vte after excluding patients diagnosed in 1999 . Our findings shed new light on the scope and impact of vte on older cancer patients . The substantial risk of hospitalization with a major hemorrhage in the first year after diagnosis of a vte emphasizes the importance of identifying opportunities for risk reduction in this population . We also found that although vte is associated with increased risk of death, this risk varied across cancer types and was, in many cases, attenuated by adjusting for underlying tumor characteristics . With the number of newly diagnosed cancer patients and cancer survivors increasing dramatically, it is imperative to further clarify the optimal approach to older cancer patients who are at risk for vte and those who have already experienced a vte
Reproductive health programming for female sex workers (fsw) has traditionally emphasized contraception, including condom use for both pregnancy and hiv prevention, and screening and treatment of sexually transmitted infections [14]. While these are critical components of reproductive health programming, fertility is of great importance in sub - saharan africa [57], and there is growing evidence that fsw, like other women, desire children [810]. Although the need for comprehensive reproductive healthcare for fsw has been raised, to date limited attention has been given to safer pregnancy planning for fsw . There has been increased awareness of the safer conception needs of people living with hiv in general [1215]; however service provision is scarce in sub - saharan africa, and safer conception counseling received by hiv - affected couples has been limited [12, 17, 18]. Safer conception approaches employ risk - reduction strategies for hiv - affected couples trying to conceive, such as biomedical approaches to ensure viral suppression of hiv - infected partner(s) and behavioral strategies to reduce and efficiently time the number of unprotected vaginal sex acts . As fsw are disproportionately affected by hiv, safer conception approaches are particularly relevant for fsw trying to become pregnant, in order to minimize periconception hiv infection and superinfection risks among fsw, to minimize onward transmission risks to partners of fsw living with hiv and to reduce risks to infants . Several barriers may prevent fsw in west and central africa from being reached by safer conception counseling . Hiv prevalence across the region has been estimated at nearly 35% among fsw, a burden far exceeding that of other reproductive - aged women in the region; however safer conception knowledge and services are likely more scarce than those available to women in more generalized epidemics . In addition to poor availability of safer conception services in general, knowledge of hiv status among fsw may be low and engagement in care limited [22, 23]. Furthermore, among fsw engaged in hiv care, women who sell sex may not feel comfortable discussing reproductive desires with healthcare providers out of fear that providers would not support their reproductive choice or provide preconception care based on their occupation or hiv status . Limited evidence is available about fsw trying to conceive, including whether hiv prevention strategies are being adopted to reduce risk of hiv acquisition or transmission to partners and infants in the periconception period . For fsw trying to conceive, engagement in the hiv treatment cascade including hiv testing and awareness of hiv status, linkage to hiv care and/or prevention of mother - to - child transmission (pmtct) services, antiretroviral therapy (art) initiation, and adherence to treatment (viral suppression) is critical for the optimization of health outcomes for fsw, partners, and infants . The objective of this analysis is to assess fertility intentions and hiv among fsw, as well as engagement in the hiv prevention, treatment, and care cascade among fsw trying to conceive in two west african countries studies with key populations (populations most at risk of hiv infection), including fsw and men who have sex with men, were conducted in four sites including kara and lome in togo and ouagadougou and bobo - dioulasso in burkina faso . Methodology has been described in detail elsewhere, but, briefly, rds is chain - referral method used to recruit hard - to - reach populations . A select number of seeds who met study eligibility criteria and were well connected within the fsw community were purposively identified in coordination with local stakeholders from the sex worker community and invited to participate in the study . Seed selection took into account seed characteristics in order to ensure that a range of ages, years engaged in sex work, countries of origin (local versus foreign - born fsw), education, marital status, and languages spoken were represented . Seeds were released gradually over time to ensure steady enrollment . Each consenting seed (n = 20) participated in all study activities and was then given three coupons to invite other eligible fsw . The invitees returned the coupons to the study site and, if eligible, were also enrolled and completed study procedures after which they also became recruiters and were given three coupons to invite other known acquaintances in their network to participate . Participants were reimbursed for average local transport costs to the study site and the equivalent of a small meal, 2,000 cfa in burkina faso (approximately usd 4) and 5,000 cfa (approximately usd 10) in togo . Secondary reimbursements to compensate for time and expenses used recruiting other fsw into the study were also provided for each eligible participant (up to three) that the participant successfully recruited . Secondary reimbursements were 1,500 cfa (approximately usd 3) per eligible recruited fsw in burkina faso and 3,000 cfa (approximately 6 usd) in togo . Reimbursements varied across countries based on differences in average local transport costs to study sites and food costs . Study procedures for all enrolled participants included a cross - sectional sociobehavioral questionnaire and hiv counseling and rapid testing . Questionnaires were administered by experienced quantitative interviewers who had completed training in research ethics and study procedures . Hiv counseling and rapid testing was done in accordance with the national guidelines in each country . Hiv counselors administered the pre- and posttest counseling, while a laboratory technician collected blood samples from the participants and processed the hiv tests ., determine hiv 1/2 ag / ab combo rapid test was used and all positive results were confirmed with hiv bispot immunocomb 11 . The determine hiv 1/2 ag / ab combo rapid test was also used; however sequential testing of positive samples was conducted using first response hiv test 120 cards (pmc medical). All participants received pretest counseling and were counseled of the benefits of knowing one's status however participants could choose whether or not to receive their hiv test results . Referrals to treatment and care were made for anyone receiving a confirmed positive hiv test result not already engaged in either pre - art care or treatment . Fsw were eligible to participate in the study if they were 18 years old and born female, reported that the majority of their income within the past 12 months was obtained from sex work, presented a valid rds coupon, and had not previously participated in the study . Study participation was anonymous, and all study - related procedures were conducted in private rooms at secure study sites . As the results focus on fertility intentions and the need for preconception care among reproductive - aged fsw, women> 49 years of age were excluded from the present analysis . Assessment of fertility intentions, particularly the indicator trying to conceive, refers to fsw who indicated that they are currently trying to become pregnant . For this analysis we considered risk taking behaviors to be behaviors which may increase risk of horizontal or vertical transmission of hiv in the periconception period, such as having untreated sexually transmitted infections (stis), unprotected vaginal or anal sex with a nonpaying partner, and having multiple nonpaying partners . Conversely, risk mitigating behaviors among women trying to conceive were behaviors which may decrease periconception hiv transmission risk, such as engaging in hiv testing, 100% condom use with clients, and talking to nonpaying partners and clients about hiv . This is a secondary data analysis of data collected to estimate hiv prevalence across sites as well as engagement in hiv prevention and treatment activities . Secondary objectives of the study included improved understanding of risks for hiv infection, reproductive health, and human rights violations among fsw . Descriptive statistics for the overall sample of women of reproductive age are provided . Throughout the analyses we do not present rds - adjusted estimates, as rds adjustment is not appropriate when sample populations across study sites are combined . Among women trying to conceive, descriptive statistics of hiv prevention and risk behaviors were compared using chi - squared statistics between women with reactive and nonreactive hiv test results at enrollment . Hiv status and engagement in steps of the treatment cascade were further compared between women who were and who were not trying to conceive using chi - squared statistics . Between january and july 2013, 1,380 fsw were enrolled at study sites in burkina faso and togo . Thirty - one women were excluded from this analysis as they were above reproductive age (> 49 years), leaving the analysis population to 1,349 . Crude hiv prevalence among women enrolled was 19.2% (95% confidence interval (ci) 17.121.3) and ranged from 8.5% (95% ci 5.511.4) in ouagadougou to 31.9% (95% ci 26.936.8) in bobo - dioulasso . The majority of women had never been married (55.7%) and had at least one child (70.4%). The distribution of years engaged in sex work was roughly split between those newer to sex work (02 years of experience), those with 25 years of experience, and fsw engaging in sex work for more than 5 years . Overall, 267 out of the 1,349 fsw (19.8%) reported that they were trying to become pregnant at the time of the study . Long - acting reversible methods (larc) of contraception were used by 24.8% of women, including 24 women (9.0%) currently trying to conceive . Fsw trying to conceive trended to more frequently report unprotected vaginal sex with nonpaying partner(s) in the past month as compared to fsw who also had nonpaying partner(s) but were not trying to conceive (77.9% versus 70.6%, resp . Limited to the subset of fsw trying to conceive, figure 1 further demonstrates the frequency of reported behaviors that elevate or mitigate hiv - related risks in the periconception period . Among fsw trying to conceive, the proportion of women reporting unprotected vaginal sex acts with nonpaying partners was similar between women living with and without hiv (76.6% versus 78.5%, p = 0.92). Around 12% of women reported unprotected anal sex with their nonpaying partners in the past month as well . Overall in the chi - squared analyses assessing periconception behaviors that increase hiv transmission, reporting multiple nonpaying partners was the only risk behavior associated with hiv infection status among women trying to conceive . Fsw living with hiv were less likely to report multiple nonpaying partners as compared to hiv - uninfected fsw . This may suggest that fsw known to be living with hiv reduce the number of their nonpaying partners to minimize onward transmission risk, while hiv - uninfected fsw may take greater personal risks in order to become pregnant . In terms of behaviors known to mitigate risk, just over one - third of women had talked to their sexual partners in the past month about their hiv infection status, and slightly more women had talked to their nonpaying partners about his hiv infection status . History of ever testing for hiv was comparable between hiv - infected (79.4%) and -uninfected (74.4%, p = 0.40) women who were trying to conceive, as was consistent condom use during sex acts with clients (82.5% and 74.5%, resp . Engagement in the hiv treatment cascade among fsw trying to conceive is illustrated in figure 2 . Solid boxes in the cascade diagram represent women retained in care, whereas dotted lines represent individuals who have fallen out of care at various points across the continuum . Among women trying to conceive, of the 56 women never tested for hiv, 19.6% were living with hiv and reportedly unaware of their status . Twenty - seven percent of the 211 women who had previously tested were also living with hiv and reportedly unaware of their status . In total, 25.5% (68 of 267) fsw trying to conceive were living with hiv, and 66.2% (45/68) of hiv - infected women reported no previous hiv diagnosis . The majority (82.6%, n = 19/23) of women aware of their hiv status reported that they had previously had a cd4 count taken, all of whom received the results and 94.7% of whom were eligible for treatment per the national guidelines . All but one fsw trying to conceive who had received cd4 testing and was art - eligible was on treatment; however, art coverage among all hiv - infected fsw trying to conceive was 25.0% (17/68). There were differences in engagement in the care continuum between women in togo and burkina faso . In togo 23.7% of hiv - infected fsw trying to conceive reported awareness of their status, as compared to 51.9% in burkina faso (p = 0.02). Art coverage among hiv - infected women was 13.2% in togo and 44.4% in burkina faso (p <0.01). In terms of comparisons in engagement in hiv care between fsw trying to become pregnant and those not trying to conceive, hiv testing history, reported awareness of hiv infection status, and reported hiv treatment uptake were comparable between the two groups (table 2). Women trying to conceive were more likely to be living with hiv (24.5%) as compared to women not trying to become pregnant (17.7%, p <0.01). The trend of higher fertility intentions among fsw living with hiv versus hiv - uninfected fsw was seen across age strata (1824 years, 2535 years, and 36 years); however this relationship was only statistically significant among women aged 2535 years (results not shown). In terms of other demographics, engagement in art treatment among fsw living with hiv and trying to conceive was similar among married / cohabitating fsw and nonmarried fsw (36.4% versus 24.1%, resp ., p = 0.71), as well as among women who were already mothers versus nonmothers (26.8% versus 25.0%, p = 0.87). A substantial proportion of fsw in the sample were trying to become pregnant and both fsw living with hiv and hiv - uninfected fsw were facing periconception hiv transmission risks . Hiv - uninfected women trying to conceive were engaged in frequent unprotected sex with their nonpaying partners, increasing the potential for exposure to hiv . Furthermore, fertility intentions were higher among women living with hiv as compared to hiv - uninfected fsw . Within the subset of women trying to conceive, one - fourth were living with hiv, but two - thirds of hiv - infected women reported being unaware that they were living with hiv . Among the subset of women aware of their hiv infection status, there was high reported engagement in hiv care, including cd4 staging and art initiation; however large gaps in infection diagnosis resulted in low overall art coverage among hiv - infected fsw trying to conceive . Treatment coverage gaps were evident in both married / cohabitating and nonmarried women, as well as fsw who were already mothers and nonmothers . Risks to fsw and their partners in the periconception period reinforce the importance of earlier hiv infection diagnosis and treatment as prevention for serodiscordant partnerships independent of cd4 count per world health organization guidelines . Low art coverage among hiv - infected fsw trying to conceive may result in high risks for hiv transmission to hiv - uninfected male partners and to infants . This may be particularly true among the fsw sampled, as the majority of fsw trying to conceive appeared to be unaware of their status and thus less likely to utilize other hiv prevention measures while trying to conceive, such as unprotected sex limited to the periovulatory period, self - insemination, or preexposure prophylaxis by the male partner to reduce transmission risks . Equally, fsw living with hiv but unaware of their status may be less motivated to attend antenatal care and engage in pmtct programs during early pregnancy . Furthermore, frequent occupational exposure through sex work to hiv and the possibility of hiv superinfection during the periconception period may result in higher viral load among fsw and greater transmission risks to hiv - uninfected partners compared to other serodiscordant relationships . Women trying to conceive in our analysis had comparable art coverage to women who were not trying to become pregnant . However, women trying to conceive were more likely to be living with hiv than women who were not trying . As this study was cross - sectional, causality cannot be determined; however there was no evidence in our study that living with hiv caused women to be more likely to want to conceive . Many fsw living with hiv in our study did not report a previous diagnosis, and prior hiv diagnosis (unlike actual hiv - infection status) was not associated with fertility intentions . Other studies with hiv - infected african women of reproductive age have reported that women known to be living with hiv have lower fertility intentions than hiv - uninfected women [31, 32]. Alternatively, our findings may indicate that attempted conception may be increasing risk for hiv infection . Outside of self - insemination and more advanced reproductive technologies, unprotected sex is required to conceive; thus hiv transmission risks may be higher among women trying to conceive further reinforcing the importance of treatment in serodiscordant partnerships and low - cost strategies to conceive while minimizing potential exposure to hiv . Indeed, among both hiv - infected and hiv - uninfected fsw trying to conceive, unprotected sex with nonpaying partners was common, and communication with partners about hiv was low . Other studies have also documented frequent sex without condoms among women trying to become pregnant . We are not aware of any published hiv prevention studies which have provided fsw with safer conception services to minimize risk for hiv acquisition and transmission . While there is increasing awareness that fsw may have fertility desires [8, 10], this appears not to have translated into safer conception service provision . Within the sub - saharan african context more generally, safer conception guidelines have been published, but services for couples affected by hiv remain largely unavailable outside of individual providers who may offer counseling . Even in the cases in which services have been offered, generating demand takes time, as many individuals may want to become pregnant but may not recognize the related need for engagement in hiv care, including safer conception services . Fsw living with hiv are harder to reach with preconception care than women who do not sell sex, due to low existing engagement in hiv care among fsw [22, 23], and perceptions or experiences of intersecting stigma related to their reproductive goals as sex workers and women living with hiv [3538]. Given that many fsw are trying to conceive, safer conception counseling services are required, alongside efforts to reduce stigma and other barriers to engaging in hiv testing and care programs . In mixed hiv epidemics like burkina faso or togo, health care providers may be less aware of safer conception methods and may be less equipped to counsel fsw affected by hiv about how to conceive safely . Development of improved clinical and cultural competencies to counsel fsw and their partners about fertility and low - cost methods for safer conception will be required to ensure that messaging is clear and appropriate . These results suggest the need for this training to be integrated into both clinical hiv and family planning curricula for health care providers treating fsw . These results represent a sample of women in two west african countries recruited using nonrandom sampling methods and thus may not be generalizable to other fsw within the study countries or elsewhere . Our sample includes 1,349 fsw in burkina faso and togo who may be very different from other fsw not reached through the rds sampling methods . Despite this limitation, truly random samples with fsw are difficult to achieve given the fact that there is no sampling frame for fsw and venue - based methods for recruitment are also limited by the study team's ability to enumerate and engage with all sex work venues . Peer recruitment methods represent a way of reaching women that may otherwise not be found as well as fsw not already engaged in existing fsw programs or services . Thus, despite the limitations of a nonrandom sample, our study sample demonstrates the potential magnitude of safer conception needs . Furthermore, although both fertility intentions and engagement in the hiv treatment cascade may differ among fsw in other sub - saharan african countries with more generalized epidemics, hiv prevalence among fsw is also higher in those settings and thus there is likely a need for safer conception services in other settings . Causal attribution of hiv transmission risks related to attempted conception cannot be made from these cross - sectional data . Information about disclosure to partners and the hiv status of fsw's partners with whom they were trying to conceive was not collected and would strengthen our understanding of hiv transmission risks in this context . Similarly, we did not collect information on safer conception counseling received or strategies employed by fsw, nor do we have objective confirmation of adherence to art and achievement of viral suppression among women trying to conceive who are living with hiv . However based on self - reported treatment data, the best case scenario of viral suppression among fsw living with hiv and trying to conceive in this sample is approximately 25% (17/68) if all hiv - infected fsw on treatment were virally suppressed . The hiv transmission risks among individuals and couples trying to conceive in the context of hiv are likely not evenly distributed in the population . High fertility intentions and unprotected vaginal sex in order to achieve conception increase the hiv acquisition risks experienced by fsw . Furthermore, low engagement in repeat hiv testing and care among fsw trying to conceive increases the hiv transmission risks to their partners and children . Improving access to and uptake of hiv prevention and treatment services for fsw, including safer conception counseling, could help to optimize hiv prevention goals and support the reproductive rights of fsw.
It refers to upper abdominal pain or discomfort, which is taught to arise from the upper gastrointestinal tract (1). Dyspepsia is a global concern, although most of the published data have arisen from western countries . It is assumed that dyspepsia in populations from developing countries is mostly organic in nature, whilst functional dyspepsia is more prevalent in western nations (2). The prevalence of this disorder varies between 3% and 40% in different studies (3, 4). This variation in the prevalence rates may be related to differences in the definition of dyspepsia in those studies . Although not life - threatening, the symptoms are long - lasting (5). Various risk factors have been found to have associated with these disorders such as helicobacter pylori infection (6, 7), psychiatric disorders (6, 8, 9) and behavioral characteristics (10). In addition, geographical distribution of functional dyspepsia is different in the world (11 - 14). Considering the negative effect of this disorder on patients' quality of life (15 - 17); understanding its prevalence in the general population in different regions of iran and other populations as well as the implementation of suitable interventions can lead to health promotion in the community . It also helps better understanding about the problem and remains a good resource for clinical researches (18 - 20). Many studies in this issue have emphasized mainly on dyspepsia and the functional type of this disorder often has been ignored . Comparing the condition of functional dyspepsia from different views such as the prevalence and risk factors among various populations is a main defect in research in the field of gastroenterology, particularly in iran . Considering the high prevalence of the dyspepsia symptoms in iran through daily clinical experiences and very low information about functional dyspepsia particularly in southeast of iran, it seems that an understanding of the prevalence and potentially relevant risk factors can help the policy makers to establish a program for training and early diagnosis of the disease in the future . The aim of this study was to estimate the prevalence of functional dyspepsia and its associated factors in the adult population in kerman (southeast of iran). In this cross - sectional study, a study population was selected using a cluster random sampling method based on the postal code divisions of kerman, a city in southeast of iran . This study was nested in a comprehensive study (kercadr: the study of coronary artery disease risk factors in kerman, 2008). Full details of sampling and methodology of kercadr study were presented elsewhere (14). Assuming the prevalence of 25% for functional dyspepsia in the previous studies and a design effect equal to 1.5 and also considering 20% drop out, we calculated a sample size of 2200 to estimate the prevalence of functional dyspepsia by the accuracy rate of 0.025 in the significance level of 0.05 . Demographic, anthropometric, psychosocial, nutritional and behavioral information had been collected by the original questionnaire in kercadr study with reliability and validity of 87% and 85%, respectively . Specific questions about the main objectives of the current study asked by a standardized questionnaire based on rome111 criteria (1). This questionnaire contains more than 20 questions about the dyspepsia symptoms and evidences of past medical history of recruits . An expert practitioner collected the data by direct face to face interview, asking specific questions and investigating relevant medical documents . The patient's privacy was respected in this study . Ethical approval, with a code of k/89/161, patients who had signs and symptoms of weight loss, anemia, lesions and wounds in previous endoscopy and also those who had used drugs for the peptic ulcer were excluded from the study . Statistical analyses were undertaken by spss version 16 (spss inc, chicago, il, usa). Chi - square and fisher's exact tests were used to compare the categorical variables, and the logistic regression model was used to calculate the association between dyspepsia and risk factors . From a total of 2320 individuals enrolled in the study, 110 cases were excluded and 2210 cases (1049 males and 1161 females) with the mean age of 43.4 16.25 years were studied . No statistically significant difference was observed between males and females regarding age (p = 0.7). Among the participants, the standardized prevalence of functional dyspepsia in kerman was 16.1% (95% confidence interval: 14.3 - 18.1). Table 1 shows the prevalence of functional dyspepsia in relation to demographic, anthropometric, behavioral and psychosocial data . Results showed that although the dyspepsia was more common in females (17.1%; 95% ci: 15.8 - 18.5), young adults (18.9%; 95% ci: 18 - 19.8), subjects with academic education (16.8; 95% ci: 12.9 - 21.6), anxiety (16.9; 95% ci: 14.8 - 19.3) and depression (18.1%; 95% ci: 14.7 - 22), none of these associations was statistically significant . Results also revealed that the prevalence of this disorder was lower among high fruit and vegetable consumers [(15.8%; 95% ci:13.6 - 18.3) and (16.1%; 95% ci: 10.3 - 24.3), respectively], the tea - coffee and fast - food consumers [(16.1%; 95% ci: 14.2 - 18.2) and (8.8%; 95% ci: 5.2 - 14.6), respectively] and patients with low physical activity (13.8%; 95% ci: 11.3 - 16.6) and abdominal obesity (7.3%; 95% ci: 3.6 - 14.5), while only the association between functional dyspepsia and abdominal obesity was statistically significant (p = 0.0002) 58.3% (95% ci: 51.8 - 64.3) had epigastric pain or burning, 37.3% (95% ci: 31.5 - 43.6) had postprandial fullness and 18.6% (95% ci: 13.9 - 24.3) complained of early satiety . Only about 3 percent (95% ci: 1.4 - 6.4) of the patients had all of the three above - mentioned symptoms . There was no statistically significant difference between males and females in the relevant symptoms (table 2). Table 3 showed that anxiety after controlling for other variables increased the risk of functional dyspepsia more than 65 percent (p = 0.004) and also depression increased that risk about 2.13 percent (p <0.0001). Although high consumption of fruit decreased the risk of functional dyspepsia (crude or = 0.66; p = 0.04), controlling other variables demonstrated no statistically significant protective effect (adjusted or = 0.92; p = 0.7). Moreover, current smoking and drinking teacoffee and alcohol increased the risk of functional dyspepsia about 5%,, 30% and 35%, respectively; however, their effects were not statistically significant (p = 0.4, 0.4 and 0.8, respectively) (table 3). Our study showed that the prevalence of functional dyspepsia was about 16% and it was significantly associated with anxiety and depressive disorder; although it was slightly greater among females, and peaked in the age group 25- 34 years old . Moreover, the prevalence of functional dyspepsia was greater to some extent in cigarette smokers, those who drank alcohol and those who consumed tea and coffee regularly compared with other subjects . The prevalence of functional dyspepsia in our study was less than other studies such as, uk (21%) (15), us (26%) (13) and jordan (60%) (16). The rates in our study were very close to the rates of the studies conducted in western iran (18%) (17), china (18.4%) (18) and another study (15.7%) (11). These discrepancies could be due to various definitions in different studies (19) and also the exclusion criteria applied especially in the current research . Although functional dyspepsia was more common in females, that association was not statistically significant . This was similar to the findings of another study in which the prevalence of dyspepsia was equally distributed between the genders (20); however, was in contrast to the most previous studies, which showed that dyspepsia was more prevalent among females (8, 18, 21). Although our study showed no significant association between age and functional dyspepsia, the prevalence of the disease was more common in 25 - 34-year age group . Similarly, in studies conducted in shiraz (6) and us (22), there was no relationship between age and dyspepsia . On the other hand, one of the studies indicated that the prevalence of dyspepsia was higher in 35 - 50-year age group (20), while other studies found the positive and reverse associations between age and functional dyspepsia, respectively (21, 23). Psychological disorders, particularly depression and anxiety have been shown to be the major risk factors for functional dyspepsia in one study (8), as indicated in the current study . In addition, another study found that anxiety seemed to be related to abnormal antral retention of food in functional dyspeptic patients . Since psychological stresses have been shown to affect gastrointestinal motility, it seems that the emotional factors, such as depression and anxiety, have also a negative effect on gastric motility . However, there is some evidence which suggests that there is an association between psychological abnormalities and impaired gastric motor function in patients with functional dyspepsia (24). Moreover, in the study (11) anxiety but not depression found to be a significant risk factor for the functional dyspepsia . Some studies have shown that anxiety was negatively associated with pain threshold and discomfort, gastric rendition, and abdominal ardor and discomfort . Moreover, the fluctuating cortisol levels, which is one of the most studied physiological responses to acute stress, seems to be related to different symptoms of functional dyspepsia (24, 25). Although abdominal obesity in dyspeptic patients was more than that of other participants in our study, applying exclusion criteria for subjects showed no association between obesity and functional dyspepsia similar to the findings of the study conducted in shiraz (6). There are different results in the previous studies which show both negative (12) and positive associations (11) between obesity and functional dyspepsia . In addition to our findings, the behavioral risk factors such as smoking and the alcohol and coffee consumption had no relationship with functional dyspepsia (6, 7). That was in contrast to the finding which indicated a harmful effect of smoking (12). Adjusting for potential confounders such as demographic and behavioral risk factors, fruits and vegetables seemed to have no association with functional dyspepsia (although the crude association was found), which was in parallel to the results of a study in southern iran (6). Abdominal pain or burning was the most common symptoms in the present study, which was similar to the results of another study conducted in iran (5). We considered a sufficient sample size and good participation rate in this study; however, the participants with evidences of other diseases were excluded from the study and thus the association among dyspepsia and risk factors was estimated based on half of our sample size . This study conducted only on urban population and also information about food consumption was based on only frequency of usage without detection of units and calories of dietary regimens . In conclusion, we estimated a prevalence of 16.1% of functional dyspepsia in kerman, southeast of iran, which was lower than that in most other studies and associated only with psychiatric disorders . More precise studies collecting detailed information about food consumption and area of residence could lead to better estimation of the effect of such risk factors.
Inflammatory bowel disease (ibd) comprises a myriad of different presentations, levels of severity and responses to treatment . It is especially known that pediatric ibd represents more extensive inflammation with a predilection for colitis . It is an immune - mediated inflammation of the gastrointestinal tract arising out of a host immune dysregulation with genetic and environmental influences . The immune pathophysiology often results in extraintestinal manifestations in ibd that involves multiple organs including the bones and joints, skin, liver and pancreas . The incidence of extraintestinal manifestations has been reported to range from 18 to 47% of pediatric and adult patients with ibd . This includes both acute and chronic pancreatitis as well as pancreatic insufficiency, the latter seen especially in adult ibd . There have been very limited and no recent published data on the incidence of elevated lipase in pediatric ibd [1, 2]. Stawarski and iwaczak conducted a 4-year study in over 100 children with ulcerative colitis or crohn's disease (cd) and found the incidence of acute pancreatitis to be 4.5% in children with cd and 5.1% in children with ulcerative colitis . Acute pancreatitis in children has been increasing in incidence over the past couple of decades and some authors have attributed this to a rise in systemic illness in children . The association of pancreatitis with ibd may be due to the systemic inflammation but may also arise from other causes such as a direct autoimmune hit to the pancreas, the effect of macromolecules of lipase and most importantly, the effect of medications . The most common agents used in the treatment of ibd are associated with drug - induced pancreatitis . This effect of drug - induced pancreatitis makes it challenging to manage pancreatitis in children with ibd, since removal of the offending drug will interfere with maintenance of remission and control of the underlying inflammation in ibd . A 13-year - old hispanic female presented to the pediatric gastroenterology clinic with complaints of hematochezia described as soft to loose stools with a passage of blood clots for the preceding 46 months . She reported occasional mild abdominal pain, but denied fever, nausea, vomiting, loss of appetite, weight loss or nocturnal symptoms and had minimal urgency and tenesmus . Her laboratory workup was significant for elevated fecal calprotectin of 225 g / g (reference 162.9 g / g) and an elevation in pancreatic enzymes with amylase of 102 u / l (reference 25115 u / l) and lipase of 1,053 u / l (reference 73393 u / l). Liver enzymes, esr, and crp were obtained to look for evidence of systemic inflammation but were normal . Following negative stool studies for infection, due to persistent hematochezia, she underwent endoscopy that revealed left - sided colitis with erythematous friable mucosa (fig 1). Pathology reported chronic active colitis with cryptitis and crypt abscesses, with most prominent changes in the left colon, paneth cell metaplasia and significant chronic inflammation . Serologic testing with an ibd-7 panel that differentiates cd from ulcerative colitis based on antibodies was consistent with ulcerative colitis . She was started on balsalazide (750 mg by mouth 3 times daily), with some improvement, but ultimately required steroid suppositories (rectacort 2 times a day for 2 weeks) for complete resolution of her rectal bleeding . U / l, which was> 3 times the upper limit of normal (lab range 73393 u / l). U / l) that showed persistent elevation to> 3 times the upper limit of normal, at 305374 u / l . After 3 months of treatment of her ulcerative colitis, her lipase began to show a downward trend approaching normal values at 73 u / l (fig 2). This correlated with clinical improvement of her ulcerative colitis, as she had normal regular bowel movements with no further bleeding or abdominal pain . Her fecal calprotectin also correlated with clinical remission and normalized to 23.9 g / g . Follow - up calprotectin increased to 474 g / g . Her flare was found to be a result of noncompliance with balsalazide and was quickly suppressed with another course of steroid suppositories that successfully resolved her hematochezia within a week . We switched her maintenance medication to lialda for ease of administration (2.4 g by mouth once daily) and counseled her on the need for compliance with medications . Acute and chronic pancreatitis as well as pancreatic insufficiency have been reported in association with ibd, although it is often difficult to diagnose pancreatitis as it may remain silent and masked by the symptoms of systemic inflammation in ibd . Most of the prior reports of pancreatitis have been in association with cd but there are also a few reports of pancreatitis with ulcerative colitis . Several potential pathophysiological processes have been suggested for hyperlipasemia with or without pancreatitis in ibd [33, 4, 5]. There might be an abnormal passage of pancreatic enzymes from the gut lumen into the blood due to increased permeability of the inflamed mucosa in ibd . More recent reports suggest that pancreatitis and the level of pancreatic enzymes may reflect the clinical course and extent of disease in ibd [2, 6, 7]. The effect of inflammatory mediators and cytokines released from the inflamed gut on the pancreas may also contribute to pancreatic damage and leak of pancreatic enzymes into the blood [6, 7, 8]. Some experts suggest that direct involvement of the pancreas as part of the spectrum of inflammation in ibd might explain a recent report of a pancreatic granuloma noted in cd . Another mechanism may be inflammatory damage to the pancreatic ducts resulting in enzyme accumulation in the abnormal ductal system leading to further pancreatic inflammation . . Found that at least 8% of ibd patients have pancreatic duct abnormalities, and that 50% of ibd patients with primary sclerosing cholangitis have pancreatographic changes . Pancreatic involvement in ibd may also be a part of a common immune disorder whereby the target cells of gut epithelium and pancreas share a similar molecular structure lending themselves to antigenic mimicry [1, 8]. There have been reports of the rarer variant, autoimmune pancreatitis, occurring more often in ibd patients . Martinelli et al . Reported correlation between pancreatic inflammation and ibd disease activity, with 91% of their patients eliciting acute pancreatitis during active disease . They also reported that patients with recurrent pancreatitis had higher disease activity indices (pcdai and pucai) for pediatric cd and ulcerative colitis, respectively . Park et al . Showed an association between decreasing amylase and lipase levels and improving pcdai and reported normalization of pancreatic enzymes 3 months after the start of treatment for cd . Another remote possibility is cryptogenic hyperlipasemia without pancreatitis where macrolipase or immunoglobulin - linked enzymes are suggested to be induced by autoimmune mechanisms resulting in reduced glomerular excretion of the large macrolipase molecules thereby increasing serum half - life of lipase [4, 9]. This may not be associated with pancreatic inflammation with no subsequent sequelae of pancreatic insufficiency . Clinical symptoms of ibd - associated pancreatitis are found in approximately 2% of patients while exocrine pancreatic insufficiency has been reported in 2180% patients and histological changes observed in 3853% of postmortem pathological exams . Subclinical or silent pancreatitis may be masked by symptoms attributed to ibd, hence underreported [1, 6]. If undetected, it may lead to subsequent chronic pancreatitis or exocrine pancreatic insufficiency, which has been reported in 50% of ibd patients with a history of acute pancreatitis in adult studies [6, 8]. This case shows that checking pancreatic enzymes during initial presentation may be important to determine if pancreatic involvement has resulted from the inflammation in ibd or as an adverse effect of therapy . One should use caution in considering change or cessation of a particular treatment regimen in ibd with pancreatic involvement, as treatment of the underlying inflammation in ibd may be the most important management for resolution of pancreatitis [3, 8]. Longitudinal observation in our patient and follow - up pediatric studies are required to see if subclinical pancreatitis or asymptomatic elevation in pancreatic enzymes results in clinically significant pancreatic disease later in life.
Robotic dispensing machines have been available for more than 10 years as an alternative storage system to conventional pull - out drawers in community pharmacies . This system is an electronically controlled automated storage system that offers the capacity of a distinctly larger, conventional storage, while taking up only a minimum of space . The robotic dispensing machines are bound for community pharmacies with a wide range of goods . The warehouse in german community pharmacies contains 8,000 to 10,000 different products . In advertising their machines, manufacturers emphasise space saving, reduced personnel costs as a result of saved time, and increased sales due to improved counselling [1, 35]. In times of falling margins in german pharmacies, such advertising claims to cost savings are tempting for pharmacists . Although acquisition costs are high, mostly in excess of 100,000 euros, they are expected to be rapidly amortised . Already, 7% of the community pharmacies in germany operate with a robotic dispensing machine . This paper aims to analyse the impact of robotic dispensing machines from a business perspective in order to identify whether the manufacturers promises can be achieved . More specifically, our objective was to explore the effects of a robotic dispensing machine on the following:- sales volume;- acquisition costs;- costs situation;- stock value;- over - the - counter (otc) sales volume;- space;- inventory . - over - the - counter (otc) sales volume; no studies have been published in the german - speaking region on the efficiency of robotic dispensing machines in community pharmacies, but such studies have been performed in the usa and great britain and also on how robotic dispensing machines are used for preparing unit dose packs . The studies in the united states evaluated the impact of robots on the machine packaging of drugs in unit doses . Unit dose means the delivery of a single, packaged, clearly identifiable drug to a specific patient . This form of drug dispensing is receiving more attention from both the qualitative and the safety perspective . An analysis was performed during 20012002 in michigan, usa . In a pharmacy belonging to the pharmacy chain evaluated the impact of a robotic dispensing machine on the preparation of unit dose packs . A retrospective study was performed on the process of preparing unit dose packs before and after the installation of the robotic dispensing machine . Overall, a significant saving in time was registered for the preparation of unit dose packs . However, the saving in time occurred only if the staffing level was adapted to the changed requirements . Franklin et al . Studied the effect of introducing unit dose robotic dispensers from two different manufacturers into british hospitals from the aspects of drug safety, efficiency and staff satisfaction . After introduction of the dose robotic dispensers, the number of dispensing errors decreased significantly for both brands, and labelling errors were also reduced, though to a lesser extent . Pedersen et al . Examined 1173 hospital pharmacies in the usa in to assess the role of the pharmacist in the respective drug distribution system, and concentrated on the respective administrative effort associated with distribution of the drugs . A differentiation was made between the conventional manual unit dose and stationary fully automatic robot technology . Evaluation by a questionnaire led to the conclusion that the use of robot technology increases drug safety, not only in filling the individual patient's drugs, but also in administering the drugs to the patient . Mobach investigated in a dutch pharmacy the extent to which time saved from logistic work is used for giving more pharmaceutical advice . During 2004 and 2005 he demonstrated a displacement of logistic work in favour of pharmaceutical duties, resulting overall in an increase in drug safety . We surveyed the economic impact of bringing a robotic dispensing machine into service in community pharmacies using a structured questionnaire . The target population consisted of independent pharmacists who have replaced their conventional drug storage system with a robotic dispensing machine during the last few years . For reasons of practicality and feasibility it was decided not to approach all 21,551 community pharmacies because only about 1600 of them (7%) work with a robotic dispensing machine . So we asked rowa to provide a list of 253 reference customers who were using a rowa robotic dispensing machine of type rowa select in 2007 . Reference customers are defined as those who agreed to have their addresses passed on to others . The 20 items on the questionnaire are intended to reveal to what extent the installation of a robotic dispensing machine changed the economic situation in the pharmacy during the first year . The construction of the questionnaire took into account that revealing business figures would be highly sensitive, especially in view of a possible liberalisation of german pharmacy law with permission to form pharmacy chains . The level of otc sales in the pharmacy depends on numerous factors price policy, amount of advertising on radio and television and in the printed media, and, last but not least, the selling skills of the pharmacy staff . For that reason, the changes in otc sales cannot simply be attributed to the impact of robotic dispensing machines . The pharmacists surveyed were therefore asked to estimate as accurately as possible the changes in sales of otc drugs that derive from the impact of robotic dispensing machines . The answer formats are multiple choice answers (closed) and open ended questions to which the respondents must phrase their own answers (open). Among others, the comprehensibility of the items on the questionnaire was tested and adapted in a pre - test on 12 pharmacists . If this was not possible, the member of staff was asked to inform the pharmacy manager about the intended research and to tell them that the questionnaire would arrive the following day . We surveyed the economic impact of bringing a robotic dispensing machine into service in community pharmacies using a structured questionnaire . The target population consisted of independent pharmacists who have replaced their conventional drug storage system with a robotic dispensing machine during the last few years . For reasons of practicality and feasibility it was decided not to approach all 21,551 community pharmacies because only about 1600 of them (7%) work with a robotic dispensing machine . So we asked rowa to provide a list of 253 reference customers who were using a rowa robotic dispensing machine of type rowa select in 2007 . Reference customers are defined as those who agreed to have their addresses passed on to others . The 20 items on the questionnaire are intended to reveal to what extent the installation of a robotic dispensing machine changed the economic situation in the pharmacy during the first year . The construction of the questionnaire took into account that revealing business figures would be highly sensitive, especially in view of a possible liberalisation of german pharmacy law with permission to form pharmacy chains . The level of otc sales in the pharmacy depends on numerous factors price policy, amount of advertising on radio and television and in the printed media, and, last but not least, the selling skills of the pharmacy staff . For that reason, the changes in otc sales cannot simply be attributed to the impact of robotic dispensing machines . The pharmacists surveyed were therefore asked to estimate as accurately as possible the changes in sales of otc drugs that derive from the impact of robotic dispensing machines . The answer formats are multiple choice answers (closed) and open ended questions to which the respondents must phrase their own answers (open). Among others, the comprehensibility of the items on the questionnaire was tested and adapted in a pre - test on 12 pharmacists . If this was not possible, the member of staff was asked to inform the pharmacy manager about the intended research and to tell them that the questionnaire would arrive the following day . The significance tests were based on a 95% confidence interval . The chi - square test was also used . Of the pharmacies surveyed, 84% (n=74) stated that they achieved an annual sales volume of more than 1.5 million euros (table 1). Robotic dispensing machine pharmacies . In most of the pharmacies surveyed (79.2%, valid n=72), the robotic dispensing machines were installed at the time the pharmacy was established . Subsequent installation incurs more costs as a result of rebuilding measures and the need to remove existing drawers . Thus the cost of installation is about 12,000 euros if the dispensing machine is installed at the time the pharmacy is first opened, compared with more than double that for subsequent installation . Of the pharmacies with an annual sales volume of more than 2 million euros, 91% installed the robotic dispensing machine in the beginning; this is the case for only 60% of pharmacies with an annual sales volume of less than 2 million euros . Of the pharmacies surveyed, 68% were located in towns with a population of more than 20,000, predominantly in the town centre (38%) or suburbs (31%). Pursuant to the shop hours act, pharmacies in germany can open for up to 84 hours per week . On average, the pharmacies surveyed are open on average for 57.5 hours per week (n=74, =7.42). This correlates with the sales volume breakdown for all the pharmacies (higher sales volumes than the typical pharmacy). The average acquisition costs are 118,400 euros (n=72, =27.01) and average installation costs are 23,000 euros . The service life for tax purposes is 15 years . Assuming a linear depreciation, the total costs of the robotic dispensing machine per accounting period can be calculated in consideration of the capital costs as follows (table 2). Costs situation figure 1 and table 3 show that 12 months after installation of the robotic dispensing machine, the costs situation in the pharmacies surveyed deteriorated in only 6% of cases . In 50% of the pharmacies thus, in 94% of the pharmacies surveyed the costs situation either remained the same or improved . Pharmacies in a sales category of greater than 2 million euros annual net sales appear to benefit more from costs savings than pharmacies with a lower annual sales volume . Costs situation broken down by sales volume category . Of the pharmacies surveyed, 64% (n=70) stated that their personnel costs had not changed during the 12-month period after installation of the robotic dispensing machine . For 36% of the respondents the personnel costs were reduced by an average of 12.27% during this period as a result of the robotic dispensing machine . In a pharmacy with a net annual sales volume of 2 million euros and assuming 11% personnel costs (eur 220,000 p.a .) With respect to a reduction in tied capital, 58.2% of the pharmacies surveyed were able to reduce their stock value by an average of 17.4% . This could be attributed to a different stocking strategy for lowering the stock level because, in contrast to manual dispensing in a conventional store, there is no advantage to be gained from large orders when the goods are stored in a robotic dispensing machine . Nonetheless, 18% of the pharmacies surveyed reported higher stock values, which are partly the result of structural changes . The stock turnover rate, which rises when the stock value is reduced at the same level of stock outgoings, increased by 6.4% on average among the pharmacies surveyed . This correlates with the change in the stock values 12 months after installation of the robotic dispensing machine pharmacies that focus on saving personnel costs when using a robotic dispensing machine also appear to reduce the stock value (table 4). Surveyed, 52% attributed increased sales in the otc sector to the impact of the robotic dispensing machine . It is possible that these increases resulted from time saved as a result of robotic dispensing being used for additional sales . The presence of the pharmacy staff at the counter during the entire sale appears to benefit the sales conversation with the customer . There are no significant differences between sales volume in the categories of <2 million euros and> 2 million euros . Pharmacies that aim to achieve substantial increases in otc sales by using a robotic dispensing machine must accept the fact that this is possible only if the staffing level remains unchanged . On the other hand, pharmacies that save on personnel costs by using a robotic dispensing machine cannot expect increases in otc sales ., rowa robotic dispensing machines were all individually dimensioned and the floor space they required varied accordingly . When installed from the beginning, the robotic dispensing machine replaces the conventional storage system mostly pull - out drawers . Before the robotic dispensing was installed the average number of columns of drawers was 20.8 . Assuming that the columns of drawers are of standard size (130 cm 40 cm= 0.52 m), this corresponds to an area of 10.8 m. if the floor area for accessing and pulling out the drawers is included, this amounts to a total floor space of 21.16 m for the drawer system . 12.1=9.06 m, i.e. 43% of the original a saving of over 40% (p=0.26). In 59% of the pharmacies the space saved is used as additional behind - the - counter (no customer access) and self service display area . The average gain in area amounts to 57.6% . The inventory in the pharmacy is done either on a specific day or continually . In the conventional store after installation of the robotic dispensing machine, the time required on average was only 15.3 hours . The average acquisition costs are 118,400 euros (n=72, =27.01) and average installation costs are 23,000 euros . The service life for tax purposes is 15 years . Assuming a linear depreciation, the total costs of the robotic dispensing machine per accounting period can be calculated in consideration of the capital costs as follows (table 2). Costs situation figure 1 and table 3 show that 12 months after installation of the robotic dispensing machine, the costs situation in the pharmacies surveyed deteriorated in only 6% of cases . In 50% of the pharmacies thus, in 94% of the pharmacies surveyed the costs situation either remained the same or improved . Pharmacies in a sales category of greater than 2 million euros annual net sales appear to benefit more from costs savings than pharmacies with a lower annual sales volume . Costs situation broken down by sales volume category . Of the pharmacies surveyed, 64% (n=70) stated that their personnel costs had not changed during the 12-month period after installation of the robotic dispensing machine . For 36% of the respondents the personnel costs were reduced by an average of 12.27% during this period as a result of the robotic dispensing machine . In a pharmacy with a net annual sales volume of 2 million euros and assuming 11% personnel costs (eur 220,000 p.a . ), this corresponds to an annual reduction of about 27,000 euros . The stock value remained unchanged for 23.9% (n=67%) of the pharmacies surveyed . With respect to a reduction in tied capital, 58.2% of the pharmacies surveyed were able to reduce their stock value by an average of 17.4% . This could be attributed to a different stocking strategy for lowering the stock level because, in contrast to manual dispensing in a conventional store, there is no advantage to be gained from large orders when the goods are stored in a robotic dispensing machine . Nonetheless, 18% of the pharmacies surveyed reported higher stock values, which are partly the result of structural changes . The stock turnover rate, which rises when the stock value is reduced at the same level of stock outgoings, increased by 6.4% on average among the pharmacies surveyed . This correlates with the change in the stock values 12 months after installation of the robotic dispensing machine . Pharmacies that focus on saving personnel costs when using a robotic dispensing machine also appear to reduce the stock value (table 4). Of the pharmacies surveyed, 52% attributed increased sales in the otc sector to the impact of the robotic dispensing machine . It is possible that these increases resulted from time saved as a result of robotic dispensing being used for additional sales . The presence of the pharmacy staff at the counter during the entire sale appears to benefit the sales conversation with the customer . There are no significant differences between sales volume in the categories of <2 million euros and> 2 million euros . Pharmacies that aim to achieve substantial increases in otc sales by using a robotic dispensing machine must accept the fact that this is possible only if the staffing level remains unchanged . On the other hand, pharmacies that save on personnel costs by using a robotic dispensing machine cannot expect increases in otc sales . This trade - off until 2007, rowa robotic dispensing machines were all individually dimensioned and the floor space they required varied accordingly . On average when installed from the beginning, the robotic dispensing machine replaces the conventional storage system mostly pull - out drawers . Before the robotic dispensing was installed the average number of columns of drawers was 20.8 . Assuming that the columns of drawers are of standard size (130 cm 40 cm= 0.52 m), this corresponds to an area of 10.8 m. if the floor area for accessing and pulling out the drawers is included, this amounts to a total floor space of 21.16 m for the drawer system . 12.1=9.06 m, i.e. 43% of the original a saving of over 40% (p=0.26). In 59% of the pharmacies the space saved is used as additional behind - the - counter (no customer access) and self service display area . The average gain in area amounts to 57.6% . The inventory in the pharmacy is done either on a specific day or continually . In the conventional store after installation of the robotic dispensing machine, the time required on average was only 15.3 hours . Comparison of the distribution of sales volumes among all german community pharmacies with the sales volume categories of the pharmacies investigated reveals that the pharmacies with a robotic dispensing machine in this study generally lie above the sales volume category of a typical pharmacy . Typical pharmacy means a pharmacy in the highest populated sales volume category among the approximately 21,500 community pharmacies in the country . The mean net annual sales of all pharmacies was 1.7 million euros . In this study, the total costs amounted to 24,303 euros per year, which roughly corresponds to the average personnel costs of a pharmaceutical technical assistant (pta) in one year . The estimated costs situation after installation of a robotic dispensing machine in the pharmacy depends to a large extent on four factors: the purchase price of the robotic dispensing machine (which, in turn, depends on the size and space requirements of the machine), the reduction in the stock value, the saving in personnel costs, and the reduction in the time required for taking inventory . In this context, the biggest influence is exerted by the personnel requirements, followed by the stock value . Whether additional self - service area will be gained does not depend on the sales volume of the pharmacy or other parameters . The strongest correlation between additional self - service and behind - the - counter display area can be made with the development of the otc sales volume (though this correlation is not significant). It does not appear to be possible to save on personnel costs and increase otc sales simultaneously by using a robotic dispensing machine . If the staffing level is held constant, the use of a robotic dispensing machine evidently leads to increased otc sales . If the staffing level is reduced, personnel costs are lowered but no increase in otc sales results from using the robotic dispensing machine . Pharmacies with low otc sales volumes benefit more than pharmacies with high otc sales volumes . However, an increase in otc sales exerts a relatively small effect on the overall costs situation in comparison to parameters such as stock value and personnel costs . In this study, in most cases the stock values could be reduced as a result of using a robotic dispensing machine . Although the time needed for the annual inventory is substantially reduced after installation of a robotic dispensing machine, this saving has only a minimal effect on the personnel costs . Nonetheless, the annual inventory normally takes place outside business hours so that the time saved reduces the need for staff to work in the evenings or on weekends, which could improve employee motivation . The savings in personnel costs are rather modest . In order to achieve cost benefits, higher otc sales volumes are possible for smaller pharmacies that keep their personnel costs stable . The space saving with a robotic dispensing machine is considerable compared with a conventional store, so that pharmacies with limited space or paying high rent can benefit especially . The present work shows that the annual costs for a robotic dispensing machine is about 25,000 euros . Since 2007, robotic dispensing machines of standard size have become available, and are about one third cheaper than custom built machines . This study focused on the impact of the robotic dispensing machine from a single supplier . The results cannot be extrapolated automatically to the impact of machines from other manufacturers because different robotic dispensing machines vary, especially with regard to price . The impact on otc sales and personnel costs was surveyed for a period of only one year after the installation of the robotic dispensing machine . It was therefore not possible to predict the long - term impact with regard to otc sales volume and personnel costs . Our results agree with the studies performed by lin et al . On the pharmacy chain punches pharma plus, where automation led to time savings in the work processes, but cost savings required adaptation of the staffing level to meet the changed staffing needs . We did not analyse the impact of automation on drug safety and employee satisfaction, as described in the work of franklin et al, pederson et al and mobach . This could be topic for future studies of the impact of robotic dispensing machines in community pharmacies . The savings in personnel costs after installation are rather modest, as is the increased sales volume in the otc sector . The saving occurs only if the staffing level is adapted to the changed staffing needs . Reducing the staffing level by one pharmaceutical technical assistant appears to completely cover the annual costs of the robotic dispensing machine.
Symptoms of cardiac involvement of tumor result from the location and impingement on adjacent structures . Conduction disturbances due to several kinds of cardiac tumor such as primary rhabdomyosarcoma,1) malignant melanoma with cardiac metastasis,2) cardiac hemangioma3) and metastatic adenocarcinoma of lung4) were reported previously . Mesotheliomas of the atrio - ventricular (av) node also cause heart block and sudden death.5) however, cardiac involvement of leiomyosarcoma is very rare . A 54-year - old man was referred to our electrophysiology laboratory because of dizziness and dyspnea . He was diagnosed with leiomyosarcoma of the right lower leg and received wide excision at another hospital 5 years ago . Recently, the patient was in good physical condition for 2 years and no abnormal findings were detected on the surface electrocardiography (ecg) 2 years prior . However, the current ecg revealed complete av block and idioventricular escaped rhythm of bifascicular block morphology suggesting infrahisian block (fig . The patient underwent cardiac magnetic resonance imaging, which revealed huge interventricular mass (6735 mm) from base to apex with low signal intensity, similar to the myocardium on the t1-weighted image (fig . 2a) and mild higher signal intensity, as compared with the myocardium on the t2-weighted image (fig . Trans - thoracic echocardiography revealed normal left ventricular ejection fraction (lvef 60%) and no hemodynamic compromise . Dual chamber permanent pacemaker was implanted on the second day of admission and we placed a right ventricular (rv) lead toward free wall of rv apex that was confirmed by fluoroscopy and echocardiography because of tumor invasion in the septum of rv apex . There were no procedure - related complications, however, ventricular tachycardia (vt) developed 3 days later . Twelve - lead ecg showed wide qrs complex tachycardia, left bundle branch block pattern with left superior axis morphology and late transition, which is compatible with vt from rv apex (fig . 3). Administered amiodarone and - blocker suppressed further events of vt and the patient was discharged uneventfully . He received pazopanib for palliative chemotherapy, which was stopped due to hepatic toxicity and poor performance status . The patient passed away after 3 months of pacemaker implantation due to progression of underlying disease and multiple organ failure . There are only a few sporadic case reports of arrhythmic presentation in patients with cardiac involvement of leiomyosarcoma . Atrial fibrillation due to metastasis to pulmonary vein and left atrium,6) ectopic atrial tachycardia with right atrial leiomyosarcoma7) and vt due to local tumor growth in the right ventricular outflow tract8) were reported previously . This was the first case report of a large metastatic mass of leiomyosarcoma located on the entire interventricular septum causing complete av block . Furthermore, there are no previous reports of cardiac involvement of tumor that caused both bradyarrhythmia and tachyarrhythmia . The patient was treated with implantation of permanent pacemaker for the management of complete av block and anti - arrhythmic drug suppressed vt successfully . Pacemaker mediated tachycardia or pacemaker - induced vt was considered because of subsequent development of vt after pacemaker implantation without prior history of vt . However, 12-lead qrs morphology of vt was different from that of paced ventricular rhythm (fig . 4) and apical septum of rv due to tumor invasion was suspected as the origin . Pacemaker interrogation excluded pacing - induced vt or pacemaker - mediated tachycardia (fig . 5). There was no histopathological diagnosis of cardiac lesion, however, we diagnosed the mass as a malignant metastasis because of the simultaneous similar pattern of widespread metastatic lung lesions with prior histopathologic confirmation of metastatic malignant leiomyosarcoma . Complete surgical resection of cardiac mass was not feasible because of infiltrative growth in the entire interventricular septum . Pacemaker implantation and anti - arrhythmic medications were started for symptomatic relief . Upgrade to implantable cardioverter defibrillator (icd) was considered regarding underlying structural substrate for vt . However, vt was well controlled with anti - arrhythmic drug and overall mean survival time in patients with metastasizing soft tissue sarcoma is approximately only 10 months despite therapy.9) thus, upgrade to icd was not performed and our patient passed away 3 month later due to disease progression cardiac metastasis should be considered when the patient with previously known leiomyosarcoma complains of cardiac symptom . Clinicians should be aware that both bradyarrhythmia and tachyarrhythmia could develop in patients with cardiac metastasis of malignant leiomyosarcoma.
Streptococcus agalactiae (group b streptococcus (gbs)) is one of the most common bacterial causes of life - threatening infection in newborns [1, 2]. Gbs was first reported as a human pathogen in 1938, but it was not until the 1970s that gbs was described as a major pathogen responsible for neonatal sepsis, pneumonia, and meningitis . Gbs neonatal infection is divided into two categories: early - onset disease, which occurs within the first week of life, and late - onset infection, which occurs between one week to 3 months of age . Gbs vaginal colonization occurs in 4% to 40% of pregnant and nonpregnant women and appears to be dependent upon geographical location [59]. The gbs bacterium also may lead to chorioamnionitis, myonecrosis of the uterus, neonatal pneumonia, premature delivery, premature labor, premature rupture of amniotic membranes, postpartum endometritis, and septic abortion [1014]. Furthermore, both mother and newborn infant may experience bacteremia, which can cause both septic shock and death . Prior to extensive prevention efforts in the 1990s, the incidence of invasive neonatal gbs infection ranged from 2 to 3 cases per 1,000 live births . In 1996, the centers for disease control and prevention (cdc) issued guidelines recommending the use of intrapartum antibiotic prophylaxis and by 1999, the incidence of early - onset gbs infection was reduced to 0.5 cases per 1,000 live births . In 2002, in order to further decrease the incidence of gbs sepsis, the cdc issued revised guidelines that recommended universal screening of pregnant women between 35 and 37 weeks of gestation . However, gbs infection continues to be a considerable problem, causing significant morbidity and mortality in mothers and their newborn infants . The cdc reported for the period 20002002, the average early - onset disease incidence was 0.49 cases per 1,000 live births . Following the revised cdc recommendations in 2002, average early - onset disease incidence decreased to 0.33 cases per 1,000 live births . The purpose of this retrospective study was to determine the screening rate for gbs, the incidence of maternal gbs colonization and early - onset neonatal gbs sepsis . In addition, we determined compliance with the cdc recommendation for the use of intrapartum antibiotic prophylaxis for gbs positive women in a private tertiary care hospital after the 2002 cdc recommendation for universal screening of pregnant women for gbs colonization . The study was conducted at the woman's hospital of texas (wht), kelsey - seybold clinic (ksc), and the kelsey research foundation (krf). The wht is a private tertiary care hospital with private physicians, a level iii nursery that accepts maternal - fetal transfers, and the only specialty hospital in houston, texas focused on the care of women and infants . Ksc is a large, multispecialty medical clinic with 300 physicians that serves an ethnically diverse population of over 400,000 patients at 18 locations in houston, texas . The krf is a 501 (c) (3) nonprofit that collaborates with healthcare and research institutions in the texas medical center to conduct health services research, provide patient education, and develop quality improvement initiatives . Since 1995, the krf has maintained a database of clinical information from both wht and ksc about the pregnancy experience of over 20,000 women at ksc . The database contains information describing the pregnancy experience and outcomes of mothers and infants who received care at wht and ksc . Data for all (9) obstetricians at ksc who routinely performed deliveries at wht are included in this study . The database was queried to identify the number of deliveries performed during the two - year period (january 1, 2003 through december 31, 2004). Outcome variables included the number and percentage of pregnant mothers screened for gbs, the gbs status of those women screened, the rate of intrapartum antibiotic usage, and the incidence of early - onset gbs sepsis . Sepsis is defined as a positive blood or spinal culture, or both, in addition to the clinical signs and symptoms of infection . Demographic variables, including age, ethnicity, and insurance status, also were collected from the administrative database . Data was analyzed using microsoft excel and access (2003, version 11.6355.6360 sp1) software . Because this is a retrospective analysis, waiver of consent was approved by the institutional review board of wht . At 35 to 37 weeks gestation, a standard cotton aerobic bacterial culture swab was gently inserted into the lower third of the vagina and then in a single motion, the perineum was swabbed and the external anal sphincter was swabbed . The swab was then placed in stuart's transport medium and sent to the laboratory at room temperature . All specimens were sent to a commercial laboratory as directed by patients' insurance providers . A screening culture was completed on arrival in labor and delivery if pregnancies were <35 weeks . If gbs bacteriuria was diagnosed during a pregnancy, the patient was considered colonized and treated during labor as per the cdc guidelines . All women who had gbs positive cultures were given antibiotic prophylaxis in labor as recommended by the cdc guidelines . For the two - year study period, 2,108 women delivered 2,135 infants . Of these 2,108 deliveries, there were 39 sets of twins (1.8%), 20 infant deaths (0.9%), and 171 (8%) premature deliveries . (prematurity is defined as infants born at a gestational age less than 37 weeks .) The population was 40% african - american, 25% caucasian, 28% hispanic, 5% asian, and 2% other . Approximately 3% of the women in the sample were less than 18 years of age, 56% were 1830 years of age, 39% were 3140 years of age, and 2% were 41 or older . The majority (71%) of women were insured through an hmo, while 27% had coverage with a ppo, 2% with medicaid, and fewer than 1% were self - insured . The demographics of mothers who were gbs positive, gbs negative or those receiving intrapartum antibiotic prophylaxis were comparable to the demographics of the study sample . Chi - square tests were performed and there were no statistical differences in the subgroups . Among the 2,108 mothers, 1,874 (89%) were screened for gbs and, of these, 1,322 (71%) tested negative and 552 (29%) tested positive for gbs . There were 231 (11%) of the 2,108 mothers with an unknown gbs status . Thirty - two (1.5%) of the 2,135 infants in the sample were evaluated for sepsis during their hospitalizations . The sepsis workup included a cbc, blood culture, and lumbar puncture for spinal fluid analysis and culture . Gestational age for 21 (66%) of these infants with sepsis was <30 weeks, for 8 (25%), gestational age was 30 to 36.7 weeks, and for 3 (9%), gestational age was 37 weeks . These infants were cultured for sepsis at birth and received antibiotics until results were available . For the majority (30) of these infants, culture results were negative and antibiotics were discontinued . For the two culture positive infants, one had positive blood cultures for gbs and the other had positive blood cultures for coagulase negative staphylococci, and none had meningitis . Thirty - one of the 32 (97%) infants were admitted to the nicu and had admissions of more than 30 days . Of these infants, of the 1,322 mothers who tested negative for gbs, 346 (26%) received antibiotic prophylaxis . Of the 346 that received antibiotics, 314 (91%) received antibiotics for surgical prophylaxis (c - section) and 32 (9%) received antibiotics for acute infection (chorioamnionitis, pyelonephritis, and respiratory infection). Among the 314 women who received antibiotics for surgical prophylaxis, 43 (14%) delivered vaginally interestingly, these 43 women, although initially gbs negative by culture at 3537 weeks, met the cdc risk - based criteria for gbs prophylaxis when admitted to labor and delivery . A total of 523 (95%) of the 552 mothers who were gbs positive received intrapartum antibiotic prophylaxis . The three gbs positive infants were born to gbs positive mothers and all three had a gestational age <36 weeks (33.5, 32.5, and 25.2 weeks). The mother who delivered her baby at 33.5 weeks had severe chorioamnionitis with artificial rupture of membranes and delivered vaginally on the day she was admitted . The second mother delivered twins at 32.5 weeks by repeat c - section on the day she was admitted . The mother had premature rupture of membranes with gbs status unknown at time of delivery and subsequently tested positive for gbs . The third gbs positive mother delivered her baby at 25.2 weeks by primary c - section . The mother was admitted eight days prior to delivery and diagnosed with preterm premature rupture of membranes, and chorioamnionitis . She received ampicillin / sulbactam, amoxicillin, and erythromycin antepartum, and cefazolin at the time the umbilical cord was clamped . The infant, in addition to being gbs positive with bacteremia, subsequently was diagnosed with klebsiella pneumonia and expired 14 days after delivery . A total of 165 (71%) of the 231 mothers with unknown gbs status received antibiotic prophylaxis for gbs based on weak risk factor assessment . Of these 165 mothers, 106 (64%) delivered babies at <37 weeks gestational age, 1 (0.9%) had rupture of membrane with labor> 18 hours, there were no mothers in this group with a temperature> 38c, and 105 (99%) had no recognized risk factors as outlined in the cdc guidelines except for unknown gbs status . The flow diagram in figure 1 shows the status of the women in the study . Our study demonstrates that in a private tertiary care hospital setting, both a high rate of screening for gbs and administration of intrapartum antibiotic prophylaxis can be achieved . The three infants with early - onset gbs sepsis born to gbs positive mothers were less than 34 weeks gestation, and they would have been treated based on risk factors alone . The results for this study population are reflective of the overall population delivering at wht . A major strength of this study is our ability to capture data on gbs screening and usage of intrapartum antibiotic prophylaxis from our obstetrical database . Similar surveillance studies conducted by the cdc have been limited due to their inability to measure health care provider compliance with screening guidelines . Despite our high rate of gbs screening, 11% of mothers had unknown gbs status at delivery . However, more than two - thirds of these women received prophylaxis for gbs based on risk factors alone . An additional strength is that the results reflect the management of gbs in a private tertiary care nonteaching obstetrical unit, with 80 practicing obstetricians, versus an academic hospital . The retrospective nature of the study may be a weakness because the study sample was not controlled and was limited to a small proportion of the total deliveries at wht . However, the study sample did represent 42% of all ksc deliveries during the two - year study period . Another weakness is that there were 11% (231) of mothers that were either not screened or data were not available . If a gbs positive rate of 29% is used, then this would contribute an additional 70 women to the gbs positive group . A confounding finding was that there were three newborns who developed early - onset gbs sepsis born to gbs positive mothers that were administered appropriate intrapartum antibiotic prophylaxis . It is not known whether or not gbs amnionitis was present at the time of admission to labor and delivery . This emphasizes the fact that universal screening and intrapartum antibiotic prophylaxis cannot eliminate the occurrence of neonatal early - onset gbs sepsis . Based on our experience reviewing gbs in mothers, we have undertaken an extensive review of neonatal outcomes in newborns of mothers receiving antibiotic prophylaxis within 4 hours of delivery . We also are developing a study to determine whether rapid screening using the pcr method in untested and gbs negative mothers will further reduce gbs infection in newborns . This study found that adherence to the 2002 cdc guidelines for screening and prophylaxis for gbs could readily be accepted, and that in the first year of implementation, 89% of women delivering in a private tertiary care hospital with a large number of practicing obstetricians were screened . This retrospective study demonstrated that universal screening for gbs is effective and can be achieved in a private tertiary care hospital.
Triploidy is a chromosomal anomaly that is seen in 12% of all conceptions and up to 10% of spontaneous abortions 1 . It is classified as diandry when the extra set of chromosomes is from the father and digyny when the extra set of chromosomes is of maternal origin . Most cases of diandry occur as a result of dispermy and less commonly are caused by nondisjunction during the first (mi) or the second meiotic division (mii) of spermatogenesis . On the other hand, most cases of digyny are caused by errors in mi or mii of oogenesis 2 . Although most triploid conceptions end in first trimester spontaneous abortions, there are few reports of live born infants with triploidy, with the longest reported survival of digynic triploidy at 10 months 3 . Based on personal communication of the authors of same report, about half of previously reported 38 triploidy cases were live - born and lived for a few minutes to 5 days 3 . 4 reported a girl with diandric triploidy who survived for 10 weeks and reviewed the data on 7 previously reported triploid patients who survived beyond 2 months of age . 5 described a girl with digynic triploidy who survived for 46 days and discussed parental of origin and clinical findings of four additional triploid (three digynic and one diandric) infants who survived 4 weeks or more . Parental origin studies of triploidy showed variable diandric to digynic ratios 2,68, reflecting ascertainment bias and different selection criteria 8 . Digyny is associated with asymmetric severe fetal growth restriction (fgr) and a very small placenta 8,9 . Diandric triploid fetuses are relatively well grown or exhibit symmetric fgr accompanied by a large cystic, often molar, placenta 9 . Since the introduction of noninvasive prenatal testing (nipt) to clinical practice, it has evolved to be a common screening test for chromosomal aberrations in pregnancies complicated by abnormal ultrasound or maternal serum screens or for advanced maternal age 10 . However, there is limited data in the utility of nipt in the detection of triploidy and the matter has never been systematically investigated . Here, we present a digynic triploidy case that escaped diagnosis despite using two methods of nipt . Our data suggest that low fraction fetal dna in nipt along with multiple congenital anomalies and fgr should alert medical professionals to the possibility of digynic triploidy and demonstrate that snp microarray can detect the parental origin of triploidy and its mechanism . The proband was born to a 24-year - old g1p0 after a pregnancy complicated by multiple fetal anomalies . There was no family history of congenital anomalies, early infant deaths, or consanguinity . The mother declined first trimester screening for fetal anomalies and chromosomal abnormalities . At 18 weeks of gestation, the fetus had been diagnosed with early - onset fgr, oligohydramnios, and bilateral brain ventriculomegaly . Severe oligohydramnios would have made the amniocentesis technically difficult and the parents declined the procedure . Nipt on cell - free fetal dna (cffdna) testing at 18 weeks of gestation could not be interpreted secondary to an insufficient fraction (1.3%). Repeat ultrasound assessment in our center at 22 weeks was notable for asymmetric bilateral ventriculomegaly, severe fgr (<5th percentile), oligohydramnios (amniotic fluid index was 5th centile), small chest, a possible right pelvic kidney, and the fetal gender could not be determined . The mother declined amniocentesis and opted to repeat cffdna testing at 22 weeks of gestation, which demonstrated the expected representation of chromosome 21, 18, and 13 materials, absence of y chromosome material, with cffdna fraction of 5.2% . At 33 weeks, the estimated fetal weight was 601 g. the mother was counseled on the poor prognosis for her fetus but opted for full obstetric intervention, including potential cesarean section with full neonatal management . After one dose of antenatal steroids on the day prior to delivery, the proband was born via cesarean section for nonreassuring fetal status with a 0/8 biophysical profile and fetal bradycardia . A partial placental abruption, very small placenta and short, thin cord were noted at the time of delivery . At birth, the neonate had no spontaneous respirations and a heart rate below 80 beats per minute, for which he was intubated and placed on a respirator, but soon required high frequency ventilation and nitric oxide . Despite the nipt results, the infant was found to have male genitalia with hypospadias . On physical examination his weight was 590 g (50th centile for 23 weeks' gestation), length was 30 cm (50th centile for 23 weeks' gestation), and occipitofrontal circumference was 25 cm (50th centile for 26 weeks' gestation). Facies showed frontal bossing, hypertelorism, a bulbous nasal tip, a depressed nasal bridge, micrognathia, and low set and posteriorly rotated ears . There was cutaneous syndactyly of 24th toes bilaterally, as well as 34th fingers of the left hand, webbing of all digits of the right hand, and adducted thumbs bilaterally . A head ultrasound demonstrated enlarged, dysplastic lateral ventricles with a dysplastic corpus callosum and possible partial thalamic fusion . A skeletal survey revealed thin clavicles, gracile ribs and humeri, and butterfly vertebral bodies in the upper thoracic spine . An echocardiogram showed a moderate perimembranous ventricular septal defect (vsd) and atrial - level shunt, a large patent ductus arteriosus (pda), and dilation of the aortic root (z score + 2.3) and ascending aorta (z score + 3.2). Renal ultrasound showed small kidneys with loss of normal corticomedullary differentiation with few scattered microcysts . Ultrasound of the bladder showed an irregular contour suggestive of trabeculations . Despite maximum ventilatory support, the neonate's condition continued to decline . The family chose to redirect plans to supportive care and the neonate succumbed to respiratory failure at 16 h of life . At autopsy, the placenta weighed 51.7 g and had microscopic features of chronic placental insufficiency including distal villous hypoplasia and increased syncytial knotting . The umbilical cord had three vessels with focal obliteration of the umbilical artery, consistent with flow change due to focal vascular thrombosis . External examination demonstrates relative macrocephaly, dysmorphic facies, hypertelorism, micrognathia, and syndactyly of the third and fourth fingers on left hand . Due to multiple congenital anomalies in the neonate and discrepancy between the male phenotype and the nipt results, fish analysis was performed on a direct preparation from a sample of peripheral blood and revealed two hybridization signals for cep x and one hybridization signal for sry in all 500 nuclei analyzed . Subsequent rapid fish analysis for select loci revealed three copies of chromosomes 13, 18, and 21 using lsi 13, cep 18, and lsi 21, two copies of the x chromosome (cep x), and one copy of the y chromosome (cep y). A chromosomal microarray analysis (cma), using the affymetrix cytoscan hd array was performed with reflex to karyotype; the latter confirmed 69,xxy pattern . The cma of the proband revealed triploidy as indicated by the presence of four - allele peak tracks on all autosomes with a normal log2 ratio . There were two copies of chromosome x (fig.2) and one copy of chromosome y as interpreted from the allele peak tracks, log2 ratio and smooth signal . Notably, an apparent copy number neutral region demonstrating a long contiguous stretch of homozygosity (lcsh) on chromosome x from bands p22.2 to q24 and spanning 106.28 mb was observed and suggests that the majority of the length of chromosome x is identical across all loci . This pattern is consistent with a triploidy of digynic origin in that the two copies of chromosome x are uniparental and as by nature the y chromosome is paternally derived, the x chromosomes are therefore maternally attributed . Regions distal to the lcsh include the pseudoautosomal regions demonstrated a log2 ratio and allele peak tracks similar to the pattern seen in all autosomal chromosomes (fig.2). Additionally, regions of meiotic recombination (cross - over) were observed on chromosome x encompassing bands p22.33 to p22.2 on the short arm and bands q24 to q28 on the long arm as determined by genomic stretches demonstrating heterozygosity . These findings, along with similar changes on the autosomes, indicate error in nondisjunction during mii as the most likely mechanism . The cma also revealed a maternally inherited 762 kb gain of chromosomal material on 9p21.2 (26,456,90427,218,960;hg19), likely of no clinical significance . Of note, because of the normalization algorithm employed during analysis, the detection of triploidy is not typically possible in array platforms containing solely copy number probes . Chromosomal microarray analysis (cma) data from the x chromosome with a normalized copy number state 2 in par1 and par2, and 1 across the remainder of the chromosome with a long contiguous stretch of homozygosity observed proximally, and regions of heterozygosity observed distally . The results of nipt in the proband make this case particularly interesting . Currently, there are four commercially available noninvasive prenatal tests in the united states: natera's panorama prenatal test (npp), sequenom's maternit21 test (sm), ariosa diagnostics's harmony test (adh), and verinata health's verifi test (vhv) 11 . For our patient's mother, the subsequent sm test revealed no y chromosomal material, consistent with a normal female fetus . One possibility for this difference is the type of technology used in each of these tests . The npp test uses multiplex pcr followed by snp - based targeted sequencing and analysis of parental genotypes 12, as opposed to the sm test which uses massively parallel shotgun sequencing and quantification of the number of reads from each chromosome 13 . While both methods allow for accurate determination of individual chromosomal trisomies, the sm test uses a comparison among the relative number of reads from each chromosome . Hence, the sm test may be at a disadvantage in detecting triploidy, which affects all of the autosomes equally . In fact, while none of the other three commercial companies make a claim regarding the detection of triploidy, the npp test in a recently published study has been able to correctly identify all four diandric trisomy cases, while all four digynic cases tested were found to have low cffdna fraction after adjusting for maternal weight and gestational age (<4%) and consequently escaped diagnosis 14 . The small size of the placenta in digynic triploidy likely contributes to low cffdna 14 . The source of cffdna in maternal blood is dying cytotrophoblastic cells 15 and its low fraction reflects low placental mass as seen in digynic triploidy 14 . The lack of observed y chromosomal material in our proband by sequenom nipt can be related to an overestimation of the cffdna fraction because of limitation in the quantification method used, which is based on differentially methylated markers in fetal and maternal dna 13 . Furthermore, the reported accuracy of detecting sex chromosome abnormalities is lower than autosomal aberrations with overall sensitivity of 96.2%, false positive rate of 0.3%, and nonreportable rate of 5% 16 . The small size of y chromosome and fewer interrogated genomic regions on y chromosome can also lead to a weaker signal - to - noise ratio 16 . These limitations were exacerbated by the triploid karyotype composition since the sex chromosome representations are evaluated as ratios of normalized read counts as compared to the total autosomal read counts . The diagnosis of digynic triploidy can also be challenging because of difficulties in performing amniocentesis due to oligohydramnios . In conclusion, snp microarray can be an effective tool in determining the mechanism and parental origin in triploidy . A low cffdna fraction in the context of fgr and multiple congenital anomalies should alert medical professionals to the possibility of digynic triploidy . The development of highly sensitive nipt technologies that combine massively parallel sequencing and snp analysis has the potential to improve the detection of digynic triploidy . Irb approved consent was obtained to publish the photographs and clinical data in the medical literature.
Reverse shoulder replacement surgery is increasing in prevalence due to the advent of better performing, longer lasting implant designs . Even with these improvements, the burden of revision surgery is inevitable, as implants become infected, loosen, or wear . Day et al . Have previously described the growth in upper extremity arthroplasty procedure rates at 7 - 13% per year, much higher than primary and revision knee arthroplasty (6% and 7%, respectively), and primary and revision hip arthroplasty (4.5% and 2.5%, respectively). Notably, they highlight the rapidly growing revision burden for the upper extremity, a concern due to the more complex nature of revision procedures . One challenge that may face the revision surgeon is when one side of the joint is well - fixed, for example, a well - ingrown glenoid component and a loose humeral component . Intraoperatively, circumstances can arise where the revising surgeon does not have access to a full range of implants from the required manufacturers . Therefore, they may have to remove a well - fixed component to ensure both sides of the bearing couple are from the same implant system or replace only the loose component and mix between implant systems and potential manufacturers . Due to the sphere - in - a - cup nature of reverse shoulder implants, some manufacturers and surgeons anecdotally suggest that this type of system mixing is allowable . However, to the authors' knowledge, the true compatibility between different reverse shoulder arthroplasty (rsa) systems has not been studied . Therefore, the primary objective of this study was to determine the intersystem geometric variability between similar - sized implants from different rsa systems . As a secondary objective, the intrasystem manufacturing variability between polyethylene inserts of the same system was also determined, as this could affect the accuracy of the intersystem measurements . It was hypothesized that across the range of systems, some implants would demonstrate high compatibility and others would demonstrate low compatibility . The polyethylene inserts for eight different rsa implant systems were obtained from six manufacturers [table 1]. For each implant system, six polyethylene inserts of the same size were obtained, either from the same or different manufacturing lots . In all cases, the smallest available diameter of the insert was acquired . For six of the implant designs, the manufacturers reported diameter was 36 mm, and for the remaining two designs, the diameter was reported to be 38 mm . List of implant manufacturer, system and sizes evaluated in this study all 48 polyethylene inserts were scanned with a laboratory microcomputed tomography scanner, using a validated protocol that has been widely utilized to study the polyethylene components from hip and knee arthroplasty . The scans were performed with an isotropic voxel spacing of 50 m, and x - ray energy of 90 kvp and 40 ma . There were 1200 views acquired per scan, with 10 acquisitions averaged per view to reduce noise . The insert geometries were extracted from the reconstructed scan volumes using isosurface rendering, at the highest surface quality settings, and saved in stereolithography format for further evaluation [figure 1a h]. The three - dimensional geometries of the polyethylene inserts from eight different reverse shoulder arthroplasty systems reconstructed from microcomputed tomography scanning . (a) arthrex, (b) biomet, (c) depuy, (d) exactech, (e) tornier fl ex shoulder system reverse, (f) tornier aequalis reversed, (g) zimmer anatomical shoulder inverse / reverse, (h) zimmer trabecular metal reverse shoulder the analysis process is shown visually in figure 2 . One insert surface geometry for each of the eight different implant systems was imported into polygonal geometry editing software (geomagic studio, 3d systems inc ., this particular geometry, for each model, was then imported into dimensioning software (geomagic qualify, 3d systems inc ., three - dimensional (3d) comparisons were then made between the six 36 mm diameter inserts and the two 38 mm diameter inserts . Intersystem comparisons were conducted by co - registering two inserts at a time, using an iterative closest points algorithm to determine the best fit between the two articular surface geometries . Once registered, the 3d deviations between the two surfaces were calculated and mapped across the entire surface using the dimensioning software . The maximum absolute deviation (either positive or negative) between the two surfaces was computationally determined from the deviation maps . Visual representation of the steps performed by analyzing the articular surface variability between systems intrasystem manufacturing variability was determined by comparing the six scanned inserts for each implant system . The six geometries from each model were co - registered, and the deviations between them mapped, using a custom software utility . This software, previously validated, functions in a similar manner to the software used to compare the geometric compatibility of the different models, but can better handle larger files and perform comparisons across more than two geometries . From each deviation map (which displayed the average deviation across the six geometries), regions of interest were selected from the articular surface, the rim, and the backside surface, where the maximum amount of deviation was visualized within those structures . From within each region of interest, the maximum deviation was recorded . Descriptive statistics (mean, standard deviation, and range) were calculated for the maximum intersystem surface deviation and the intrasystem manufacturing variability . Differences between implants and regions of interest were compared using a one - way anova with tukey post hoc test . The average maximum deviation demonstrated by the six 36 mm diameter insert systems in the articular surface comparisons [figure 3a] ranged from 53 12 m with the zimmer anatomical inverse / reverse to 71 12 m with the arthrex univers revers . There was no significant difference between any of the implant models (p = 0.61). The overall maximum deviation between any two articular surfaces was 60 16 m on average, with a range of 30 - 80 m . The maximum articular surface deviation between the two 38 mm diameter models was 51 m . The pattern of the deviations across the articular surface varied between each comparison [figure 4]. The measured deviations from the polyethylene inserts tested . (a) the deviation in micrometers between the articular surfaces of the different reverse shoulder arthroplasty systems . (note that the depuy and exactech inserts were the only 38 mm models, so represent a single comparison). (b) the intrasystem manufacturing variability for the different reverse shoulder arthroplasty systems tested exemplar articular deviation maps, with the torner flex shoulder system reverse insert used as the reference model compared to the 36 mm diameter polyethylene inserts from the other models and manufacturers . (a) intact tornier aequalis reversed insert geometry with the articular surface that was isolated for comparison highlighted in red, (b) arthrex, (c) biomet, (d) tornier flex shoulder system reverse, (e) zimmer anatomical shoulder inverse / reverse, (f) zimmer trabecular metal reverse shoulder . Registrations were centered at the base of the geometry, where the glenosphere would make contact with the polyethylene insert the average maximum deviation between different inserts from the same system (i.e., intrasystem manufacturing variability) ranged from 30 6 m with the depuy delta xtend reverse to 71 24 m with the biomet comprehensive reverse [figure 3b]. The only significant difference (i.e., one system having significantly higher manufacturing variability) was between the depuy and biomet systems (p = 0.03), with all other systems demonstrating the same magnitude of intrasystem manufacturing variability . The overall mean of the deviations, across all systems, was 49 17 m, with a range of 23 - 99 m . There was no difference (p = 0.38) in deviation magnitude based on the location of the measurement (articular surface, rim, or backside surface). The articular surfaces of the same - sized polyethylene inserts from the 8 rsa systems tested in this study were found to be highly compatible . The magnitude of deviations between implant models was on average 60 m, roughly the thickness of a strand of human hair, and there was no appreciable pattern to the deviations . This is likely due to the hemispherical nature that all systems implement, for articulation with the round glenosphere . Although it is not the purpose of this paper to make surgical recommendations, similarly sized humeral inserts from the different implant systems tested in this study could theoretically be interchanged . In certain circumstances, this could reduce the complexity and the associated complications of revising a well - fixed implant, which could potentially benefit the patient . Such data in the orthopedic literature have been for applications in which geometric accuracy is a requirement, including wear measurements in retrieved implants and radiographic motion tracking in radiostereometric analysis or fluoroscopy . Previous studies have reported the manufacturing variability for polyethylene inserts used in total knee replacement to be on the order of tens of microns, similar to the findings of this study for reverse shoulder inserts (52 17 m). This intrasystem manufacturing variability is likewise on the same order of magnitude as the geometric variability between inserts of different systems (60 16 m). Therefore, the geometric variability between the articular surfaces of different implant systems is equivalent to the manufacturing variability of those same implants . These observed tolerances are also relevant to researchers who will undertake retrieval studies of failed reverse shoulder implants, by establishing the measurement uncertainty for estimating the unworn implant geometry . Limitations of this study are that only a single size of polyethylene insert (the smallest) was studied from each system, and not all implant models or manufacturers were included in the study . However, it seems likely that the results would be applicable to other polyethylene insert sizes within the systems studied . The focus of this study was the articular surface, which is the most important aspect of interdesign compatibility . However, many of the implant systems have different rim geometries [figure 1], which are an additional variable and could potentially have an effect on edge wear and impingement . The glenospheres offered by different manufacturers were also not examined, however by their inherent nature the variation between their articular surfaces, as was found with the polyethylene inserts, is likely to be similar . Our method to directly examine the entire articular surface is more thorough than simply measuring the diameter of liners from different manufacturers, which would reveal whether the difference in diameter (if any) was consistent throughout the entire geometry . In the case of a slightly narrower liner articulating against a slightly broader glenosphere, the curvature of the glenosphere should still enable bearing contact, but with reduced surface area coverage . In the reverse case of a broader liner and a narrower glenosphere, there would be greater penetration of the glenosphere providing increased surface area coverage . The articular surfaces of similarly sized polyethylene inserts used in rsa systems examined in this study were found to be equivalent, with a mean intersystem variability of 60 16 m . This variability is on the same order of magnitude as the intrasystem manufacturing variability of these polyethylene inserts, which was 52 17 m . Although it is not the purpose of this paper to make surgical recommendations, our findings suggest that revision surgeons can theoretically interchange same - sized implant components from the different rsa systems tested, when faced with a well - fixed implant on one side and no way to implant a new component from that same system on the opposite member of the bearing couple
Push - up exercise (pue) is a representative closed kinetic chain exercise (ckce) that can strengthen shoulder and truncus muscles in daily living1,2,3,4,5 . Ckce is performed by applying resistance to the proximal and distal parts of limbs simultaneously while the distal is kept immobile6 . It is frequently used in exercise programs for dynamic stability of joints and upright posture maintenance because it causes co - contraction of various muscles, in addition to enhancing muscle strength and endurance, and provides more proprioception by stimulating afferent receptors around joints7 . In addition, pue is mainly used for muscle strength evaluation of truncus muscles8, and frequently used in rehabilitation programs for patients with shoulder damage9, 10 . In pue, because muscle activity varies depending on the position of the hands, the palmar width is a key factor in the exercise . Among previous studies on palmar width in pue, cogley et al.11 compared the muscle activity of a group with a large palmar width with that of one with a small palmar width, but the muscles examined in the study were limited to the pectoralis major and triceps brachii . Another study by rho et al.12 classified palmar width into three types (narrow, neutral, and wide positions) and examined electromyography (emg) of the pectoralis major and latissimus dorsi, but it did not mention the precise width between the hands . In addition, a study by oh et al.13 study compared emg of the infraspinatus, serratus anterior, upper trapezius, and triceps brachii across different palmar widths in pue but had a limitation in research design in that palmar width was kept uniform (20 cm), as it was not considered in their study . These studies suggest that research on palmar width in pue as clinically practiced ckce is lacking, and the claims of the studies fall short in terms of sufficient supporting evidence . In particular, when providing training programs to strengthen shoulder and truncus muscles or rehabilitation programs for shoulder joint patients, palmar width in pue is expected to play a significant role in the rehabilitation period and performance of patients . Thus, this study aimed to identify the optimal palmar width for each muscle in pue by determining the differences in muscle activity of shoulder and truncus muscles in pue with three types of palmar width applied separately . We hypothesized that pue performed with narrow and wide palmar widths induces higher muscle activities than pue with a neutral palmar width . Twelve right - handed male korean university students majoring in physical education participated in this study as subjects . All subjects were healthy and had no previous history of damage in the wrist, shoulder, or elbow joint . All of them participated in the study voluntarily after being fully informed of the study objective . Subjects characteristics are shown in table 1table 1.subject characteristics (n = 12)variablesage (yrs)21.40.5height (cm)173.11.1body mass (kg)70.51.4shoulder width (cm)42.10.6values are means ses . Values are means ses palmar width was defined as the distance between the acromion in subjects . For each subject, the palmar width was considered the neutral position (100%), half the palmar width was considered the narrow position (50%), and 1.5 times the palmar width was considered the wide position (150%). Before beginning the experiment, subjects were given information on push - up movements with the three palmar widths, and underwent a light warm - up to enable natural push - up movements . Each subject performed a total of nine push - ups with each of the palmar widths, and among the nine, five (from 3rd to 7th) push - ups were selected . One push - up was defined as one with complete flexion and extension of the arms . For emg analysis, t246h ag - ag / cl surface electrodes (bio - protech inc ., wonju, republic of korea) were attached to each muscle on the right side of subjects, and the sites for electrode attachment were determined based on the instructions of merletti et al.14; specifically, they were attached to the deltoideus p. acromialis (da), pectoralis minor (pmi), pectoralis major (pma), serratus anterior (sa), biceps brachii (bb), triceps brachii (tb), latissimus dorsi (ld), and infraspinatus (is). For synchronization of 2d image data and emg data of subjects push - up movements, one raptor - e camera (motion analysis inc ., usa) and a desk dts emg system (noraxon usa inc, scottsdale, az, usa) with eight channels were used . All emg signals were amplified, band - pass filtered at 10250 hz, and sampled at 1,024 hz using mr - xp program (noraxon usa inc, scottsdale, az, usa). The root mean square (rms) values of each muscle were measured for three seconds in the pue with the neutral palmar width . Muscle contractions were calculated relative to the mean emg signal for two seconds in the middle portion of the emg recording, excluding the measurements in the first 0.5 seconds and last 0.5 seconds . The muscle activities resulting from one push - up were calculated as the relative voluntary contraction (% rvc). Armonk, ny, usa) was used for statistical analysis, and the statistical significance of differences was examined by one - way repeated anova, with significance accepted at a value of p <0.05 . In the present study, emg activity was greater in the pmi, tb, and is muscles during push - ups performed with the 50% palmar width compared with the 100% and 150% palmar widths (p<0.001, p<0.01, p<0.001). Pma muscle activity was greater during push - ups performed with the 50% and 100% palmar widths compared with the 150% palmar widths (p<0.001). Sa muscle activity was greater during push - ups performed with the 150% palmar width compared with the 100% and 50% palmar widths (p<0.001). However, the da, bb, and ld muscles did not show any significant differences (p>0.05) (table 2table 2.mean of the average muscle activity during push - up with various palmar widthsmusclesmean se (% rvc)50%100%150%da89.35.6100.07.197.56.3pmi*131.74.0100.03.184.32.5pma115.04.3100.03.492.32.9sa71.22.6100.04.4119.24.9bb105.56.9100.06.9121.69.1tb116.57.0100.05.986.05.3ld92.45.6100.06.0103.57.1is**162.710.3100.05.175.74.2values are means ses . 50%: narrow position; 100%: neutral position; 150%: wide position; da: deltoideus p. acromialis; pmi: pectoralis minor; pma: pectoralis major; sa: serratus anterior; bb: biceps brachii; tb: triceps brachii; ld: latissimus dorsi; is: infraspinatus . * * p<0.001; * * p<0.01 . 50%: narrow position; 100%: neutral position; 150%: wide position; da: deltoideus p. acromialis; pmi: pectoralis minor; pma: pectoralis major; sa: serratus anterior; bb: biceps brachii; tb: triceps brachii; ld: latissimus dorsi; is: infraspinatus . * * * p<0.001; * * p<0.01 . This study aimed to present the optimal palmar width for each muscle in pue when implementing pue with three palmar widths (50%, 100%, and 150% of the palmar width in the neutral position) applied separately in 12 healthy men in their 20s by measuring the activity of the da, pmi, pma, sa, bb, tb, ld, and is . The results showed that the highest level of muscle activity in the pmi, pma, tb, and is was observed for pue with the 50% palmar width . Previous studies reported findings consistent with the results of the present study . Specifically, cogley et al.11, who compared the results of emg of the pma and tb for a group with a wide palmar width (mean 85.8 cm) with those of a group with a narrow palmar width (mean 66.3 cm) in pue reported that the group with the smaller palmar width showed significantly higher emg activity for both muscles . Gouvali et al.15 also found that the emg activity of the pma was high when the palmar width was small, and rho et al.12 reported that pue with a small palmar width resulted in the highest pma activity . In addition, according to a study by oh et al.13, the activity of the tb increased significantly when pue was performed with a small palmar width, supporting the experimental results of the present study showing that pue with the 50% palmar width is more effective in muscle strength performance of pmi, pma, tb, and is . However, pue with the 150% palmar width resulted in statistically significantly higher activity in the sa . A study by kang et al.16 stressed that the sa is the key muscle for increasing stability of scapula . They reported that when there is an imbalance in the stabilizing muscles of the scapula, exercise focusing on the sa is beneficial and that the relative contribution of the sa is also higher than those of other muscles17 . Similarly, oh et al.13 reported that the activity of the sa increased significantly when the palmar width was large during pue, which is consistent with the experimental results of the present study showing that pue with the 150% palmar width is more effective in muscle performance of sa . This suggests that the activity of the truncus muscles varies with different palmar widths in pue; accordingly, for effective pue, a narrow palmar position is recommended for the pmi, pma, tb, and is, and a wide palmar position is recommended for the sa . Thus, the findings of this study are expected to serve as reference materials for pue applications in training programs for truncus muscle strengthening or rehabilitation programs for scapula patients . However, generalization of the study findings may be limited due to the restricted sample selection and size . Therefore, further research with a wider range of subjects including women and scapula patients in addition to healthy male adults is needed in the future.
Census regions are presented for selected calendar years (1977, 1981, 1983, 1986, and 1987) to reflect regional variations in short - stay hospital (ssh) inpatient discharges and discharge rates (total and surgical) for aged medicare beneficiaries (table 1). For the 25 leading surgical procedures for aged enrollees, we focus on 1987 data to show regional variations in the number of surgical discharges and surgical discharge rates per 1,000 enrollees (table 2), total charges and average charges per discharge (table 3), and the number of total days of care and average length of stay per discharge (table 4). The article includes a technical note with definitions for the major surgical classifications and the 25 leading surgical procedures in 1987 . Procedure codes for the leading procedures were derived from the international classification of diseases, 9th revision, clinical modification, (icd-9-cm) (public health service and health care financing administration), and only those codes designated as operating room procedures by the health care financing administration (hcfa) were included in the leading procedures . Thus, some diagnostic and therapeutic surgical procedures may have been more frequently reported than were the leading procedures but were excluded from the list of the leading procedures because they were not performed in an operating room . Although as many as three icd-9-cm procedure codes are reported on the hcfa form 1450, only the principal procedure code has been used in this article . The proportion of ssh discharges involving inpatient surgery for the aged medicare population has increased substantially since the inception of the medicare hospital insurance (hi) program . During the first 10 years (1967 - 76) of medicare, for example, the proportion of surgical discharges for aged medicare beneficiaries was about 32 percent of all discharges (helbing, 1980). However, the proportion of surgical discharges for the elderly residing in all areas increased from 33 percent in 1977 to 38 percent in 1983, rose to 53 percent in 1984, and climbed steadily to 61 percent in 1987 . Among the regions, the proportion of surgical discharges in the northeast region increased from 36 percent in 1977 to 68 percent in 1987 the south region, which had the lowest proportion of surgical discharges (31 percent) in 1977, moved into third place (58 percent) among the regions in 1987 . Large geographic variations in the incidence of surgery raise a great many questions regarding local differences in the criteria of appropriate use of surgical services or differential access to surgical specialties that cannot be addressed directly because of the lack of definitive data for measuring potentially pertinent variables . The increased proportion of reported discharges with surgery since the late 1970s appears to be related to the introduction of the icd-9-cm diagnostic and procedural coding system and the implementation of the medicare prospective payment system (pps) established by public law 98 - 21 and implemented on october 1, 1983 . The increase in the percent of discharges with surgery from 1978 to 1979 was due largely to the changes in coding and reporting practices instituted in 1979 . The icd-9-cm was used to code procedures beginning in 1979, and it was organized differently than earlier versions of the classification system, resulting in a broader definition of surgical procedures; the icd-9-cm also included further, for services rendered to medicare enrollees, changes in the way sshs are paid under pps encouraged hospitals to become more diligent in adhering to and applying icd-9-cm medical coding techniques and conventions . Prior to pps, there was no monetary incentive for hospitals to promote either complete reporting of patient history or accurate coding of patient diagnoses and surgical procedures on medicare claims . After pps, however, the financial position of hospitals could be improved by promoting both reporting and coding precision . Under pps, payment to hospitals for the care of medicare patients is based mainly on the coding of the principal diagnosis (condition) that caused the admission of the patient to the hospital . In addition, the reporting and coding of a surgical procedure is a major factor used to determine the preset pps payment . When hospitals began to recognize that the medicare payment would be larger for most admissions requiring surgical procedures, the reporting and coding of these procedures began to receive top priority, thus generating the increase in reported surgeries . Pps also precipitated changes in ssh admission practices changes that resulted in an overall decrease in admissions . Thus, the 7-percent decrease in the total discharge rate per 1,000 aged hi enrollees (table 1) resulted in an increase in the proportion of discharges with surgery . The hospitals' response to pps also shifted patient care, in some cases, to alternative treatment sites, such as ambulatory surgical centers . Thus, some procedures previously done on an inpatient basis are now being done in the outpatient setting . For instance, kozak (1989) reports that the decline in [inpatient] eye surgery was almost all from decreases in cataract surgeries . The rate of lens extraction fell 90 percent, and the rate of insertion of prosthetic lens dropped 88 percent from 1983 to 1987 . In addition to the increase in reported surgeries induced by the new coding system and incentives provided by pps, a true increase in the number of surgical procedures for aged medicare beneficiaries may have resulted from advances in medical technology . Such advances have made it possible to operate on elderly patients for whom surgery would previously have been considered too risky; this surgery now would necessarily be performed on an inpatient basis . For example, the national center for health statistics reports that the rate of elderly patients undergoing cardiovascular surgery increased 63 percent from 1983 to 1987: the number and rate of bypass anastomosis for heart revascularization more than doubled . The number of discharged patients 65 years of age and over who had one or more bypass procedures increased from 66,000 in 1983 to 117,000 in 1987 medicare trend data for selected calendar years 1977 - 87 are presented in table 1 to highlight utilization patterns by area of residence (u.s . Census regions) for aged beneficiaries with surgery performed on an inpatient basis in sshs . The data show that the proportion of surgical discharges for aged hi enrollees increased from 33 percent of all discharges for aged enrollees in 1977 to 61 percent in 1987 (figure 1). The surgical rate per 1,000 aged hi enrollees increased 74 percent, indicating that the number of discharges for aged beneficiaries with surgery increased at a faster rate than the total aged hi enrollment population (figure 2). For all aged medicare beneficiaries receiving ssh inpatient services, the total number of discharges rose from 7.8 million in 1977 to 9.0 million in 1987, an increase of 15 percent . However, for aged beneficiaries with ssh inpatient surgery, the number of discharges rose from 2.6 million discharges in 1977 to 5.5 million in 1987, an increase of 114 percent . The total ssh discharge rate per 1,000 hi enrollees declined from 334 in 1977 to 312 in 1987, a decrease of 7 percent . In contrast, the corresponding surgical discharge rate per 1,000 aged hi enrollees climbed from 110 in 1977 to 191 in 1987, an increase of 74 percent . Among the regions during the 1977 - 87 period, the increase in the surgical rate in the west (71 percent) and south (73 percent) regions was similar to that for all areas (74 percent). The northeast region showed the largest increase (95 percent) in the surgical rate, from 108 discharges per 1,000 enrollees in 1977 to 211 discharges per 1,000 enrollees in 1987 . The north central region, however, showed an increase (61 percent) in the surgical rate per 1,000 hi enrollees that was substantially less than the increase in the surgical rate for all areas (74 percent). This lower increase reflects the fact that the north central region had the highest surgical rate among the regions in 1977 (115 discharges per 1,000 hi enrollees5 percent above the rate for all areas) and the lowest rate in 1987 (185 discharges per 1,000 enrollees3 percent below the rate for all areas). For the 25 leading procedures (excluding those procedures not defined as operating room procedures by hcfa), data in table 2 focus on regional variations in the types of surgery most frequently performed on aged medicare beneficiaries in the ssh inpatient setting . Table 2 data include the number of surgical discharges and surgical rates per 1,000 hi enrollees for aged medicare enrollees undergoing specific surgical procedures in 1987 . Surgical rates for icd-9-cm code 60.2 (transurethral prostatectomy) an based on the number of male hi enrollees . Similarly, the number of female hi enrollees is the basis of the surgical rates for icd-9-cm code 68.4 (total abdominal hysterectomy). During 1987, discharges for the 25 leading procedures accounted for 23 percent (1.3 million) of all surgical discharges (5.5 million). Among the leading procedures, the largest number of discharges (236,000) was reported for icd-9-cm code 60.2 (transurethral prostatectomy). For every 1,000 aged male hi enrollees residing in all areas, 20.3 discharges for transurethral prostatectomies were recorded . Males living in the northeast region (18.3 of every 1,000) were slightly less likely to have this surgery, and males living in the south region (23.6 per 1,000 male hi enrollees) were more likely to have a transurethral prostatectomy . After transurethral prostatectomy, the highest rates of ssh inpatient surgical procedures per 1,000 aged hi enrollees were reported for: icd-9-cm code 51.22 (total cholecystectomy4.3 of every 1,000 enrollees). Icd-9-cm code 79.35 (open reduction of fracture with internal fixation3.7 of every 1,000 enrollees). On the other hand, the lowest rates (fewer than 1 of every 1,000 hi enrollees) were reported for: icd-9-cm code 03.09 (other exploration and decompression of the spinal cord). It should be noted that during this period, the place of service for most lens surgery shifted from inpatient settings to outpatient settings of hospitals or to free - standing surgical centers . For example: the surgical rate per 1,000 enrollees for icd-9-cm code 13.59 (other extracapsular extraction of lens) ranged from 0.2 in the west region to 2.4 in the northeast, a difference of 1100 percent . This variation may be in part the result of the relative scarcity of ambulatory surgical centers in the northeast region . Of all the ambulatory surgical centers (897) in the united states as of january 1988, only 97 centers were located in the northeast region . The surgical rate for icd-9-cm code 53.02 (repair of indirect inguinal hernia) ranged from 0.5 in the west region to 1.3 in the northeast, a difference of 160 percent . Similarly, the surgical rate per 1,000 enrollees for icd-9-cm code 53.01 (repair of direct inguinal hernia) varied 120 percent among the regions, ranging from 0.5 in the west region to 1.1 in the northeast region . Data presented in table 3 highlight regional variations in hospital total charges and average charges per discharge . In 1987, the ssh total charges for all inpatient surgical procedures performed on aged medicare hi enrollees amounted to $46.6 billion . More than 27 percent ($12.8 billion) of the total charges was for the 25 leading surgical procedures . The average charge per discharge for aged medicare beneficiaries discharged from sshs in 1987 ranged from $8,120 in the south region to $9,574 in the west region, a difference of 18 percent . Regional variations in the average charge per discharge were substantial for some of the leading surgical procedures . For example: for icd-9-cm code 36.14 (aortocoronary bypass of four or more coronary arteries), the highest average charge per discharge ($37,907) was recorded for the west region and was 36 percent higher than the lowest average charge ($27,881) for enrollees residing in the south region . A difference of 33 percent in the average charge per discharge was shown for icd-9-cm code 86.22 (excisional debridement of wound, infection, or burn), which ranged from $11,861 in the south region to $15,801 in the west . For icd-9-cm code 81.62 (other replacement of head of femur), the average charge per discharge ranged from $9,305 in the north central region to $12,223 in the northeast region, a difference of 31 percent . Icd-9-cm code 79.35 (open reduction of fracture with internal fixation of femur) had an average charge ranging from $9,384 in the north central region to $11,956 in the northeast, a difference of 27 percent . In table 4, we present the number of total days of care and the average length of stay (alos) for aged medicare beneficiaries with inpatient surgical procedures performed during 1987 . Differences in the alos per discharge among the census regions were substantial for many of the leading surgical procedures . For example: the alos per discharge for aged medicare beneficiaries with surgery ranged from 8.2 days in the west region to 12.2 days in the northeast, a difference of 49 percent . The largest regional variation in alos for the leading surgical procedures was shown for icd-9-cm code 60.2 (transurethral prostatectomy)an alos that ranged from 4.8 days in the west region to 8.9 days in the northeast, a difference of 85 percent . For icd-9-cm code 79.35 (open reduction of fracture with internal fixation, femur), the alos ranged from 11.7 days in the west region to 21.0 days in the northeast, a difference of 79 percent . Similarly, the alos for icd-9-cm code 81.62 (other replacement of head of femur) ranged from 12.0 days in the west region to 21.5 days in the northeast, a difference of 79 percent . Short - stay hospitalgeneral and special hospitals certified as participating facilities under medicare and reporting average stays of fewer than 25 days.dischargethe formal release of an inpatient from a hospital . All discharges including those persons who died during their hospitalization are included.hospital chargesthe hospital's charges for room, board, and ancillary services as recorded on the billing form (hcfa-1450).surgeryincludes any operative procedures recorded on the patient's billing form defined as surgery in the international classification of diseases, 9th revision, clinical modification, volume 3 . This includes procedures involving incision, excision, amputation, introduction, endoscopy, repair, destruction, suture, or manipulation . For the purposes of this article, only the procedures classified as operating room procedures by hcfa were selected to appear in the list of the 25 leading procedures.annual surgical rate per 1,000 enrolleesa ratio of the total number of discharges with inpatient surgery (multiplied by 1,000) to the number of persons entitled to benefits as of july 1 of that year . General and special hospitals certified as participating facilities under medicare and reporting average stays of fewer than 25 days . The formal release of an inpatient from a hospital . All discharges including those persons who died during their hospitalization are included the hospital's charges for room, board, and ancillary services as recorded on the billing form (hcfa-1450). Includes any operative procedures recorded on the patient's billing form defined as surgery in the international classification of diseases, 9th revision, clinical modification, volume 3 . This includes procedures involving incision, excision, amputation, introduction, endoscopy, repair, destruction, suture, or manipulation . For the purposes of this article, only the procedures classified as operating room procedures by hcfa were selected to appear in the list of the 25 leading procedures . A ratio of the total number of discharges with inpatient surgery (multiplied by 1,000) to the number of persons entitled to benefits as of july 1 of that year . The data shown in this article were derived from the health care financing administration (hcfa) short - stay hospital inpatient stay record file . This file is generated by linking information from three hcfa master program files for medicare beneficiaries . Thus, the statistical stay record provides information on the patient, the hospital, and the hospitalization . The data are based on short - stay hospital stay records contained in the 20-percent inpatient stay record file . Therefore, the data are subject to sampling variability . The data were extracted from the short - stay hospital inpatient records received and processed in hcfa as of december 1988 . Therefore, 1987 discharges recorded after that date were not included . The surgical procedure information recorded on the sample discharge records used to prepare this article were coded based on the international classification of diseases, 9th revision, clinical modification, volume 3 . Three- or four - digit codes were assigned for the principal surgical procedure of each sample bill record . The incompleteness of the medpar (medicare provider analysis and review) stay record files used to prepare this article is a result of the inherent administrative time lag between the time when a bill (hcfa-1450) is submitted for payment and when it is posted to the central records . A complete count of medicare discharges from short - stay hospitals in 1987 will probably amount to about 3 percent more than the total figures used in this study.
Hepatic stem - like (hsl) cells have a potential of self - renewal and an ability to differentiate to hepatocytes and biliary duct cells (nagai et al . (2005) described that hsl cells converted to pancreatic neuroendocrine cells when incubated with sodium butyrate (nab) and betacellulin . While many tissue stem cells are cultured in vitro in the medium with some growth factors, cytokines or extracellular matrix, hsl cells proliferate in dulbecco s modified eagle s medium (dmem) with 10% fetal calf serum (fcs) without differentiation or tumorigenesis . If the mechanisms underlying continuous proliferation of hsl cells without differentiation become clear, many tissue stem cells will be applicable to cell transplantation therapy for many types of diseases . A histone deacetylase inhibitor, nab, acts as a differentiation - promoting agent for a wide variety of cell types . In mice, nab induces differentiation of immature hepatocytes into mature parenchymal cells and reduces their proliferation (iwai et al . Recently, it was shown that human and murine embryonic stem cells were able to differentiate into hepatocyte - like cells by treatment with nab (zhou et al . When hsl cells were cultured with nab, they converted to flattened cells with large cytoplasm (yamada et al . There are many studies about nab induced differentiation, while there is little information about anti - proliferative effect of nab on hsl cells . To reveal the molecular mechanisms that sustain proliferative activity of hsl cells with pluripotency, it is necessary to identify the molecular event induced by nab treatment . In this study, we performed two - dimensional electrophoresis (2-de) to search for proteins statistically more abundant in undifferentiated hsl cells than in nab - treated cells, and identified those proteins by matrix - assisted laser desorption / ionization - time of flight mass spectrometry (maldi - tof - ms) analysis . These proteins might be participated in the molecular mechanism that causes the vigorous proliferative activity of the hsl cells . Hsl cells were cultured in dmem (asahi technoglass co., tokyo, japan) with 10% fcs (biofluids, rockville, md, usa) at 37 c in a 5% co2 atmosphere . An hsl pellet about 70 mg in weight (2 dishes of 100 mm culture dish, nearly confluent) were lysed in 350 l of extraction solution (8 m urea, 1 m thiourea, 0.5% 2-mercaptoethanol, 3% nonidet p-40) with 50 l of protease inhibitor cocktail (complete mini edta - free; roche diagnostics gmbh, mannheim, germany) by microhomogenizer (ina optica, osaka, japan). The homogenate was centrifuged at 15,000 g for 20 min, 4 c and the clear supernatant was subjected to the zoom strip isoelectric focusing (ief) gel . Total protein concentration of the supernatant was determined with coomassie protein assay kit (pierce, rockford, il, usa) according to the bradford method (bradford 1976). The ief gel strips (zoom strip ph 3 - 10nl, invitrogen corp ., carlsbad, ca, usa) were rehydrated at room temperature for 1 h with ca . 30 g of protein for analytical assay or 150 g of protein for in - gel digestion by trypsin . After rehydration, ief was carried out at 175 v for 20 min, followed by voltage ramping to 2,000 v for 45 min, focusing for 30 min, with total focusing for 1,800 vh . Following ief, the ipg strips were thawed and incubated for 15 min in 5 ml lithium dodesyl sulfate (lds) reducing solution containing 1.25 ml nupage lds sample buffer and 0.5 ml nupage sample reducing agent followed by incubation with s - alkylation solution containing 116 mg iodoacetamide and 1.25 ml nupage lds sample buffer for 15 min at room temperature . Finally, the ipg strips and precision plus unstained protein standards (bio - rad laboratories, hercules, ca, usa) were positioned on top of nupage 4 - 12% bis tris zoom gel (invitrogen corp ., gels were run in the xcell surelock mini - cell system (invitrogen corp ., carlsbad, ca, usa), at 200 v for 50 min . Following electrophoresis, the gels were stained using the deep purple total protein stain (amersham biosciences corp ., piscataway, gel images were acquired using the typhoon 9200 variable image analyzer (amersham biosciences corp ., the density of protein spots were calculated using image quant software (amersham biosciences corp ., the spots were selected randomly with the goal of detecting as many proteins as possible . The electrophoresis was repeated 10 times, the densities of 198 spots on each gel were compared using student s t - test, and differentially expressed spots (p preparative electrophoresed gel for in - gel tryptic digestion was fixed in 45% methanol and 10% acetic acid overnight and stained by coomassie brilliant blue r-250 . The proteins were washed, dehydrated and digested in situ within the gel with 1 l of 5 ng trypsin in 50 mm ammonium bicarbonate overnight at 37 c . The resulting peptides were extracted from the gel matrix with 3 l of 0.1% trifluoroacetic acid/50% acetonitrile . The gel pieces were extracted again with 3 l of 0.1% trifluoroacetic acid/50% acetonitrile . The supernatants were combined with the first extraction and the pooled digests were lyophilized and resuspended in 3 l of 0.1% trifluoroacetic acid . Following peptide extraction, the samples were prepared for maldi - tof - ms analysis with a ziptip c18 (millipore, billerica, ma, usa) according to manufacturer s instructions . The peptides were eluted into 3 l of 5% acetonitrile in water with 0.1% trifluoroacetic acid and spotted on the prespotted anchorchip (bruker daltonics inc ., the peptide mass spectra analyzed by auto flex maldi - tof - ms system (bruker daltonics inc ., billerica, ma, usa) were searched against the national center for biotechnology information (ncbi) nonredundant database using mascot search engine (perkins et al . 1999). Cultured hsl cells were washed three times with sterile ice cold phosphate buffered saline (pbs: 137 mm nacl, 2.7 mm kcl, 10 mm na2hpo4 12h2o, 2 mm kh2po4). Total rna was prepared from scraped hsl cells (10 cells) using micro - to - midi total rna purification system (invitrogen corp ., hsl cell cdna was prepared from total rna 5 g by revertra ace (toyobo co. ltd .,, 1 l of cdna solution was added by 5 l 2 gotaq green master mix (promega co., madison, wi, usa), 1 l of 10 m forward and reverse primers and 2 l nuclease free water . Pcr was made as below: pre - denaturation 95 c, 2 min, 30 cycles of denaturation 95 c, 30 s, annealing 60 c, 30 s, elongation 72 c, 3 min, and post - elongation 72 c, 10 min . The primers used were: ezrin forward; 5-cagagagtcatggaccagca-3, ezrin reverse; 5-cggggtcaacttgtcatctt-3, prohibitin forward; 5-ggcatgcctgagtagaccttg-3, prohibitin reverse; 5-tcacggttaagagggaatgg-3, vimentin forward; 5-agatcgatgtggacgtttcc-3, vimentin reverse; 5-cacctgtctccggtattcgt-3, annexin a3 forward; 5-ttgatgccaagcaactgaag-3, annexin a3 reverse; 5-caggctttcgtctcttccac-3, glyceraldehyde-3-phosphate dehydrogenase (gapdh) forward; 5-ccatcaccatcttccaggag-3, gapdh reverse; 5-cctgctcaccaccttcttg-3. The primer sequence was selected using amplified dnas were separated on 1% agarose gel electrophoresis and stained with 0.5 g / ml ethidium bromide . Hsl cells were cultured in dmem (asahi technoglass co., tokyo, japan) with 10% fcs (biofluids, rockville, md, usa) at 37 c in a 5% co2 atmosphere . An hsl pellet about 70 mg in weight (2 dishes of 100 mm culture dish, nearly confluent) were lysed in 350 l of extraction solution (8 m urea, 1 m thiourea, 0.5% 2-mercaptoethanol, 3% nonidet p-40) with 50 l of protease inhibitor cocktail (complete mini edta - free; roche diagnostics gmbh, mannheim, germany) by microhomogenizer (ina optica, osaka, japan). The homogenate was centrifuged at 15,000 g for 20 min, 4 c and the clear supernatant was subjected to the zoom strip isoelectric focusing (ief) gel . Total protein concentration of the supernatant was determined with coomassie protein assay kit (pierce, rockford, il, usa) according to the bradford method (bradford 1976). The ief gel strips (zoom strip ph 3 - 10nl, invitrogen corp ., carlsbad, ca, usa) were rehydrated at room temperature for 1 h with ca . 30 g of protein for analytical assay or 150 g of protein for in - gel digestion by trypsin . After rehydration, ief was carried out at 175 v for 20 min, followed by voltage ramping to 2,000 v for 45 min, focusing for 30 min, with total focusing for 1,800 vh . Following ief, the ipg strips were thawed and incubated for 15 min in 5 ml lithium dodesyl sulfate (lds) reducing solution containing 1.25 ml nupage lds sample buffer and 0.5 ml nupage sample reducing agent followed by incubation with s - alkylation solution containing 116 mg iodoacetamide and 1.25 ml nupage lds sample buffer for 15 min at room temperature . Finally, the ipg strips and precision plus unstained protein standards (bio - rad laboratories, hercules, ca, usa) were positioned on top of nupage 4 - 12% bis tris zoom gel (invitrogen corp ., carlsbad, ca, usa). Gels were run in the xcell surelock mini - cell system (invitrogen corp ., carlsbad, ca, usa), at 200 v for 50 min . Following electrophoresis, the gels were stained using the deep purple total protein stain (amersham biosciences corp ., piscataway, gel images were acquired using the typhoon 9200 variable image analyzer (amersham biosciences corp ., the density of protein spots were calculated using image quant software (amersham biosciences corp ., the spots were selected randomly with the goal of detecting as many proteins as possible . The electrophoresis was repeated 10 times, the densities of 198 spots on each gel were compared using student s t - test, and differentially expressed spots (p <0.05) were identified . Preparative electrophoresed gel for in - gel tryptic digestion was fixed in 45% methanol and 10% acetic acid overnight and stained by coomassie brilliant blue r-250 . The proteins were washed, dehydrated and digested in situ within the gel with 1 l of 5 ng trypsin in 50 mm ammonium bicarbonate overnight at 37 c . The resulting peptides were extracted from the gel matrix with 3 l of 0.1% trifluoroacetic acid/50% acetonitrile . The gel pieces were extracted again with 3 l of 0.1% trifluoroacetic acid/50% acetonitrile . The supernatants were combined with the first extraction and the pooled digests were lyophilized and resuspended in 3 l of 0.1% trifluoroacetic acid . Following peptide extraction, the samples were prepared for maldi - tof - ms analysis with a ziptip c18 (millipore, billerica, ma, usa) according to manufacturer s instructions . The peptides were eluted into 3 l of 5% acetonitrile in water with 0.1% trifluoroacetic acid and spotted on the prespotted anchorchip (bruker daltonics inc ., the peptide mass spectra analyzed by auto flex maldi - tof - ms system (bruker daltonics inc ., billerica, ma, usa) were searched against the national center for biotechnology information (ncbi) nonredundant database using mascot search engine (perkins et al . Cultured hsl cells were washed three times with sterile ice cold phosphate buffered saline (pbs: 137 mm nacl, 2.7 mm kcl, 10 mm na2hpo4 12h2o, 2 mm kh2po4). Total rna was prepared from scraped hsl cells (10 cells) using micro - to - midi total rna purification system (invitrogen corp ., hsl cell cdna was prepared from total rna 5 g by revertra ace (toyobo co. ltd ., osaka, japan) with oligo (dt)20 (toyobo co. ltd .,, 1 l of cdna solution was added by 5 l 2 gotaq green master mix (promega co., madison, wi, usa), 1 l of 10 m forward and reverse primers and 2 l nuclease free water . Pcr was made as below: pre - denaturation 95 c, 2 min, 30 cycles of denaturation 95 c, 30 s, annealing 60 c, 30 s, elongation 72 c, 3 min, and post - elongation 72 c, 10 min . The primers used were: ezrin forward; 5-cagagagtcatggaccagca-3, ezrin reverse; 5-cggggtcaacttgtcatctt-3, prohibitin forward; 5-ggcatgcctgagtagaccttg-3, prohibitin reverse; 5-tcacggttaagagggaatgg-3, vimentin forward; 5-agatcgatgtggacgtttcc-3, vimentin reverse; 5-cacctgtctccggtattcgt-3, annexin a3 forward; 5-ttgatgccaagcaactgaag-3, annexin a3 reverse; 5-caggctttcgtctcttccac-3, glyceraldehyde-3-phosphate dehydrogenase (gapdh) forward; 5-ccatcaccatcttccaggag-3, gapdh reverse; 5-cctgctcaccaccttcttg-3. The primer sequence was selected using primer3 software (rozen and skaletsky 2000). Gapdh was used as internal control as described before (miura et al . Amplified dnas were separated on 1% agarose gel electrophoresis and stained with 0.5 g / ml ethidium bromide . Hsl cells proliferated with a cobblestone appearance (fig . 1) and did not show any changes in shape spontaneously even in long - term culture (nagai et al . 2002). In this study, morphological changes occurred in response to 5 mm nab in some population of cells on dishes, and 20 mm nab was more effective (fig . 1). In the following study, hsl cells were incubated with 20 mm nab for 18 h. to clarify the proteins involved in proliferation of hsl cells, we compared the proteins expressed in hsl cells before and after nab treatment by 2-de . At the first glance, quite similar expression patterns with little difference were observed in both samples (fig . 2). We selected 198 spots on the gel, and the spot concentrations were calculated by image quant software . One was the ratio of the concentration of one spot to the total spot intensity of selected 198 spots (fig . The other was the ratio of the concentration of one spot to the randomly selected spot intensity as standard . The numbers of selected spots for standard were 21, 46, 57, 77 and 116 (fig . 3). We performed statistical analysis by using student s t - test and the spots with statistically different intensity (p <0.05) using all 6 data sets, were selected . We found 5 proteins up - regulated after nab treatment and 14 proteins down - regulated after nab treatment . The down - regulated proteins were the candidate proteins which might have some roles in the hsl cell proliferation . Then those proteins were in - gel digested by trypsin and analyzed by maldi - tof - ms . Mascot search identified 6 proteins, namely prohibitin, vimentin, ezrin, annexin a3, acidic ribosomal phosphoprotein p0 and grp75 (fig . We compared the protein spots on 2-de gels stained by deep purple fluorescence dye between control hsl cells extract and nab treated hsl cells extract, whereas the protein spots on the 2-de gels for maldi - tof - ms analysis were stained by coomassie brilliant blue . The amounts of the 8 proteins were too low even under deep purple staining to see under coomassie brilliant blue staining or detect by mass spectrometer . To subconfluent hsl cells cultured in dmem, final 2.5, 5, 10 or 20 mm nab were added and incubated for 0, 12, 15, 18, 21 and 24 h. cells were observed by phase - contrast microscope . Significant morphological changes were observed 18 h after 20 mm nab treatment hsl cellsfig . Cell extracts prepared from hsl cells (a) or nab treated hsl cells (b) were separated first by isoelectric focusing on ph gradient between 3 and 10, and then on 412% gradient polyacrylamide gel electrophoresis . Proteins were stained by fluorescent dye, deep purple, and detected by laser scanner typhoon 9200 as described in experimental procedure . The expression patterns were almost the same between control and nab - treated hsl cells . To obtain statistically reliable results, 3statistically conferred protein spots . To confer expression intensity between control and nab treated cells statistically, 198 spots were selected randomly . The spots, those intensity were used as standard, were shown as the number . The proteins down - regulated by nab treatment were identified by mascot searchtable 1the proteins identified by mascot searchspot no.giidentified proteinscoremasspicover (%) matched peptidetotal peptide2962664759prohibitin6027,7575.44439283551980303annexin a314736,5695.966321383611693176acidic ribosomal phosphoprotein p06134,3655.914810324414389299vimentin16053,7575.06643553501000439grp758573,9845.873619357917902245ezrin6254,2546.16391938gi: accession number, mass: calculated molecular weight, pi: calculated iso - electric point, total peptide: number of the peptide used to search the data base, matched peptide: number of the peptide contained the hit protein, cover: sequence cover ratio with detected peptide on complete amino acid sequence, score: highest hit score of several mascot searches morphological changes in hsl cells treated with nab . To subconfluent hsl cells cultured in dmem, final 2.5, 5, 10 or 20 mm nab were added and incubated for 0, 12, 15, 18, 21 and 24 h. cells were observed by phase - contrast microscope . Significant morphological changes were observed 18 h after 20 mm nab treatment hsl cells two - dimensional electrophoresis . Cell extracts prepared from hsl cells (a) or nab treated hsl cells (b) were separated first by isoelectric focusing on ph gradient between 3 and 10, and then on 412% gradient polyacrylamide gel electrophoresis . Proteins were stained by fluorescent dye, deep purple, and detected by laser scanner typhoon 9200 as described in experimental procedure . The expression patterns were almost the same between control and nab - treated hsl cells . To obtain statistically reliable results, 2-de was executed 10 times for each sample statistically conferred protein spots . To confer expression intensity between control and nab treated cells statistically the spots, those intensity were used as standard, were shown as the number . The proteins down - regulated by nab treatment were identified by mascot search the proteins identified by mascot search gi: accession number, mass: calculated molecular weight, pi: calculated iso - electric point, total peptide: number of the peptide used to search the data base, matched peptide: number of the peptide contained the hit protein, cover: sequence cover ratio with detected peptide on complete amino acid sequence, score: highest hit score of several mascot searches prohibitin is expressed in wide variety of tissues such as heart, liver and neurons . It has been shown that prohibitin localizes in mitochondrial inner membrane and may play a role in regulating mictochondrial respiration activity and in aging (rajalingam and rudol 2005; mishra et al . A recent study indicated that prohibitin is required for raf activation induced by ras, a downstream signal activated by epidermal growth factor (rajalingam et al . 2005). It is expected that prohibitin has some role in activating classical mitogen - activated protein (map) kinase cascade in intact hsl cells to continuous proliferation . Vimentin is the cell matrix proteins which constructs cell structures (clarke and allan 2002; wangi and stamenovic 2002). Vimentin is well known as an intermediate filament found in various nonepithelial cells, especially mesenchymal cells . It was also reported that vimentin interacted with a phosphorylated erk and protected it from dephosphorylation (perlson et al . (chen et al . 2007), in hsl cells, nab down regulates prohibitin and vimentin expression . Ezrin is thought to be involved in connections of major cytoskeletal structure of the plasma membrane (bretscher et al . The expression levels increase in the fetal rat gut epithelium between day 15 and day 20 of gestation and during the first week after birth (baril et al . Ezrin also serves as a substrate for protein tyrosine kinases and mediates adhesion - mediated events (srivastava et al . It is reasonable to suppose that those cell matrix proteins are involved in the alteration of morphology of hsl cells after 18 h culture with nab (fig . 1). Annexin a3 is one of the annexin family proteins that is described as an inhibitor of phospholipase a2, also possesses anti - coagulant properties . However, the biological role of annexin a3 is not totally clear . In the liver, annexin a3 rna interference study suggested that annexin a3 has a role in dna synthesis (niimi et al . Nab is known as an inhibitor of histone deacetylase that modulates the expression of genes by causing in histone acetylation (heinz et al . 2002). To confirm whether prohibitin, vimentin, ezrin and annexin a3 expression was regulated transcriptionally, 4, the mrna expression levels of the proteins selected were not changed 18 h after nab treatment . Because a slight difference in protein expression level was observed between in the control hsl cells and in the nab treated hsl cells, mrna transcription activity did not change dramatically . Other possible mechanisms were those, the protein degradation was stimulated, the expression level of the protein was regulated by translation or the protein was modified by some post - translational modifications . Total rnas were extracted from control hsl cells (c) or nab treated hsl cells (n). The sizes of the amplified bands of prohibitin, vimentin, ezrin and annexin a3 were about 200 bp as expected . No significant differences were observed between control and nab treated hsl cells expression of mrna for prohibitin, vimentin, ezrin and annexin a3 . Total rnas were extracted from control hsl cells (c) or nab treated hsl cells (n). The sizes of the amplified bands of prohibitin, vimentin, ezrin and annexin a3 were about 200 bp as expected . No significant differences were observed between control and nab treated hsl cells grp75 is known as mitochondrial stress-70 protein belonging to the heat shock protein 70 family (wadhwa et al . High level expression of grp75 in hsl cells suggested that hsl proliferation is controlled not only under growth signal but also under protection from aging . Acidic ribosomal phosphoprotein p0 forms a pentameric complex by interaction with dimers of p1 and p2, and constructs ribosomal large subunit ., our study demonstrates that the proteins regulating the map kinase cascade, cytoskeletons, chaperon and protein synthesis system, have some role to continue proliferation of hsl cells without differentiation . These results suggest that the map kinase cascade and related pathways are activated continuously in proliferating hsl cells . Further study is required to elucidate how these proteins regulate continuous proliferation of hsl cells.
Pancreatic cancer carries a challenging prognosis since it is most often diagnosed at an advanced stage of disease [1, 2]. At the time of diagnosis, 35% of patients have locally advanced pancreatic cancer (lapc)/stage iii disease with abutment or encasement of the celiac axis and/or the superior mesenteric artery (sma) and/or unreconstructable portal venous encasement [3, 4]. Recent options for attempts at enhanced local control have been external beam radiation therapy, high frequency ultrasound, and ethanol injections . Recently thermal based ablative techniques, radiofrequency ablation (rfa), and microwave have been used but have been found to induce a high incidence of injury to adjacent vital structures: blood vessels and nerves [611]. As a result, these techniques have not been shown to provide any benefit and are limited in their effectiveness for palliation [7, 8]. Irreversible electroporation (ire) is a nonthermal technique which utilizes electrical pulses to destroy cells by causing membrane permeability ultimately leading to apoptosis . Ire has been shown to have good efficacy and safety in the management of patients with lapc . Furthermore, the potential for improved survival rates has been reported in patients treated with ire ablation combined with chemotherapy and/or chemoradiation therapy compared with patients treated with chemotherapy or chemoradiation therapy without ire ablation . These promising findings have led to increased utilization of ire both intraoperatively and percutaneously [14, 15]. Since ire is relatively new to the field of locoregional therapy, post - ire imaging findings are scant [16, 17]. The purpose of this study is to characterize the expected computed tomography (ct) imaging findings in the surveillance of patients with locally advanced pancreatic cancer (lapc) treated with irreversible electroporation (ire). A review of our prospectively maintained soft tissue ablation registry from may 2010 to may 2013 was performed . Institutional review board approval and patient informed consent five representative patients with lapc treated with ire in situ (ire ablation without surgical resection for adenocarcinoma) were selected for this interim report . The ct imaging for these patients includes an immediate pre - ire scan (within 7 days of procedure), a discharge ct scan (approximately 710 days after procedure) then serial ct scans every 3 months for the next 2 years with serum ca19 - 9 . If the ct scan was equivocal or concerning for recurrence, a ct - pet scan was obtained for confirmation of the patient's current disease status . The selection of patients for this study was based on the availability of at least one preprocedural ct image and at least one or several surveillance ct images . This was necessary because, to discriminate between the target lesion and the initial postablation bed, preoperative images and immediate postoperative images (usually done within 1 month) were needed . By the same token, to compare changes in the ablation zone, the immediate postoperative scan and at least one - follow - up ct scan were needed . A three - phase ct scan was performed less than 7 days before ire and after induction chemotherapy to ensure the persistence of locally advanced pancreatic cancer and to rule out macroscopic metastatic disease that may have ensued in the interim (pancreatic cancer typically does not respond to induction chemotherapy). Also, patients with masses greater than 3.5 cm were excluded as candidates for therapy based on our institutional protocol . High definition intraoperative ultrasound was utilized to reassess tumor size and to confirm that the pancreatic tumor in question truly meets the criteria for lapc . Are introduced and spaced approximately 2 cm (0.3 cm) apart, bracketing the tumor with the adjacent vital structures . A 0.5 cm to 1 cm margin is included in the ire field . Ire is performed with the nanoknife system (angiodynamics, lanthan) utilizing 1,500 volts / cm for a delivery of> 90 pulses (pulse width 7090 ms). A repeat ultrasound with power doppler is performed to confirm preserved flow and patency to the vital structures . We decided to follow our patients with ct scan because there is better availability and cost benefit compared to pet - ct and mri . Standard workup for our patients includes a three - phase ct scan with pancreatic protocol at the time of diagnosis, which allows us to both diagnose and stage the patients appropriately . For most patients three - phase ct scan was also obtained in the immediate postoperative period (less than 1 month postoperatively) to assess the patency of vital structures and to establish a baseline of the postablation bed . No true ablation efficacy can be confirmed at this immediate ct scan since ongoing electroporation effects (most commonly cellular apoptosis) are seen in this immediate phase . All ct images were obtained with a helical scanner (somatom sensation 64, siemens) before and after a bolus injection of 100 ml of nonionic contrast (ioversol [optiray 350]; mallinckrodt inc ., hazelwood, missouri) intravenously at a rate of 3 ml / sec . After contrast injection, two spiral ct scans were obtained during the arterial phase and portal venous phase at 30 and 70 seconds, respectively, after the initiation of the injection . Precontrast and arterial phase acquisitions were performed on the upper abdomen to include the pancreas and liver; portal venous phase imaging included the abdomen and pelvis . Contiguously reconstructed sections were obtained through the pancreas (5 mm 5 mm for noncontrast and 2 mm 2 mm for arterial and venous); coronal reconstructions for each phase were performed . We do not believe in cross comparing ct scan to mri or ct scan to potential pet scan unless there are abnormal signs of recurrence; most of these patients were followed with serial triphasic ct scan alone and were not comprehensively evaluated with both ct and mri . Given that a majority of these patients did not have preoperative mri most commonly because of the metal biliary stents in place, we have found that triphasic ct scan is the most consistent, most reliable way to image this unique subset of patients who were treated with ire . Pre - ire and post - ire ct studies were reviewed by three radiologists (tvm, bs, and oa). These physicians were not involved with the ablation process and were blinded to the final clinical assessment and serum tests at the time of interpretation . Therefore, for the sake of description, we will describe the treated lesion in the immediate postablation scan as the ablation bed; the same area in subsequent surveillance studies will be termed the ablation zone . Post - ire imaging of the pancreas has been reported to be quite challenging due to the acute inflammatory changes seen from the first through tenth postoperative day . Further, an ongoing apoptotic process that persists for up to 68 weeks after electroporation has been reported [19, 20]. The lesions we measured had poorly defined margins, which made measurement cumbersome and may affect the accuracy and reproducibility of the measurement . Ablation success was defined as the ability to deliver the intraoperative therapy and within 3 months to have no evidence of macroscopic tumor by ct imaging . According to previously reported data, local recurrence after ablation most often presents as new tissue in the periphery of the treated lesion and/or apparent enlargement of the ablation defect, in other words a change in size or morphology from a previously those imaging criteria along with a rising ca 19 - 9 value and the clinical presentation (return of back pain, jaundice, or worsening gastric emptying) were used to determine incomplete electroporation or local recurrence . The arterial phase study provided the best images because it was easier to separate the relatively hypoattenuating ablation zone from the adjacent vasculature . Also, it provided the opportunity to scrutinize the adjacent vessels for any new or increasing stenosis . As figures 15 demonstrate, the immediate postablation bed and zone are invariably larger than the original ablated tumor . We remain descriptive because the entire bed was extremely difficult to measure owing to the amorphous, irregular nature of the ablation . Moreover, the ablated tissue is not within an encapsulated organ; therefore, the ablation zone does not have defined borders as seen after, for instance, liver ablation . Four patients that showed continued stable disease are highlighted in figures 1through 4 . An amorphous, hypoattenuating region with irregular shape persisted in subsequent ct scans in all patients with stable disease . Moreover, the ablation zone was typically smaller (due to decreased edema, hyperemia, and granulation tissue) than the immediate postablation bed in the following months and remained stable provided there was no recurrence . Along with persistent irregular shape the ablation zone showed increased tumor bulk and extension as well as new mass effect (new narrowing of a blood vessel). Enhancement of the ablative bed was variable and often showed increased enhancement in the three - month and longer follow - up images . As it is nonthermal, it is less susceptible to perfusion - mediated tissue cooling (heat sink) potentially allowing the ablative zone to be more predictable . . Locally advanced pancreatic cancer (lapc) is a good candidate to demonstrate this novel ablative technique because surgery and thermal ablative techniques are challenging in the patient subset . Since ablative therapies for pancreatic carcinoma are becoming quite prevalent, it is important that radiologists are aware of the imaging findings so as to better direct the referring physician . Some papers have been published on imaging findings after post - ire ablation of the liver [16, 17]; however, we cannot apply those principles to pancreatic cancer ablation . The main reason for this difference is the significant heterogeneity of the pancreatic tumor and tissue . Given the location, all stage pancreatic cancers are not surrounded by normal pancreatic parenchyma and the retroperitoneal tissue, surrounding vessels, and duodenum all create variability on imaging, which is further challenged by the electroporation effects . In our study, we found that the postablation bed is larger in volume than the initial mass . The postablation bed and zone appear irregular, amorphous, and hazy without margins or true boundaries . The ablation zone may decrease in size from the initial post - op bed to the initial surveillance study as the surrounding edema / fluid and inflammation resolve revealing the true ablation zone; however, as mentioned above, since there have been reports of an ongoing apoptotic process that persists up to 68 weeks after ablation, it will not be unusual to see some increase in volume in surveillance [19, 20]. Therefore, size is considered secondary in the ct evaluation for this reason and because the postablative bed / zone has poorly defined margins, making objective imaging assessment (size, attenuation) cumbersome . This may undoubtedly affect the accuracy and reproducibility of the measurement . Nonetheless, any increase in volume after stabilization of the postablation zone is considered worrisome for recurrence (figure 5). Other clues that may suggest recurrence are any new encasement or narrowing of adjacent vessels or any subjective extension of soft tissue outside the boundaries of the previously established baseline ablation zone . However, in patients that have undergone prior radiation therapy or undergo post - ire radiation therapy, persistent isolated narrowing (without other worrisome findings) is not always recurrence and must be followed with serial imaging, clinical evaluation, and ca19 - 9 serum tumor markers . Vessels within and adjacent to the ablation bed may show narrowing immediately after the procedure, but this should resolve or at least remain stable in subsequent studies . Often, if narrowing of a vessel is seen with the index tumor it will often persist after ablation . For equivocal cases, pet / ct may play a role in differentiating postablative changes from recurrence . First is our small sample size; however, the ongoing study is the single largest evaluation of this population to date . Second, it was difficult to make an objective imaging assessment for the reasons detailed above . We believe that this pictorial interim report has value because it is the first paper on this topic and may provide a foundation for future studies . Further, as we gather more data at our institution, we will publish our results when our findings mature . Another limitation was that the efficacy of the ablative therapy relies only on imaging characteristics . Our imaging findings are related to the presence or absence of macroscopic disease and there is no way to prove by imaging whether or not small volume disease remains at the ablation bed . However, what we cannot see through imaging cannot be treated; therefore our goal is to provide a guide as to when the imaging characteristics are worrisome enough to intervene . Despite these limitations, we still believe that this is a worthy introduction to the understanding of postablative pancreatic lesions . These therapies are becoming prevalent and it is important that we understand the imaging characteristics . It is difficult to predict if these postablative changes will apply to other ablative technologies such as rfa or microwave; therefore it will be interesting to compare our findings with subsequent studies or case series . We conclude that ct imaging may be a useful tool to assess post - ire ablation changes . Although it is not ideal, its wide availability makes it prudent that we understand its utility in this patient subset . It cannot be overstated that serial imaging over at least 26 months must be employed to detect recurrence by comparing with prior studies in conjunction with clinical and serum studies . A single ct image time point is not sufficient to document a recurrence in patients with locally advanced pancreatic cancer given the heterogeneity of the tissue being imaged, the ongoing ire effects, and the limitations of the modality as described in this paper . Further studies are needed to evaluate a more ideal imaging modality for this unique patient population.
The colonization of land by plants was a major event in plant evolution, transforming the environment on land.1,2 knowledge of the origin of land plants is a prerequisite for understanding the transition from the aquatic to the terrestrial habitat of plants . The green algae are basically divided into charophyte and chlorophyte algae, and it is agreed that the charophyte algae are the closest algal relatives of land plants.3 analyses of both morphological and molecular data have established that land plants evolved within charophyte algae more than 450 million years ago.4,5 the charophyte algae are mostly freshwater green algae with diverse morphologies, comprising six distinct groups: mesostigmatales, chlorokybales, klebsormidiales, charales, coleochaetales, and zygnematales . Of these, the latter three (charales, coleochaetales, and zygnematales) have been considered the ancestors of land plants (fig . However, which group of charophyte algae is most closely related to land plants has remained controversial over the past decade . In recent years, large amounts of molecular data are available and methodological developments are increasing at a fast pace, thus investigating the origin of land plants becomes more feasible and tractable . In this review, we integrate recent phylogenetic developments on the origin of land plants, discuss the limitations in the phylogenomics era, and provide potential directions for further research on the land plants origin . Next - generation sequencing techniques have changed the prospects for molecular evolution, and it is feasible to obtain more data at a reasonable cost . In the field of phylogenomics, which is the use of genomic data to establish and clarify evolutionary relationships, more data indeed are essential to accurately estimate phylogenetic trees (eg, reducing sampling error by increasing the amount of information; including new taxa that beak up long branches). However, it is certainly to be expected that deeper divergences will become increasingly difficult to address as we go further back in time, because the markov models we use for sequence evolution are expected to saturate and lose some information at the most ancient divergences.6 at shorter times, there are other potentially misleading processes happening with real populations, and a possible ancient rapid radiation at the time of terrestrial colonization by the descendants of charophyte algae7 could be a major factor impeding the accurate inference on the origin of land plants . Charales, perhaps the most developmentally complex green algae, were initially suggested in an earlier period as a sister group of land plants8 (fig . 1a), and the early molecular phylogenetic analyses using four (two plastid, one mitochondrial, and one nuclear) or six (four plastid, one mitochondrial, and one nuclear) genes uncovered this topology.9,10 this hypothesis was an appealing result, in that charales appeared to have similar morphologies and growth patterns to land plants, and it supported an evolutionary scenario toward increasing cellular complexity . However, the charales are macrophytic and coenocytic algae with multiple nuclei in large cells.11 in contrast, coleochaetales and zygnematales are true multicellular algae (with plasmodesmata) that have separate cells, each with a single nucleus . In this cytological sense, coleochaetales or zygnematales may represent more appropriate sister groups to land plants, based on the transition from unicellular to multicellular organization . Indeed, the genome - scale molecular data consistently reject the charales as sister to land plants and support alternative charophyte groups . Previous phylogenomic analyses of chloroplast genomes have yielded topology with coleochaetales as sister to land plants12,13 (fig . 1b), but the taxon sampling of charophyte algae from these analyses was limited, possibly resulting in a less reliable topology . In addition, if evolutionary models do not describe the biological properties of the data, then tree building can be incorrect.14,15 worst of all perhaps, while the use of more data could reduce sampling errors, it simultaneously makes systematic errors more apparent . Thus, not all phylogenetic problems can be easily resolved with genome - scale analyses, and more attention must be given to systematic errors when large datasets are used for phylogenetic inference . Considering both sampling and systematic errors in genome - scale data, zhong et al.16 reported three new chloroplast genomes from charophyte algae and used a site pattern sorting method17 as well as site- and time - heterogeneous models1820 to reduce both classes of errors and address the branching order among charophyte algae and land plants . The chloroplast phylogenomic results strongly rule out earlier hypotheses placing charales or coleochaetales as sister group to land plants . Furthermore, this analysis indicated that more realistic models have a better fit to the data with more confidence and better infer the origin of land plants . Cox et al.21 also supported the zygnematales closest to land plants by reducing the compositional bias in chloroplast - genome data . Because of the highly variable structure of algae mitochondria, there are few studies investigating the origin of land plants using mitochondrial genomes . Turmel et al.22 analyzed 40 mitochondrial protein - coding genes from charophyte algae, but did not clearly resolve the relationship among the zygnematales, coleochaetales, charales, and land plants . Recently, the multilocus nuclear data have been commonly used to infer the origin of land plants . Phylogenomic analyses of a large number of nuclear genes have supported topologies with either zygnematales23,24 or the branch subtending zygnematales and coleochaetales25,26 as closest to land plants . However, sparse taxon sampling of charophyte algae (6 taxa23, 8 taxa24, and 10 taxa25,26) from these nuclear genome analyses cannot yet unambiguously provide the accurate phylogenetic topology . To increase the taxon sampling, wickett et al.27 applied rna - seq technology to sequence 92 transcriptomes of green plants including 18 charophyte algae and found high support for a sister relationship between zygnematales and land plants . The large nuclear genomic data have been recently used to investigate land plant origins,2325 but there is considerable variation (relatively low probabilities) between gene trees from different nuclear genes . The concatenation method combines different genes into a single supergene tree that is then considered to be equivalent to the species tree . This method was suggested to give more accurate trees than a consensus approach that summarizes congruence among individual gene trees.28 the assumption of the concatenation method is that it assumes all genes have the same (or at least similar) gene trees,29,30 but it has become clear that individual gene trees appear to conflict with one another and gene tree heterogeneity is ubiquitous.31,32 thus, the concatenation method may yield misleading inferences of species relationships in the presence of a high level of gene tree heterogeneity.33,34 if selecting the genes with strong phylogenetic signals (high average internode support), concatenation method may still accurately reconstruct the specie tree.35 high gene tree heterogeneity from nuclear genes has been a significant issue in phylogenomics.31,36 there are many reasons for gene tree heterogeneity and gene trees versus species trees conflict, including horizontal gene transfer, natural selection, and incomplete lineage sorting (ils) (fig . 2a): it is well accepted within evolutionary studies that there is a continuum from individuals, populations, races, varieties, sibling species, species, species complexes, subgenera, genera, etc . Along this continuum we expect introgression and hybridization to be quite normal, even if these two processes decrease at deeper divergences.natural selection (fig . 2b): it has been generally assumed that most, if not all, mutations were neutral and that genetic drift was the dominant effect . In practice, we know very little about the factors of natural selection that might be operating in related lineages . If the mutational process is random (and is not related to any needs of the organism) and is occurring all the time, then there is no surprise if related lineages independently happen upon similar mutations that are advantageous.incomplete lineage sorting (fig . 2c): it takes time for two variants in a population to coalesce, especially for larger populations . The failure of two or more lineages in a population to coalesce leads to the possibility that at least one of the lineages coalesces first with a lineage from a less - closely related population.36 this factor is currently best studied and modeled as to why gene trees are distinct . The probability of inferring the wrong species tree due to ils has been calculated theoretically for four individual species,37 and later pamilo and nei38 confirmed that ils is a general case and proposed that adding more gene sequences will still provide the correct relationship 2a): it is well accepted within evolutionary studies that there is a continuum from individuals, populations, races, varieties, sibling species, species, species complexes, subgenera, genera, etc . Along this continuum we expect introgression and hybridization to be quite normal, even if these two processes decrease at deeper divergences 2b): it has been generally assumed that most, if not all, mutations were neutral and that genetic drift was the dominant effect . In practice, we know very little about the factors of natural selection that might be operating in related lineages . If the mutational process is random (and is not related to any needs of the organism) and is occurring all the time, then there is no surprise if related lineages independently happen upon similar mutations that are advantageous . 2c): it takes time for two variants in a population to coalesce, especially for larger populations . The failure of two or more lineages in a population to coalesce leads to the possibility that at least one of the lineages coalesces first with a lineage from a less - closely related population.36 this factor is currently best studied and modeled as to why gene trees are distinct . The probability of inferring the wrong species tree due to ils has been calculated theoretically for four individual species,37 and later pamilo and nei38 confirmed that ils is a general case and proposed that adding more gene sequences will still provide the correct relationship . In terms of investigating the origin of land plants, an ancient rapid radiation can lead to short internal and long external branches, which can increase the potential for both ils and gene tree heterogeneity . The multispecies coalescent model is designed to approximate variation in a species tree topology derived from ils, and it chooses ancestors from the population backward through time for multiple sequences but places some constraints on how recently the coalescences occur . Because gene trees are allowed to vary in the multispecies coalescent model, coalescent methods can consistently estimate species trees in spite of the presence of heterogeneous gene trees.3941 using a data set of 289 nuclear genes from 32 green plant taxa, zhong et al.42 applied the multispecies coalescent model for the first time to revisit the origin of land plants . In this study, the coalescent method across different subsets of data consistently suggested that the ancestors of zygnematales are the closest relatives of land plants (fig . In contrast, concatenation methods yield misleading inferences of species relationships in the presence of a high level of gene tree heterogeneity for the origin of land plants and support inconsistent relationships across different subsets . This analysis also shows that the multispecies coalescent model could greatly accommodate gene tree heterogeneity in deep - level phylogenies . Later, wickett et al.27 used similar coalescent methods with increasingly larger number of taxa and arrived at the same results . Thus, figure 1c appears the best estimate for the origin of land plants the zygnematales are the closest group to land plants . In molecular phylogenomics, markov models are used to describe substitutions among dna or protein sequences, and therefore to reconstruct phylogenetic trees and understand evolutionary events . When selecting the best model for specific data, there is always a balance between the oversimplified and overfitted models . Oversimplified models often describe the evolutionary property with only a few parameters and have the same model for all sites, possibly leading to biased conclusions . In contrast, evolutionary models that use too many parameters may have all sites to vary consistently in their rates and substitution types and overfit the data resulting in errors for estimating a large number of parameters . So it is important to evaluate whether the data can be adequately explained by evolutionary models and to identify the misfitting parts in the data . We anticipate that a goodness - of - fit test between models and data will become a standard step in phylogenomic analyses . Similarly, we suggest that the use of more complex (well - fitted) models that incorporate heterogeneity of the substitution process will significantly improve the accuracy of phylogenetic inference . Further, with the increase of genomic data, gene tree versus species tree incongruence is becoming even more obvious, implying that biological factors may lead to incorrect gene trees and blur the treelike relationships . Ils appears to be the main biological mechanism resulting in gene tree heterogeneity in empirical data sets, and the multispecies coalescent model should be considered as the useful tool to efficiently accommodate gene tree variation . In general, there has been little theoretical work on the ability of methods to recover deeper divergences (eg, origin of land plants), although we cannot say that it is impossible to recover very deep phylogeny accurately, neither has it been shown that we can . In the future, we need to better understand deeper and deeper phylogeny beyond the limit of markov models that were applied to primary sequences . We are now living in very exciting times, and the power of phylogenomics can be combined and integrated with many other aspects of biology to be able to study a wide range of questions . This has started that the origin of land plants is likely the ancestors of zygnematales . It appears to be the single - nucleus multicellular lineage of green algae (rather than the lineage of the charales) that led to the multicellular land plants . Most of the charophyte algae have motile sperms, but the current members of zygnematales do not have motile sperms, which is assumed to be a derived feature within them . This scenario implies that there is an independent loss of motile sperms that occurred in the sister lineage of land plants.43 we indeed need additional genome - scale data from some lineages of charophyte algae, especially breaking up some long branches . Given that congruence of results from multiple and independent lines of evidence is a key approach for the validation of phylogenetic estimation, it is also desirable to investigate which topologies are supported with indels, gene order, and retrotransposon data . We can also include cytological features on the optimal tree with sequence data and study the evolution of the cell structure of charophyte algae.
This cross - sectional descriptive study was conducted in the divisions of pediatric metabolism, ankara children s hospital, ankara, dokuz eylul university, izmir, krkkale university, krkkale and erzurum local research hospital, erzurum, turkey between january and july 2014 . Among 65 eligible families, 4 parents refused to participate due to unsuccessful contact . The respondents and non - respondents did not differ according to demographic features of the families . A total of 61 patients and 36 healthy controls with similar demographic characteristics were enrolled in the study . Most of them have been started their diet during the newborn period and were on phe - restricted diet for at least one year . Five of the children were diagnosed at the second months of life and started the diet . The exclusion of this study were patients who were using bh4 (sapropterin dihydrochloride) or large neutral amino acid medications, while most patients were receiving 0.20 - 0.40 g / kg / day natural protein with their diet; hyperphenylalaninemia patients with free diet; parents who had a chronic disease requiring dietary or medical treatments or having a child with chronic or acute disorder other than pku . We searched the pubmed database for articles using the following keywords phenylketonuria, depression, anxiety, diet, inborn errors of metabolism, parent, mother, father to reach the previous studies on this topic and to decide the methods of the study . After describing the materials content and the study protocol to the parents, one of them (mother / father) if the patient was taken to hospital with one of the parents, that parent became the person who completed the scales . When the patient was taken with both of the parents, the parent who became volunteer to complete the scales was selected . First section was on the sociodemographic characteristics of the child and the family (children s age, gender and dietary compliance, parent s age, gender, marital status, educational level, occupation). The second section was on the stressors affecting the family such as illnesses, experience of financial troubles due to household income, history of medical or psychiatric disease or drug usage, domestic losses and family problems . The third part was on the supply of specific formulas and low - protein products, experiences, and interactions with medical staff and their satisfaction . This is a self - report questionnaire consisting of 2 sub - scales each including 20 items evaluating the level of anxiety.19 the turkish validity and reliability studies were performed by oner and le compte20 in the turkish adult population . State anxiety describes the person s feelings at a particular time and under particular conditions, whereas trait anxiety is independent of conditions and reflects personality characteristics and general feelings.19 the responses to each item in the anxiety questionnaire are given a score from 1 to 4 . The total scores can be in range from 20 (lowest possible anxiety score) to 80 (highest possible anxiety score), with higher scores indicating more anxiety.20 although no cut - off indicating clinically significant levels of anxiety is established in the technical or administrative materials for the stai, cut - offs of 4021,22 and 4523,24 were used in prior studies . In our study, the scores 45 for stai - s was accepted as the presence of state and stai - t was accepted as the presence of trait anxiety . This inventory consists of 21 items, each rated on a 0 - 3 point likert scale, and reflects the presence and severity of depression.25 the questions of the inventory represent symptoms commonly associated with a depressive disorder such as mood, crying spells, guilt, self - hate, sleep and appetite disturbances, and weight loss . The highest score of this scale was 63 and the high total scores indicate a high severity level of depression . The turkish validity and reliability studies of this inventory were performed by hisli.26 the cut - off score for bdi was 17.26 the study protocol was designed in compliance with the declaration of helsinki . The study was started after the approval of the ethics committee of ankara children s education and research hospital . Statistical analyses were performed using the statistical package for social sciences version 12.0 (spss inc ., the mean was compared by using the student s t - test or mann - whitney u - test . The percentage was calculated in the presence and absence group by pearson s chi - square test . Correlation between continuous variables were categorized as low (correlation coefficient was between 0.100.29), moderate (between 0.30 - 0.49) and high (> 0.50) according to their correlation coefficient values . To identify the independent variables (neurological impairment of the patient, gender and educational level of the parents, and the difficulties in the supply of low - protein products due to the poverty) that contribute to depression and anxiety, multiple logistic regression analysis was performed and values were expressed as odds ratios (ors) and 95% confidence intervals (cis). This cross - sectional descriptive study was conducted in the divisions of pediatric metabolism, ankara children s hospital, ankara, dokuz eylul university, izmir, krkkale university, krkkale and erzurum local research hospital, erzurum, turkey between january and july 2014 . Among 65 eligible families, 4 parents refused to participate due to unsuccessful contact . The respondents and non - respondents did not differ according to demographic features of the families . A total of 61 patients and 36 healthy controls with similar demographic characteristics were enrolled in the study . Most of them have been started their diet during the newborn period and were on phe - restricted diet for at least one year . Five of the children were diagnosed at the second months of life and started the diet . The exclusion of this study were patients who were using bh4 (sapropterin dihydrochloride) or large neutral amino acid medications, while most patients were receiving 0.20 - 0.40 g / kg / day natural protein with their diet; hyperphenylalaninemia patients with free diet; parents who had a chronic disease requiring dietary or medical treatments or having a child with chronic or acute disorder other than pku . We searched the pubmed database for articles using the following keywords phenylketonuria, depression, anxiety, diet, inborn errors of metabolism, parent, mother, father to reach the previous studies on this topic and to decide the methods of the study . After describing the materials content and the study protocol to the parents, one of them (mother / father) if the patient was taken to hospital with one of the parents, that parent became the person who completed the scales . When the patient was taken with both of the parents, the parent who became volunteer to complete the scales was selected . First section was on the sociodemographic characteristics of the child and the family (children s age, gender and dietary compliance, parent s age, gender, marital status, educational level, occupation). The second section was on the stressors affecting the family such as illnesses, experience of financial troubles due to household income, history of medical or psychiatric disease or drug usage, domestic losses and family problems . The third part was on the supply of specific formulas and low - protein products, experiences, and interactions with medical staff and their satisfaction . This is a self - report questionnaire consisting of 2 sub - scales each including 20 items evaluating the level of anxiety.19 the turkish validity and reliability studies were performed by oner and le compte20 in the turkish adult population . State anxiety describes the person s feelings at a particular time and under particular conditions, whereas trait anxiety is independent of conditions and reflects personality characteristics and general feelings.19 the responses to each item in the anxiety questionnaire are given a score from 1 to 4 . The total scores can be in range from 20 (lowest possible anxiety score) to 80 (highest possible anxiety score), with higher scores indicating more anxiety.20 although no cut - off indicating clinically significant levels of anxiety is established in the technical or administrative materials for the stai, cut - offs of 4021,22 and 4523,24 were used in prior studies . In our study, the scores 45 for stai - s was accepted as the presence of state and stai - t was accepted as the presence of trait anxiety . This inventory consists of 21 items, each rated on a 0 - 3 point likert scale, and reflects the presence and severity of depression.25 the questions of the inventory represent symptoms commonly associated with a depressive disorder such as mood, crying spells, guilt, self - hate, sleep and appetite disturbances, and weight loss . The highest score of this scale was 63 and the high total scores indicate a high severity level of depression . The turkish validity and reliability studies of this inventory were performed by hisli.26 the cut - off score for bdi was 17.26 the study was started after the approval of the ethics committee of ankara children s education and research hospital . Statistical analyses were performed using the statistical package for social sciences version 12.0 (spss inc ., the mean was compared by using the student s t - test or mann - whitney u - test . The percentage was calculated in the presence and absence group by pearson s chi - square test . Correlation between continuous variables were categorized as low (correlation coefficient was between 0.100.29), moderate (between 0.30 - 0.49) and high (> 0.50) according to their correlation coefficient values . To identify the independent variables (neurological impairment of the patient, gender and educational level of the parents, and the difficulties in the supply of low - protein products due to the poverty) that contribute to depression and anxiety, multiple logistic regression analysis was performed and values were expressed as odds ratios (ors) and 95% confidence intervals (cis). There were no significant differences between the case and the control groups regarding the children s age and gender, parental age and gender, the educational level of the parents and number of children in the family (table 1). All parents were married; marital status was not included, m - male, f - female, pku - phenylketonuria depression and anxiety scores were significantly higher in the case group than controls (table 2). Mothers of pku patients had significantly higher depression, and trait- and state anxiety scores compared with the other parental groups (table 3). Fathers of control children had significantly lower trait anxiety scores compared with the other groups (table 3). Mental disorders and current state - trait anxiety inventory (stai) and beck depression inventory (bdi) scores of parents of the phenylketonuria (pku) patients and healthy controls . Bdi - scores of beck depression, stai - state - trait anxiety patients mother versus other groups, p<0.05 (mann - whitney u - test); healthy controls father versus other groups, p<0.05 (mann - whitney u - test) seven parents had more than one pku patients at home (2 families had 3 children and 5 families had 2 children with pku). Number of affected children in the family, age and gender of the patient were not related with the depression or anxiety scores of the case group . There was no correlation between parental anxiety and depression scores and patient s age (beck: r= -0.158, p=0.258, stai - s: r=0.173, p=0.178, stai - t: r=0.182, p=0.111). Parents with lower educational level had significantly higher depression and anxiety scores in the case group (table 4). Parents with mentally retarded children had significantly higher beck (21.08.9) and stai - s scores (46.77.0) than the parents of neurologically unaffected pku patients (10.27.9, p=0.001) and stai - s scores (36.39.1, p=0.001). The state - trait anxiety inventory scores were 47.37.2 in mentally retarded group and 42.26.4 in unaffected group and there were no significant differences between the 2 groups (p=0.062). Fourteen (22.9%) of 61 pku patients had 2 unemployed parents and their family income consisted of state aid . The vast majority (42 families, 68.8%) of families stated that it was a financial burden to supply of low - protein products because of inadequate or absent health insurance . Parents who failed to provide low - protein products had significantly higher beck (14.29.0), stai - s (41.58.9), and stai - t scores (44.86.3) than parents who easily provided low - protein products (beck [7.27.6] p=0.004, stai - s [31.27.6] p=0.000, and stai - t scores [39.46.5] p=0.01). Association of parental education levels and stai and bdi scores in phenylketonuria group . In the case group, the parental depression score was positively correlated with stai - s (r= 0.523) p=0.000 and stai - t scores (r=0.440), p=0.001 . As expected, stai - s score was correlated with stai - s score (r=0.516) p=0.000 . Many parents (58 parents, 95%) reported that their interactions with medical staff were excellent . Logistic regression analysis revealed that lower educational level of the parent was the only independent factor for parental depression (p=0.007) and state anxiety (p=0.030). Gender of the parent, difficulty in the supply of low - protein products and presence of mental retardation of the children were not independent risk factors for depression and anxiety in this model (table 5). Logistic regression analysis between risk factors for the presence of depression, state- and trait anxiety in phenylketonuria group . Depression and anxiety scores of pku patient parents were higher than the parents with healthy children . Moreover, mothers of these patients had significantly higher depression and anxiety scores than fathers . Parents with low educational level had higher depression and anxiety scores than parents with higher educational degree . The limits caused by the strict diet and the potential risk of mental retardation makes pku a burden for patients and their families, affecting their quality of life, and psychosocial well - being . Dietary restriction makes the diet monotonous.27 a monotonous diet of one family member was shown to restrict all family members feeding style, which could be one of the reasons of the maternal anxiety.27 in addition to this highly restrictive diet, possible factors contributing family stress may include restriction of daily activities, cognitive disabilities and learning problems of patient, regular hospital visits and frequent blood sampling procedures for monitoring phe levels and lastly, financial problems.6,14 one of the most striking results of this study was the significantly higher depression and anxiety scores of mothers than fathers of the pku patients . Similarly, in previous studies18,28,29 it was found that mothers had worse psychological and physical quality of life and higher anxiety and stress levels than fathers of children who treated with restrictive diet . Suffering from a chronic metabolic disease with the necessity of caregivers control of patient s diet often results in overprotection of patients by their parents.30 on the other hand, mothers traditionally have greater role in buying and preparing foods and therefore, to be a primary coordinator of family s sick child makes the mother more stressful and anxious compared with other family members.30 fathers often spend a significant part of their day at work (out of home) and possibly feel less disease - specific parenting stress compared with mothers.31,32 children s neurological impairment was found to be another important predictor of the parents depression and anxiety in our study . Previous studies33,34 detected higher levels of anxiety and depression scores of parents who had mentally disabled children when compared with controls . Moreover, more severe forms of retardation were related with higher levels of anxiety in the caregivers, especially in mothers.35 this association is caused by the child dependency on the mother for daily activities of children, for example, toileting, bathing, feeding, clothing and transport.35 besides all these intensive activities, preparing the highly restrictive diet increases the maternal caregiving hours and affect the quality of life in families with a pku patient . In addition, the caregiver s awareness on the irreversibility of the mental retardation of their sick child is another important causative factor for the parental stress.36 difficulties in the supply of low - protein products due to the low economical status of the family could be another important stress - related factor in our study . Management of a pku patient is both time - consuming and expensive for the family, even in developed countries.37,38 low - protein food products are indispensable part of the pku diet and they responsible 99% of this extra expenditure.38 only phe - free formulas are paid totally by our health insurance system,37 therefore, all families with moderate to low income are under pressure on the supply of other specific dietary requirements of their children, because of very limited reimbursement of these products . In our study, low educational level of the parents was associated with higher depression and anxiety scores . Moreover, logistic regression analysis showed that a decrease in the literacy level was the only independent factor for depression and anxiety in parents of pku children . Low educational level was found to be associated with the increased stress and depression rates both in general population and in caregivers of sick child.39 - 43 moreover, educational and cultural factors of the parents were also shown to be the main determinants of dietary compliance and good clinical outcomes in children with pku in some studies.7,44,45 relationship between educational degree and mood disorders is important especially in women.46 previous studies have shown that, because of having fewer socioeconomic resources such as power, authority and earnings, women appear to be especially dependent on education for emotional well - being.46 in our study, most of the responders was consisted of mothers (43 mothers, 70.4%), therefore our results were compatible with the literature.7,44 - 46 parental education is related with multiple sources of psycho - social support, positive home environments, particularly more positive and sensitive parenting, which are likely to be protective against depression.47 this study has some limitations . Firstly, the evaluations depended on self - reported questionnaires and scales, and lacked clinical interviews . A second limitation was the relatively small sample size of the patients and control groups . A prospective study can be made for showing the mood status of same parents in different age periods of their children . Lastly, we did not compare both fathers and mothers of each patients for mood disturbances . However, its strength is that; it can represent turkey as it was conducted in 4 different regions of this huge country . In conclusion, these findings add to the literature s surrounding cultural, social, and various differences such as age and gender in parental anxiety, and provide a basis for future research to identify the need for early preventive measures to combat the emotional and behavioral difficulties experienced by families of pku patients . Although there is no consensus on how to manage pku patients parents anxiety and depression, it is essential that the health care team should establish a supportive and continue therapeutic communication with families . In the light of the results of this study, a future study in order to explain and specify the origin of these difficulties should be conducted . On the other hand, regional services endowed with health staff aware of the cultural and environmental characteristics of the province should be established in order to support parents in surmounting difficulties of this chronic disease.
Implant placement in posterior maxillary jaw is more complicated because of lack available bone compared to mandible jaw bone . Success of implant in the posterior maxillary bone is complex because of type iii and iv bone . After the extraction of the tooth residual ridge resorption occur in buccolingual and occlusoapial direction . There is a significant decrease in the height of the bone available in the posterior section of the upper arcade . Consequently, we often end up with a residual bone height of <3 - 5 mm between the alveolar ridge of the crest and the sinus floor . It is difficult to place> 11 mm of implant in 2 - 3 mm bone without sinus lift in posterior maxillary bone . As such this area requires bone augmentation beneath sinus to increase vertical height of bone . There are currently two principle techniques of penetrating crestal bone and in order to elevate maxillary sinus depending upon the availability of bone height . The close window technique use variety of tools and materials such as bone grafts, sinus elevators, balloon, sinus condenser, sinus curettes, and collagen to lift the sinus . Lateral access window shows a more consolidation outcome in literature; however, it is very traumatic and complex technique to perform . The osteotome sinus floor elevation (osfe) procedure, introduced by summers may be less invasive, less time - consuming, cost - effective, and reduces postoperative discomfort to the patient . The procedure consists of elevating the schneiderian membrane with osteotomes through a crestal approach, placing simultaneously platelet - rich fibrin (prf), mineralized freeze - dried human bone allograft (mfdba), autogenous bone and the implant at the osteotomy site . These prf and bone grafts are thought to provide a cushion during membrane elevation and reduce sinus perforation . The use of prf and bone graft material during simultaneous sinus lift helps to promote natural bone regeneration . The key point of this new method is to maintain the schneiderian membrane in the highest possible position, increasing simultaneous thin residual bone height> 9 - 10 mm and width 1 - 2 mm . Although summers did not define a minimum presurgical residual bone height in the original article, other author have made recommendations ranging from 7 - 9 to 6 mm . The aim of this case report was to evaluate a modified osfe technique for increased posterior maxillary bone height and width using prf that is rich in growth factor, autogenous bone that is full of live endosteal osteoblast cells and mbdba that has osteoinductive property . A 67-year - old female patient reported to the department of periodontology and oral implantology with the chief complaint of having missing teeth in right and left side of the posterior maxilla . On enquiring about previous dental treatment, it was found out that the tooth was lost because of caries and periodontal disease . Preoperative computerized tomography (ct) scans were performed to obtain an accurate measurement of the bone before surgery . Immediate and 8 months postoperative ct scans were performed to check the proper placement and success rate of the implants, bone formation, and sinus membrane position . Patient had maxillary posterior bone - 1.49 mm on the right side and 1.47 mm on the left side between the alveolar crest and maxillary sinus as seen in figure 1a . Preoperative computerized tomography (ct) scan was taken and postoperative ct after 8 months of implant placement . (a) bone height present is 1.49 mm between membrane and ridge preoperatively (b) bone height presents 15.43 mm between membrane and ridge after 8 months of implant placement preoperative and postoperative computerized tomography presurgical preparation included medical, dental, and computerized axial tomography (cat) scan radiographic evaluations and basic dental therapy to alleviate preexisting medical - dental problems . Prior to implant surgery, informed consent for bone graft and sinus lifting of the implant, cat scan consent was obtained from the patient . Patient received 625 mg augmentin (amoxicillin and clavulanate potassium) twice in a day before surgery . Patient was treated with 5 mg of triazolam orally and then draped with sterile surgical barrier . Before a surgical procedure start, a full mouth prophylaxis was done on surgery patient . The posterior quadrant of the maxilla was anesthetized via local anesthesia injection 2% lidocaine hcl and epinephrine 1:100,000 in a 30-gauge needle . A direct, full thickness midcrestal incision with a #15 blade was made through the mucoperiosteum to the crest of the ridge . Full thickness reflection of buccal and palatal tissues exposed the alveolar ridge . To reflect the flap, the implant position was marked on the alveolar crest with a small trephine drill (2.0 mm). After locating the implant position, the preparation was widened with two sizes internally irrigated trephine (3.5 mm and 4.25 mm) drill . Minimal pilot drilling (2.0 mm) intra oral radiograph of the osteotomy site was taken to determine the position of the sinus membrane as seen in figure 2a . (a) preoperative radiograph before implant placement (b) bone grafting during surgery with an osteotome technique (c) eight months after implant placement at the same time, a 4 - 5 vials of blood was collected from the patient's vein, and blood is spun by a special machine called a centrifuge and spun for approximately 12 min . The drops of clindamycin and cefazolin are added into the pieces of the prf clot . These small pieces of the membrane are placed inside the osteotomy socket as a cushion during sinus lifting . A mixture of the mfdba were taken in the separate container saturated it with saline . After 10 min, the excess fluid was drained, and clindamycin and cefazolin powder added into it . Autogenous bone grafts from maxillary tuberosity area or bone removed during the site preparation from trephine were used to fill out the osteotomy site . Osteotome site was packed with bone and membrane after gradually adding bone particles and tapping it with osteotome and mallet . If bleeding does not occur from the site, it shows that perforation happened in the sinus membrane . Trial implant is placed inside the osteotome site to check adequate width of osteotome site . After checking with trial implant, ten to twelve small holes were made with surgical quarter round bur on the buccal surface of the posterior maxilla to initiate fast healing at the implant site . Crushed bone pieces and the rest of mfdba particles are placed around the implant mainly on the buccal surface . Occlusal clearance between maxillary ridge bone and mandibular teeth is <5 mm than cover screws are placed on the implant . After repositioning the soft tissues, primary closure was attained using 4 - 0 chromic gut suture . After 4 months, abutments were placed on the implants and restorative procedure was initiated . No pathologic conditions in the implants site were seen on radiographic follow - ups after 8 months as seen in figure 2c . Implants were stable at the time of abutment connection which was performed after a healing period of 3 - 4 months . Patient reported full satisfaction for function, phonetics, and esthetics . At the osteotomy site, combination use of prf and bone grafts, it served as a cushion below the maxillary sinus floor, reducing the risk of perforation of the sinus membrane . The elevation of the sinus membrane was one of the most delicate parts of the technique, and it was performed using osteotomes, with the prf and bone graft itself . The ct scan carried out 8 months postinsertion showed a dense mineralized bone surrounding the implants . In the case, it was difficult to delineate the border line between sinus floor and newly formed tissue . The original bone height below the sinus floor as measured on the preoperative ct scan was 1.47 and 1.49 mm at two sites and on second ct scan evaluation after 8 months postsurgery, the bone height achieved was of 15.42 mm and 16.94 respectively [figures 14]. (a) cross - sectional view of the site of the right molar showing approximately <2 mm bone height (b) cross - sectional view of the site of the right molar after 8 months showing approximately> 15 mm bone height (a) preoperative computerized tomography scan showing top view of the sinus membrane (b) postoperative computerized tomography scan after 8 months of implant placement showing sinus floor elevation without perforation and bone formation radiographic assessment of bone levels before and after implant placement between sinus membrane and ridge the ct scan carried out 8 months postinsertion showed a dense mineralized bone surrounding the implants . In the case, it was difficult to delineate the border line between sinus floor and newly formed tissue . The original bone height below the sinus floor as measured on the preoperative ct scan was 1.47 and 1.49 mm at two sites and on second ct scan evaluation after 8 months postsurgery, the bone height achieved was of 15.42 mm and 16.94 respectively [figures 14]. (a) cross - sectional view of the site of the right molar showing approximately <2 mm bone height (b) cross - sectional view of the site of the right molar after 8 months showing approximately> 15 mm bone height (a) preoperative computerized tomography scan showing top view of the sinus membrane (b) postoperative computerized tomography scan after 8 months of implant placement showing sinus floor elevation without perforation and bone formation radiographic assessment of bone levels before and after implant placement between sinus membrane and ridge summer's osteotomes modified techniques may allow performing a safe and effective osteotome - related sinus membrane elevation with simultaneous implant placement in posterior maxillary area, thereby drastically reducing the total treatment time, expense and also improve healing time . Sinus membrane perforations using a conventional osteotomy the proposal for using a material to weaken the impact of the sinus membrane from perforations was suggested by lazara et al. ;(1998) however, the type of material was not specified . Therefore, in the present clinical case the choice was to use prf and bone graft materials, mainly because its biocompatibility, resilience, and availability . The survival rate was 90% when autogenous bone alone was used, and the survival rate was 87% . When freeze - dried demineralized bone (fddb) bone alone was used . The survival 98% when fddb bone and autogenous bone were both used together . Based on data, the authors suggested implant survival rates can increase if the combinations of materials are used in an appropriate way . This method has proven to be highly predictable to gain> 10 mm bone height in posterior maxillary area . No complications have occurred in the patient treated thus far, and no implant failures have occurred . Compare with other technique, which requires minimum 6 - 9 months of healing, this technique typically need only 3 - 4 months of healing . Main advantage of this technique is that implant and osteotomy site gets blood supply from buccal, lingual, mesial, and distal surfaces of blood vessels while in lateral window technique implant and bone grafts gets its blood supply largely from buccal surfaces of blood vessels . In lateral window approach risks, the possibility of breaching the blood supply, since arteries supplying the area are situated very close to mucoperiosteal region of the potential window site . Second reason could be prf, which promotes bone regeneration and soft tissue healing, improving bonding between bone and implant surface . It also serves as protection barrier to the sinus membrane, it is much less expensive than commercial membrane . Prf may allow gentle elevation of membrane, and represent an excellent source of growth factors . The platelets and the growth factors included in the fluid released by membrane may locally enhance bone regeneration as they come in contact with schneiderian membrane . It also has consistent regenerative power as it combine with osteoprogenitor cells from mfdba and autogenous bone . Third reason could be autogenous bone graft from maxillary tuberosity area which has an osteogenic potential related to the number of surviving osteoblasts and potential osteoinductive effect brought about by the release of bone morphogenic proteins and other growth factors and it also accelerates the bone production sequence . Fourth reason could be mfdba, which gives the signal from their proteins to neighboring mesenchymal cells and differentiate them into bone producing cells . Several studies have demonstrated that the failure rate is higher when the bone crest is inferior to 5 mm . Nevertheless, according to li, the osteotomy technique can be used even in residual ridges with heights of 3 - 4 mm, if primary stability has been achieved . In the present clinical case, the height of remaining bone was 1 - 2 mm, implant success rate was higher . Further investigation is needed to establish the actual contribution of prf, bone graft materials at osteotomy site to the positive outcome obtained with this modified technique, and to determine whether less residual bone height in maxilla can be successfully achieved with this modified technique . The use of prf, mfdba, and autogenous bone during osfe technique and implantation is a secure and reliable option . This autologous and inexpensive material can be considered as appropriate materials for adequate natural bone regeneration and soft tissue healing . However, in this technique the alveolar bone ridge height and its width is of prime importance and considerations . The use of prf during a sinus lift, with a combination of mfdba and autogenous bone, may be beneficial, particularly for the posterior maxillary bone growth . This modified method reduced total treatment time, expense of the patients and should be analyzed in further studies.
The prevalence of lower urinary tract symptoms (luts) in men has been clearly shown to increase with age . Although the etiologies of luts in aged men are well understood, relatively little research effort has been devoted to studying luts in adolescents and young men . Obtaining a clinical diagnosis on the basis of the history of symptoms and physical examination alone is often not possible for chronic luts in adolescents and young men . However, detailed investigations are often not performed in the belief that they will be inconclusive . Therefore, diagnosis is often empiric, and most patients are diagnosed with nonbacterial prostatitis, simple overactive bladder syndrome, or psychogenic voiding dysfunction . Recent research has focused on bladder and urethral dysfunction as a cause of luts and pelvic pain in young men, and urodynamic investigation has enhanced awareness of possible etiologies of chronic luts in adolescents and young men . To date, some specific urodynamic etiologies have been described as a cause of chronic luts in adolescents and young men . Of these, primary bladder neck dysfunction (pbnd) has been reported to be the most frequent, followed by dysfunctional voiding (dv), detrusor overactivity (do), and detrusor underactivity (du)/acontractile detrusor (ad). Because the management strategy differs for each urodynamic diagnosis, accurate diagnosis with urodynamic testing may be helpful in counseling the patient on the most appropriate treatment . Previous studies concerning this issue considered only small populations and focused on young men who had previously been diagnosed with chronic prostatitis . We investigated the etiologies of luts and compared the urodynamic characteristics between different diagnostic groups in young men with chronic luts who did not have symptoms suggestive of chronic prostatitis . To the best of our knowledge, there have been no large studies of luts in young men without symptoms suggestive of chronic prostatitis . We reviewed the medical records of male patients aged 18 to 50 years who had undergone a urodynamic study for work - up of luts at our institution between may 2003 and may 2013 . All of the cases were reviewed, and patients who had experienced symptoms for less than 6 months and those with a neurogenic abnormality, diabetes mellitus, or interstitial cystitis that affected micturition function; a history of surgery on the lower urinary tract; anatomical deformation of the lower urinary tract such as urethral stricture; or impairment of general health that affected voiding, such as having recently undergone surgery, were excluded . In addition, patients with pelvic or inguinal pain or bacterial infection or more than 10 leukocytes in expressed prostatic secretions (eps) at any time before the urodynamic study were excluded from the study population . The initial evaluation of patients presenting with luts at our institution consists of taking a history of the luts and performing a physical examination, followed by having the patient document an international prostate symptom score (ipss), overactive bladder symptom score (since 2008), and a 3-day frequency - volume chart (fvc), including the urinary sensation scale at every voiding . Episodes of urgency were defined as those with a urinary sensation scale rating of 3 . Free uroflowmetry (daba, endo tech, seongnam, korea) and measurement of the postvoid residual volume (bladderscan bvi-3000, verathon inc ., bothell, wa, usa) were performed before urodynamic evaluation . The result showing a higher maximum flow rate (qmax) was selected from two sets of uroflowmetry measures with a voided volume over 150 ml . After discontinuation of all drugs that could possibly affect micturition function for at least 3 to 7 days, a multichannel urodynamic study (ud-2000, medical measures system b.v ., enschede, the netherlands), including a pressure - flow study (pfs), was carried out in accordance with the guidelines of the international continence society . A 6-fr double - lumen catheter and a 9-fr balloon catheter were used to measure the transurethral intravesical and abdominal pressures in all of the urodynamic studies . Pelvic floor electromyography was performed by using surface electrodes attached near the anus at the 3 and 9 o'clock positions . Intravesical pressure was recorded under conditions of room - temperature saline infusion at 50 ml / min . However, the filling rate was decreased to 20 ml / min in patients with severe storage symptoms or a lower functional bladder capacity according to the results of the 3-day fvc . Bladder compliance was considered reduced when the v/pdet was 20 ml / cm h2o . During the pfs, the patient was instructed to void in a standing or sitting position under quiet and relaxed circumstances . If the first voiding trial failed, an additional trial was performed to allow for the possibility that the failure was due to cortical inhibition . Because the present study was conducted retrospectively, the treatment efficacy data retrieved had not been collected according to uniform criteria . The clinical urinary symptoms were divided into 3 categories: storage luts (8 episodes of frequency per day, 2 episodes of nocturia per night, or 3 episodes of urgency or urgency urinary incontinence on a 3-day fvc), voiding luts (intermittency score 3, weak stream score 3, or straining score 3 on the ipss), and postmicturitional luts (incomplete emptying score 3 on the ipss). Pbnd, dv, du, and ad were considered to represent voiding phase dysfunction and do, small cystometric capacity, and reduced bladder sensation were considered to represent storage phase disorders . The urodynamic diagnosis was made on the basis of the retrospective interpretation by the two investigators (s.j.j and s.c.l .) Who were blinded to the patients' clinical characteristics . Pbnd was diagnosed if bladder outlet obstruction, defined as an abrams - griffith (ag) number of 40 or greater or 20 - 39.9 with a slope of the linear passive urethral resistance ratio of> 2 cm h2o / ml / s, where the ag number was calculated as the detrusor pressure at qmax (pdetqmax)-2qmax, was present concomitant with electromyography evidence of external sphincter relaxation, and neither urethral stricture nor prostatic enlargement was observed in urethrocystoscopy and transrectal ultrasonography (trus), which were performed only in suspected cases in our young population . Dv was diagnosed on the basis of the electromyography activity of the external sphincter / pelvic floor during voiding in the absence of abdominal straining . If dv was diagnosed during a pfs, a free uroflow measurement was performed in a private setting to identify undulating intermittent increases and decreases in flow . If patients exhibited dv in a pfs and free uroflow, we repeated the same procedure in an effort to reduce false positive results caused by performance anxiety or bashful voiding during the urodynamic evaluation . We performed an additional voiding cystourethrography (vcug) in cases with equivocal findings of pbnd or dv during pfs . Du was diagnosed when the ag number was less than 20 and the qmax was less than 12 ml / s during a pfs and no obstruction was recognized in urethrocystoscopy or trus . For the purpose of classification of cases with the absence of a detrusor contraction during 2 consecutive pfs, patients with a measureable uroflow during free uroflow were considered to have du and those who did not generate a measureable uroflow in free uroflow were regarded as having an ad . Patients were regarded as positive for idiopathic do if a spontaneous or provoked involuntary detrusor contraction was observed during the filling cystometry . Maximum cystometric capacity was defined as the volume at which the patient felt that he could no longer delay micturition and small bladder capacity was defined as a maximum cystometric capacity of <350 ml . Reduced bladder sensation was defined as a diminished bladder sensation during filling cystometry . After assessment of the urodynamic etiologies of luts, comparisons among specific urodynamic etiologies were analyzed by a one - way analysis of variance with scheffe's method for multiple comparisons or by linear by linear association depending on the type of variable . The collected data were presented as the meanstandard deviation or as the number (percentage). The ibm spss ver . 19.0 (ibm co., armonk, ny, usa) was used, and a 2-tailed p value of <0.05 was determined to indicate statistical significance . A total of 308 young men who did not meet any of the exclusion criteria were included for analysis . Mean age was 40.4 (10.1) years and mean symptom duration was 38.8 (49.2) months . Storage luts were present in 247 men (80.2%), voiding luts in 166 men (53.9%), and postmicturitional luts in 129 men (41.9%). Table 1 shows the urodynamic diagnoses categorized by voiding phase and storage phase dysfunctions in all patients . Seventy - nine patients (25.6%) had normal urodynamic findings . Among the total patient population, the incidence of pbnd was highest among those in their 30s (p=0.04) and that of do was highest among those in their 40s (p=0.03) (fig . Small bladder capacity was more prevalent among men under 40 years of age (p=0.03). Dv was more frequent among those under 40 years of age, but the difference was not significant (p=0.18). The clinicodemographic and urodynamic characteristics of men with urodynamically defined voiding phase and storage phase dysfunction are shown in tables 2, 3 . The demographics and types of clinical symptoms were not significantly different between the diagnostic groups except for storage luts in distinguishing the type of storage urodynamic dysfunctions . Whereas 53.9% of patients (103/191) with voiding phase dysfunction had concomitant storage dysfunction (table 2), 69.6% (78/112) of those with storage dysfunction sixty - five and 73.2% of patients with dv and do had concomitant other storage phase and voiding phase dysfunctions (p<0.01). Qmax did not differ significantly between men with dv and those with normal urodynamics, but was significantly lower in men with pbnd or du / ad than in those with normal urodynamics . Men with dv or du / ad exhibited lower maximum cystometric capacity than did those with normal urodynamics (p<0.001, respectively). Of the storage phase dysfunctions, only small bladder capacity was associated with a lower free qmax than in men with normal urodynamics; however, the qmax during a pfs was not significantly different . The free postvoid residual was higher in patients with do than in those with small bladder capacity . Men with do were shown to have higher detrusor pressure during voiding than that in men with other storage phase dysfunctions . Medications were administered in 83.1% of men and -blockers were the most frequently prescribed, followed by anticholinergics, muscle relaxants, and cholinergics . Interestingly, no treatment was chosen in 46.8% of men with normal urodynamics, as abnormal findings were not demonstrated in their urodynamic evaluation despite their symptoms . However, the other urodynamically normal patients received treatment for luts . Although chronic luts are not uncommon in young men, they have received little scientific attention . Because the incidence and clinical implications of each urodynamic etiology for chronic luts of young men remain largely unknown, the proper diagnosis and management of each condition is challenging for physicians . Furthermore, these young men frequently experience the recurrence of symptoms despite various medications such as antibiotics, -blockers, and anticholinergics . Our study showed that most demographics and types of clinical symptoms did not differ among patients with specific urodynamic etiologies of luts . Insisted that in young men with pbnd, dv, do, or du, clinical features frequently overlap and are not as defining as they are often presumed to be . Therefore, treatment of young men on the basis of a presumed diagnosis from presenting luts is less likely to be successful, because the sensitivity of luts for the prediction of any specific urodynamically defined condition is not strong . Urodynamic investigation seems to be justified in adolescents and young men with chronic luts to confirm the correct diagnosis and avoid unnecessary and ineffective treatment . The incidence of pbnd has been reported as 40% to 50% among young men with refractory luts . Table 4 compares the incidence of each urodynamic etiology between our study and previously published studies . In our study, incidences of pbnd and dv were 26% and 23.4%, showing a relatively lower rate among young men with chronic luts . We believe that this was because our population differed from those in the previous studies . Unlike previous studies that focused on men who had been previously diagnosed with nonbacterial chronic prostatitis, we studied young men with chronic luts who did not have positive eps findings or pelvic or perineal pain responsible for nonbacterial chronic prostatitis . . The etiology of nonbacterial chronic prostatitis may be similar to the etiology of luts in young men . Abnormalities of pelvic floor muscle relaxation and poor relaxation of bladder neck during voiding have been suggested as the etiology of nonbacterial chronic prostatitis and chronic luts in adolescents and young men . Therefore, young men with nonbacterial chronic prostatitis appear to have more voiding phase dysfunctions than storage phase dysfunctions . Our results demonstrated that pbnd was the most frequent single specific dysfunction, followed by dv and du / ad . As a whole, pbnd and dv accounted for about 50% of the specific diagnoses . While approximately half of patients with voiding phase dysfunction had concomitant storage dysfunction, storage phase dysfunction similarly, nitti et al . Reported that the majority of men with do (85%) also had voiding phase abnormalities and stated that primary do without voiding phase dysfunction would appear to be unusual in young men . Men with dv or du / ad exhibited lower maximum cystometric capacity than that in men with normal urodynamics, and men with do displayed higher detrusor pressure during voiding than did those with other storage dysfunction . Although the plausible reasons for these findings need more investigations, similar results were demonstrated in our previous research with female overactive bladder patients . The etiology of du in aged persons is degeneration of muscle cells and axons . However, the mechanism of du in young men is not fully understood . Therefore, it is somewhat interesting to find that 12.7% of our young men had du / ad . While cholinergic agonists may be used to facilitate voiding efficacy in aged patients who have du without bladder outlet obstruction, the impact of such treatment on voiding efficacy has not been explored in young men with du without neurogenic abnormalities . The reported prevalence of do among young men with refractory luts is found to be 6% to 18% . Although the diagnosis of overactive bladder syndrome is based on subjective symptoms rather than objective criteria, overactive bladder syndrome is frequently described clinically as a condition associated with urodynamic do, with the latter potentially responsible for the clinical symptoms . In general, urodynamic do is detected in approximately 50% of women with symptoms of overactive bladder . In the present study, first, the present study was a retrospective study and therefore had several potential shortcomings, in particular being prone to several forms of selection bias . Second, we did not perform fluoroscopic assessment when diagnosing pbnd or dv . The robust method for the diagnosis of pbnd may be video - urodynamic . However, pbnd can also be determined indirectly by the urodynamic findings of bladder outlet obstruction with obstructive symptoms in the absence of urethral stricture, prostatic enlargement, and striated sphincter dyssynergia . Our patients were aged 50 years or less and we performed an additional vcug in cases with equivocal findings of pbnd during a pfs (26 cases). The diagnosis of pbnd was eventually made when vcug demonstrated narrowing only at the bladder neck . Similarly, for the cases with equivocal results for the diagnosis of dv in a pfs (13 cases), dv was finally diagnosed when vcug demonstrated brief and intermittent closing at the level of the membranous urethra during voiding . Third, for the purpose of classification of cases with the absence of a detrusor contraction during two consecutive pfs, patients with a measureable uroflow in free uroflow were arbitrarily considered to have du, and those who did not generate a measureable uroflow in free uroflow were regarded as having an ad . However, the number of these patients was small (6.0%). We recommend that it is essential to perform urodynamic evaluation to investigate the possible etiologies of luts in young men with chronic luts, especially in those with refractory symptoms . A high index of suspicion for possible etiologies for luts may be important for accurate and timely diagnosis of treatable luts in young men . We need to learn more about the natural history of pbnd, dv, and other possible etiologies of luts in young men . Chronic luts among young men have a variety of underlying etiologies . As a single specific dysfunction, urodynamic investigation in this population is helpful in making an accurate diagnosis and may guide adequate treatment, because clinical symptoms are not useful in predicting a specific urodynamic etiology.
The immune response in metastatic melanoma is not well known and this makes it difficult to use therapeutic approaches to stimulate the response against the tumor very hard . This case is of importance because it shows a spontaneous immune response in situ around the tumor that can give some insights to try to improve the arsenal against metastatic melanoma . The lesion had been present for at least 4 years, and become painful 4 months before presentation . Physical examination revealed a dermal mass, without pigmentation and mildly tender . A skin excisional biopsy for hematoxylin and eosin (h and e) staining, as well as immunohistochemical analysis was performed . The patient was then examined using radiological studies, and follow - up surgery was performed on the primary tumor site and on sentinel lymph nodes . Immunohistochemistry (ihc) analysis was performed as previously described. [13] staining was performed with antibodies to s-100, hmb-45, mart-1/melan - a / cd63, pnl2, tyrosinase, cd8, cd45, d2 - 40, proliferating cell nuclear antigen (pcna), antihuman plasma cell, antibody myeloid histoid antigen, imp3, insulin - like growth factor ii, mrna binding protein 3, bromodeoxyurine, topoisomerase ii alpha, cyclin d1, brca1, p21, p27, p53, epidermal growth factor receptor (egfr), cytokeratin ae1/ae3, and von willebrand factor . The epidermal findings were histologically unremarkable . Within the dermis and subcutaneous adipose tissues, a single, well circumscribed, nodular proliferation of atypical melanocytes was present . Within the proliferation, individual atypical melanocytes were of small to large size, and displayed epithelioid and spindled / fusiform morphologies; these cells displayed variable cytologic atypia . The atypical melanocytes contained round to oval nuclei with coarse, clumped chromatin and prominent, eosinophilic nucleoli . No ulceration, or satellitosis was appreciated [figure 1]. The lesional breslow thickness measured at approximately 11 mm; frequent tumoral mitoses were quantified at approximately 12 mitoses / mm . A melanoma clinical stage of iib ihc stain shown diffusely positive, membranous and cytoplasmic staining was noted on the tumor cells on review of the s-100, mart-1/melan a / cd63, pnl2, hmb45 and tyrosinase special stains . Focally positive, membranous and cytoplasmic staining was noted on these cells on review of the cyclin d1 and p53 special stains . Tumoral dermal lymphatic invasion and tumoral lymphatic angiogenesis were appreciated on review of the d2 - 40, von willembrand, cd31 and cd34 special stain [figure 1]. Around the central tumor mass, tyrosinase, pnl2, pcna, and hmb-45 were focally positive as single cells, and nests of cells . Topoisomerase ii alpha was well as p53, cyclin d1 and brca1 were also focally positive in areas immediately surrounding the tumor [figure 1]. Bromodeoxyurine was negative within the tumor but positive in one spot in the normal epidermis above the tumor . Staining for cd8, cd45, and myeloid histoid antigen were positive around and between the melanoma cells . Metastatic staging workup included a chest radiograph and positron emission tomography / computed tomography (pet / ct) scan; both failed to demonstrate tumoral masses . The patient underwent lymphoscintillography using tc99-m sulfur colloid on the day of surgery, highlighting three sentinel nodes in the right axilla . Axillary sentinel node biopsies were negative for metastatic disease by s-100 and mart-1/melan a / cd63 staining, and no residual melanoma disease to be present in the right forearm . Shows the h and e, as well as some positive ihc staining of the melanoma as well as the cells around the tumor . Upper column left to right: positive mart 1, cd45, and cd8 (dark staining). Our case was challenging, bringing together a multidisciplinary group of physicians to study and treat the clinical, immunopathological and radiological features of this dermal melanoma mass . The (1) lack of any apparent metastatic tumor primary site, (2) lack of disease within junctional melanocytes of suprajacent skin above the tumor, and (3) the presence of cd8, cd45 and myeloid histoid antigen positive cells surrounding the tumor cells represented unusual case features . The dermal foci suggestive of tumoral vascular and lymphatic invasion favored a clinical diagnosis of metastatic melanoma over that of primary dermal melanoma. [47] our findings represent novel discoveries in the immune response in situ and may suggest that the spontaneous immune response we detected could already attack and destroy the primary melanoma that we were unable to find . This may explain why no primary distant site melanoma was found despite extensive studies . As noted, we found positive staining with cd8/cd45 and myeloid histoid antigen antibodies surrounding tumor cells, forming a silhouette - shadow pattern relative to the melanoma cells . We are not sure if this staining pattern could be related to previously reported cannibalism of live lymphocytes by human metastatic (but not primary) melanoma cells . Such cannibalism has been described in other malignant tumors; its biological significance is still largely unknown . Further, possible cannibalism of melanoma cells by cd8, cd45 and myeloid histoid antigen positive cells has never been documented in primary malignant melanoma . These ihc findings created arabesques figures to form a we conclude that it is possible that in some metastatic melanomas can occur some type of in vivo recruitment of immune cells including cd45/cd8 and myeloid histiocytic antigen cells and plasma cell antibody cells that may represent a specific type of immune response against this tumor, and this may lead to the potential of a systemic and or local therapeutic response.
Is important during orthodontic treatment planning, which will ultimately affect patients satisfaction of treatment results . The desire to improve facial esthetics is one of the main reasons people seek treatment by an orthodontist or maxillofacial / plastic surgeon . Adolescents and young adults are major part of orthodontic patients with typical age characteristics for their psychological behavior . Some studies have reported that psychological factors have certain impacts on the perception of dentofacial esthetics . Self - perception of facial esthetics is the main reason for adolescents and young adults to undergo orthodontic treatment . Despite this, sometimes, patients are convinced to undergo orthodontic treatment based on orthodontic judgment of their clinician . However, patients perception of an attractive profile may differ from that of the clinician . The aim of this study was to evaluate self - perception and satisfaction of the facial profile among male adolescents and adults . Overall, 84 male patients referred to the orthodontic department of mashhad dental school participated in this study . The patients were divided into two groups: 39 adolescents (1318 years old) and 45 adults (above 18 years old). Patients with severe crowding and history of orthognathic surgery, congenital anomalies, physical and emotional abnormalities, psychological disorders, and trauma were excluded from the study . Patients were assigned to three groups: straight, convex, or concave profile based on the angle of convexity of their facial profile (g - sn - pog) according to the jacobson's soft tissue analysis . The angle of convexity of 816 indicates a straight profile while an increased or decreased angle is an indication for convex or concave profile, respectively [figure 1]. To determine the position of the maxillary or mandibular soft tissue, a constructed horizontal plane (chp) was drawn through the distance of the subnasal (sn) and pog from this vertical line determined the position of maxilla and mandible, respectively . The average distance of sn from this vertical line should be 6 3 mm and 0 4 mm for pog. A line was drawn from sn to pog to evaluate maxillary and mandibular dentoalveolar position . For facial balance, the most prominent point of the upper lip (ls) should be 24 mm anterior to this line . Likewise, the most prominent point of the lower lip (li) should be 13 mm anterior to the line . After signing a consent form, a questionnaire was filled by patients to assess their satisfaction with their own profile . The reliability of the questionnaire was also confirmed by cronbach's alpha coefficient (= 0.85). The questionnaire contained five questions, and the patients were asked to assign a number of 15 to each question; one represented the least satisfaction while five reflected the highest satisfaction with the profile . One ideal silhouette of the lower facial profile based on jacobson's soft tissue analysis, was designed by adobe photoshop cs2 software (kansas, usa). Then, the position of the maxillary, and mandibular soft tissue, and also upper and lower lip was changed in 2 mm increments by photoshop and eight silhouettes representing different relations of maxilla and mandible were constructed [figure 2]. The patients were then asked to assign a number of 110 to rank the profiles in the order of their attractiveness (1 = the least attractive and 10 = the most attractive), and to choose a silhouette that best closely resembled their own profile . Cephalometric tracings were evaluated independently by two orthodontists to determine the accuracy of the subjects in describing their own profile based on the silhouettes . (1) ideal profile, (2) prognathic maxilla, (3) retrusive maxilla, (4) bidentoalveolar protrusion, (5) bidentoalveolar retrusion, (6) prognathic mandible, (7) retrognathic mandible, (8) retrognathic maxilla, prognathic mandible, (and 9) prognathic maxilla, retrognathic mandible the dimensions of all silhouettes were similar (21 cm 29.7 cm) to minimize the risk of bias . The anova was used to compare the satisfaction of profile among the three groups . The independent t - test was also used to compare scores of satisfaction with profile in the adult and adolescent groups (= 0.05) patients were assigned to three groups: straight, convex, or concave profile based on the angle of convexity of their facial profile (g - sn - pog) according to the jacobson's soft tissue analysis . The angle of convexity of 816 indicates a straight profile while an increased or decreased angle is an indication for convex or concave profile, respectively [figure 1]. To determine the position of the maxillary or mandibular soft tissue, a constructed horizontal plane (chp) the distance of the subnasal (sn) and pog from this vertical line determined the position of maxilla and mandible, respectively . The average distance of sn from this vertical line should be 6 3 mm and 0 4 mm for pog. A line was drawn from sn to pog to evaluate maxillary and mandibular dentoalveolar position . For facial balance, the most prominent point of the upper lip (ls) should be 24 mm anterior to this line . Likewise, the most prominent point of the lower lip (li) should be 13 mm anterior to the line . After signing a consent form, a questionnaire was filled by patients to assess their satisfaction with their own profile . The reliability of the questionnaire was also confirmed by cronbach's alpha coefficient (= 0.85). The questionnaire contained five questions, and the patients were asked to assign a number of 15 to each question; one represented the least satisfaction while five reflected the highest satisfaction with the profile . One ideal silhouette of the lower facial profile based on jacobson's soft tissue analysis, was designed by adobe photoshop cs2 software (kansas, usa). Then, the position of the maxillary, and mandibular soft tissue, and also upper and lower lip was changed in 2 mm increments by photoshop and eight silhouettes representing different relations of maxilla and mandible were constructed [figure 2]. The patients were then asked to assign a number of 110 to rank the profiles in the order of their attractiveness (1 = the least attractive and 10 = the most attractive), and to choose a silhouette that best closely resembled their own profile . Cephalometric tracings were evaluated independently by two orthodontists to determine the accuracy of the subjects in describing their own profile based on the silhouettes . (1) ideal profile, (2) prognathic maxilla, (3) retrusive maxilla, (4) bidentoalveolar protrusion, (5) bidentoalveolar retrusion, (6) prognathic mandible, (7) retrognathic mandible, (8) retrognathic maxilla, prognathic mandible, (and 9) prognathic maxilla, retrognathic mandible the dimensions of all silhouettes were similar (21 cm 29.7 cm) to minimize the risk of bias . The anova was used to compare the satisfaction of profile among the three groups . The independent t - test was also used to compare scores of satisfaction with profile in the adult and adolescent groups (= 0.05) in adolescents and adults, patients with straight and concave profile showed the greatest and the least satisfaction with their own profile, respectively [table 1]. The anova test showed a significant difference in satisfaction of patients own profile among adolescents with different types of profiles (p = 0.004, f = 6.4). The post hoc tukey test showed that adolescents with straight profile were significantly more satisfied with their profile compared to patients with convex (p = 0.036) and concave profiles (p = 0.001). The satisfaction with ones own profile in adolescents with convex profile was also significantly greater than patients with the concave profile (p = 0.045). The score of patients satisfaction with their own profile classified based on facial angle into convex, concave, and straight the anova test did not show a significant difference in satisfaction of own profile among adults with different types of profiles (p = 0.062, f = 2.8). The independent t - test found no significant difference in ranking of facial profile between adults and adolescents with different types of profiles regarding satisfaction of their own profiles [table 2]. Comparison of satisfaction of profile in adolescents and adults both adults and adolescents selected retrognathic maxilla and prognathic mandible as the least attractive profile . Straight and bimaxillary dentoalveolar retrusion were chosen as the most attractive silhouettes in adolescents and adults, respectively [table 3]. Profile attractiveness based on adolescents opinion (scored 1 - 10) esthetic perception of the profile was not significantly different among the adolescent with three different types of profile (p = 0.24, f = 1.4). However, adults with different profiles chose different silhouettes as the most attractive ones (p = 0.04, f = 3.3). The post hoc test showed that there was a statistically significant difference between straight and convex profile adults (p = 0.04), and also between convex and concave profile groups (p = 0.03). The independent t - test showed no significant difference between adults and adolescents with different type of profiles in selecting the most attractive silhouettes (p> 0.05). Only 42.9% of adolescents and 22% of adults were able to correctly diagnose the silhouette that best closely resembled their own profile [table 4]. Adolescents with concave and straight profiles had the greatest and the least percentage of correct diagnosis of their own profile, respectively . However, the difference was not statistically significant (p = 0.63). In contrast, adults with straight profiles were more accurate in diagnosing their own profile, while adults with concave profile had the least percentage of correct diagnosis . Distribution of patients according to ability of diagnosis a silhouette that closely resembled their profile the chi - square test showed that adolescents were more accurate in selecting their own profile in comparison to adult patients (p = 0.044, df = 2). The correct selection among patients with straight and convex profile was not significantly different between adult and adolescent groups (p = 0.17). However, adolescents with concave profile selected their own profile more accurately than adults with similar profile (p = 0.04). In adolescents and adults, patients with straight and concave profile showed the greatest and the least satisfaction with their own profile, respectively [table 1]. The anova test showed a significant difference in satisfaction of patients own profile among adolescents with different types of profiles (p = 0.004, f = 6.4). The post hoc tukey test showed that adolescents with straight profile were significantly more satisfied with their profile compared to patients with convex (p = 0.036) and concave profiles (p = 0.001). The satisfaction with ones own profile in adolescents with convex profile was also significantly greater than patients with the concave profile (p = 0.045). The score of patients satisfaction with their own profile classified based on facial angle into convex, concave, and straight the anova test did not show a significant difference in satisfaction of own profile among adults with different types of profiles (p = 0.062, f = 2.8). The independent t - test found no significant difference in ranking of facial profile between adults and adolescents with different types of profiles regarding satisfaction of their own profiles [table 2]. Both adults and adolescents selected retrognathic maxilla and prognathic mandible as the least attractive profile . Straight and bimaxillary dentoalveolar retrusion were chosen as the most attractive silhouettes in adolescents and adults, respectively [table 3]. Profile attractiveness based on adolescents opinion (scored 1 - 10) esthetic perception of the profile was not significantly different among the adolescent with three different types of profile (p = 0.24, f = 1.4). However, adults with different profiles chose different silhouettes as the most attractive ones (p = 0.04, f = 3.3). The post hoc test showed that there was a statistically significant difference between straight and convex profile adults (p = 0.04), and also between convex and concave profile groups (p = 0.03). The independent t - test showed no significant difference between adults and adolescents with different type of profiles in selecting the most attractive silhouettes (p> 0.05). Only 42.9% of adolescents and 22% of adults were able to correctly diagnose the silhouette that best closely resembled their own profile [table 4]. Adolescents with concave and straight profiles had the greatest and the least percentage of correct diagnosis of their own profile, respectively . However, the difference was not statistically significant (p = 0.63). In contrast, adults with straight profiles were more accurate in diagnosing their own profile, while adults with concave profile had the least percentage of correct diagnosis . Distribution of patients according to ability of diagnosis a silhouette that closely resembled their profile the chi - square test showed that adolescents were more accurate in selecting their own profile in comparison to adult patients (p = 0.044, df = 2). The correct selection among patients with straight and convex profile was not significantly different between adult and adolescent groups (p = 0.17). However, adolescents with concave profile selected their own profile more accurately than adults with similar profile (p = 0.04). Recent research has shown that attractive people are regarded as to be more successful and are treated more positively . Different physical, psychological and social factors affect patients esthetic perception . For example, female asians prefer retrusive jaws . In the present study, esthetic perception and satisfaction of profile have been evaluated among iranian male adolescents and adults . Although patients esthetic perception have been studied in regard to sex, the level of education and patients cultures, the impact of age has been neglected in these studies . In this study, patients with straight and concave profile showed the greatest and the least satisfaction with their own profile . In the study of espeland and stenvik patients with ideal occlusion had the greatest satisfaction with their own profile . Showed that the severity of malocclusion affected patients satisfaction of their own profile . In our study, a significant difference in satisfaction of own profile was found among adolescents with different facial convexities . This could be as a result of characteristic features and emotions of the adolescent period that affect patient satisfaction . Obrien et al . Showed that the presence of malocclusion in adolescents reduced their satisfaction with their profile . These findings may explain lack of significant difference in satisfaction of profile among adults with different types of profile . In this study, both adults and adolescents selected retrognathic maxilla, prognathic mandible as the least attractive profile . Straight and bimaxillary dentoalveolar retrusion were chosen as the most attractive profile in adolescents and adults, respectively . Similarly, in the study of yin et al . Which was conducted on 1624-year - old males and females, the straight profile was chosen as the most attractive one . Cala et al . Also showed that adolescents preferred straight profile which is in agreement with our study . In their study, patients selected prognathic maxilla, retrognathic mandible as the least attractive profile which is not in line with our results . Review of previous literature shows that patients are increasingly aware of their concave profiles (class iii) and seek treatments such orthognathic surgery to normalize it . . Showed that straight and retrognathic mandible silhouettes were the most and the least attractive profile among patients, respectively . In our study, adults with straight profile selected prognathic maxilla as the most attractive silhouette . This finding is in agreement with the study of khosravanifard et al . Which concluded that prognathic maxilla in adult males was more preferable . Soh et al . Also found that bialveolar retrusive profile was more attractive among patients . Enhancement of esthetic perception as the patient gets older may be a contributory factor for this difference . Furthermore, enhancement of esthetic perception with increasing age has been reported by tole et al . In this study, only 24 patients (31.6%) were able to correctly diagnose their own profile (15 adolescents and 9 adults). This result is expected because patients get most of the information about their appearance by looking at the mirror and the profile view is usually considered as less important . Adolescents with concave and straight profiles had the greatest and the least correct selection of their own profile, respectively . Again, this signifies that adolescents with concave and straight profiles are aware of their facial profile type . Adults with concave profile had the least correct selection . In the study of yin et al . Bullen et al . Reported that the adolescents selected their own profile more accurately than adults which were in agreement with the result of our study . In this study, silhouettes were used to assess esthetic perception . This would omit the impact of sex, hair style, and skin color on patient's esthetic perception . Considering the above - mentioned results, it is critical that orthodontists listen to the esthetic preferences of the patients, and their self - perception, and also to consider the differences in esthetics perception of adolescent patients with their parents at the time of treatment plan . Most of the male adolescents and especially adults have an incorrect perception of their profile . In addition, it is vital that orthodontists be aware of their patients satisfaction of their profile considering their own profile esthetics before treatment.
Occipital neuralgia (on) is a rare neurological disorder characterized by paroxysmal shooting or stabbing pain in the dermatomes of the greater occipital nerve (gon) or lesser occipital nerve (lon)5). Clinical presentation and a temporary improvement with local anesthetic diagnostic block of the gon or lon confirm the diagnosis5). The primary treatment of on is conservative and focuses on reducing secondary muscle tension and improving posture . In cases of continual pain, nerve blocks may be applied or invasive procedures such as occipital nerve stimulation, neurolysis of the occipital nerves, or dorsal root entry zone rhizotomy may be performed20,21). However, the effects of nerve blocks are temporary, and surgical treatments are invasive and have risk and complications1,13,23). In this study, we present the clinical outcome of 10 patients with on treated by pulsed radiofrequency (prf). From 2010, january to 2011, march, ten consecutive patients with on were treated by prf at our institute . All patients in this study were diagnosed with on according to the international classification of headache disorders classification criteria5). All patients presented with complaints of severe sharp stabbing pain in the occipital area . The duration of pain attacks varied from several minutes to several days, and the frequency varied from several times per month to several times per day . Palpation of the occipital area or upper neck on the affected side usually revealed muscle tenderness and in most cases precipitated pain attacks . All patients provided complete histories and underwent physical examinations followed by diagnostic tests such as computed tomography and magnetic resonance imaging . All patients received gon and lon blocks with 1.0% lidocaine and dexamethasone twice by one week interval . Temporary pain relief of 50% or more was considered a positive response to the nerve blocks, and was one of the most important factors in patient selection for prf, but the effects were temporary . These patients underwent prf neuromodulation of the gon and lon for medically intractable on (fig . 1). We performed fluoroscopically guided gon and lon prf using a neurotherm nt1000 (neurotherm, inc ., middleton, ma, usa) radiofrequency generator . In the operating room, each patient was placed in a sitting position on the procedure table and the posterior neck area was prepped with betadine and aseptically draped with sterile towels . A disposable 22-gauge, 5 cm radiofrequency cannula (model s-505, neurotherm, inc ., middleton, ma, usa) with a 5 mm active tip was inserted at the levels of both the gon and lon (fig . The introducer needle was withdrawn and a disposable rf electrode (model rfde-5, neurotherm, inc ., selective sensory nerve stimulation (50 hz) showed concordant pain below 0.5 v, which confirmed localization of the prf electrode . After stimulation, prf was performed at 42 for a total of 240 pulses at each site . In all patients, pain was measured before the primary diagnostic block and every month after prf neuromodulation by the same blinded physician who had made the initial assessments . Pain was assessed using a visual analog scale (vas) (0 cm - no pain; 10 cm - worst possible pain imagined). The tpi is an incorporated pain scale of the weighted intensity and duration of headache attacks . Mean values of the vas and tpi for pain before the diagnostic block and after prf neuromodulation were compared using paired t - tests at alevel of 0.05 . Two - tailed t - test probabilities reported with p values <0.05 were considered statistically significant . From 2010, january to 2011, march, ten consecutive patients with on were treated by prf at our institute . All patients in this study were diagnosed with on according to the international classification of headache disorders classification criteria5). All patients presented with complaints of severe sharp stabbing pain in the occipital area . The duration of pain attacks varied from several minutes to several days, and the frequency varied from several times per month to several times per day . Palpation of the occipital area or upper neck on the affected side usually revealed muscle tenderness and in most cases precipitated pain attacks . All patients provided complete histories and underwent physical examinations followed by diagnostic tests such as computed tomography and magnetic resonance imaging . All patients received gon and lon blocks with 1.0% lidocaine and dexamethasone twice by one week interval . Temporary pain relief of 50% or more was considered a positive response to the nerve blocks, and was one of the most important factors in patient selection for prf, but the effects were temporary . These patients underwent prf neuromodulation of the gon and lon for medically intractable on (fig . We performed fluoroscopically guided gon and lon prf using a neurotherm nt1000 (neurotherm, inc ., middleton, ma, usa) radiofrequency generator . In the operating room, each patient was placed in a sitting position on the procedure table and the posterior neck area was prepped with betadine and aseptically draped with sterile towels . The skin was anesthetized with 5% lidocaine gel . A disposable 22-gauge, 5 cm radiofrequency cannula (model s-505, neurotherm, inc ., middleton, ma, usa) with a 5 mm active tip was inserted at the levels of both the gon and lon (fig . The introducer needle was withdrawn and a disposable rf electrode (model rfde-5, neurotherm, inc ., middleton, ma, usa) was advanced . Selective sensory nerve stimulation (50 hz) showed concordant pain below 0.5 v, which confirmed localization of the prf electrode . After stimulation, prf was performed at 42 for a total of 240 pulses at each site . In all patients, pain was measured before the primary diagnostic block and every month after prf neuromodulation by the same blinded physician who had made the initial assessments . Pain was assessed using a visual analog scale (vas) (0 cm - no pain; 10 cm - worst possible pain imagined). The tpi is an incorporated pain scale of the weighted intensity and duration of headache attacks . Mean values of the vas and tpi for pain before the diagnostic block and after prf neuromodulation were compared using paired t - tests at alevel of 0.05 . Two - tailed t - test probabilities reported with p values <0.05 were considered statistically significant . The age of the patients ranged between 34 to 70 years with a median age of 52 years . The patient sample consisted of seven (70%) women and three (30%) men . The mean total follow - up time of the patient series was 7.5 months, ranging from 6 to 10 months . The majority of patients presented with bilateral symptoms (60%, 6 of 10 patients) and the remaining four (40%) patients suffered from unilateral on: two on the right and two on the left side . Mean vas scores and mean tpi scores of the 10 treated patients are presented in table 1 . Significant improvements in pain (vas, tpi) were found in months 1 - 6 compared with the pre - diagnostic block period (p<0.05) (fig . The mean vas score before the pre - diagnostic block period was 6.9 and declined to 1.2 and 0.8 at post prf and last follow - up period, respectively (p<0.001, and p<0.001). The mean tpi score before the pre - diagnostic block period was 232.7 and declined to 53.7 and 40.6 at post prf and last follow - up period, respectively (p<0.001, and p<0.001) (fig . One patient (10%) reported a substantial reduction in analgesic requirements and pharmacotherapy was maintained in one patient who had partial recurrence of headaches (table 1). There were neither intraoperative nor postoperative complications that would lead to any type of significant morbidity or mortality . The age of the patients ranged between 34 to 70 years with a median age of 52 years . The patient sample consisted of seven (70%) women and three (30%) men . The mean total follow - up time of the patient series was 7.5 months, ranging from 6 to 10 months . The majority of patients presented with bilateral symptoms (60%, 6 of 10 patients) and the remaining four (40%) patients suffered from unilateral on: two on the right and two on the left side . Mean vas scores and mean tpi scores of the 10 treated patients are presented in table 1 . Significant improvements in pain (vas, tpi) were found in months 1 - 6 compared with the pre - diagnostic block period (p<0.05) (fig . The mean vas score before the pre - diagnostic block period was 6.9 and declined to 1.2 and 0.8 at post prf and last follow - up period, respectively (p<0.001, and p<0.001). The mean tpi score before the pre - diagnostic block period was 232.7 and declined to 53.7 and 40.6 at post prf and last follow - up period, respectively (p<0.001, and p<0.001) (fig . One patient (10%) reported a substantial reduction in analgesic requirements and pharmacotherapy was maintained in one patient who had partial recurrence of headaches (table 1). There were neither intraoperative nor postoperative complications that would lead to any type of significant morbidity or mortality . On is a neuralgiform disorder defined as a shooting or stabbing pain originates in the suboccipital region and radiates over the vertex1). Hypo- or dysesthesia in the dermatome of the gon or lon, as well as tenderness to pressure over the course of the gon or lon can accompany the pain, and constant pain can persist between paroxysms . The gon is more frequently involved (90%) than the lon (10%). In 8.7% of patients, vision impairment / ocular pain (67%), tinnitus (33%), dizziness (50%), nausea (50%), and congested nose (17%) can be present because of connections to cranial nerves v, viii, ix, x, and the cervical sympathetic ganglion7,15). The most common treatment modality for on is blockade of the gon and lon, which interrupts the pain cycle and reflex muscle spasms and relieves the symptoms . Many studies have shown that occipital nerve block is an effective treatment modality for on . In a small (n=10) retrospective study by kuhn et al.7), the gon was infiltrated with corticosteroids after a positive test block with bupivacaine . The authors observed pain relief of less than 1 week in 10% of patients, 1 week in 30%, 2 weeks in 30%, 1 month in 10%, and more than 2.5 months in 20% of patients . Hammond and danta4) observed short - term effects (less than 1 week) in 64% of patients after 1 infiltration with local anesthetic; 36% of the patients reported effects lasting longer than 1 month . Injection of depot methylprednisolone into the gon and lon regions produced complete headache relief for periods of only 10 - 77 days15). When nerve block failto relief on, cervical dorsal root ganglion prf, and occipital nerve stimulation can be performed for medically intractable on4,7,14,15,22). Weiner and reed first reported 13 patients who underwent 17 occipital nerve stimulation procedures for medically intractable on . With follow - ups ranging from 18 months to 6 years, good to excellent results were seen in 12 of 13 patients as defined by greater than 50% pain relief and requiring little or no pain medications . Slavin et al.12,13) carried out trial stimulations in 14 patients with medically intractable on . Definitive neurostimulators were implanted subcutaneously in 10 patients who had reductions in pain of greater than 50% . After a mean follow - up of 22 months, 70% of these patients still had good results . However, occipital nerve stimulation may be associated with possible complications such as infection, lead migration, hardware erosions, electrode fractures, disconnections, and sepsis13). When a prospective review explored the long - term effects of prf treatment adjacent to the cervical dorsal root ganglion, the authors found that cervical dorsal root ganglion prf also have serious complications occurred when a radiofrequency cannula was inserted at the level of the cervical dorsal root ganglion from posterior to frontal under the c - arm fluoroscopic guidance due to insertion into the cervical subarachnoid space or into vessels in this region . Potential risks include infection, stroke, paralysis and cerebrospinal fluid leakage23). To date, one case report and one prospective trial have been published concerning prf neuromodulation in on10,16). In the case report, after recurrence of pain, the prf neuromodulation procedure was repeated with again 70% pain relief lasting 5 months . Of the 19 patients included in the prospective trial, 68.4%, 57.9%, and 52.6% reported improvement of 50% or more 1, 2, and 6 months after prf neuromodulation, respectively . There were significant improvements in the use of medication and in quality - of - life parameters . Compared with the results of other treatment modalities prf treatment is safer, and therefore, should be the preferred treatment for on16). We used prf for the gon and lon, and we observed pain relief for a minimum of 6 months . Meanwhile, none of the patients in our study have exhibited any side effects related to our procedure . When performed prf for on, electrode approximation of the target nerve is very important . In our study, the puncture sites of the gon and lon were determined by external landmarks as described below2,8,9,17). On average, the gon is situated 3.8 cm lateral from the midline and one quarter of the distance along a line connecting the external occipital protuberance to the mastoid (or 2 cm lateral and 2 cm inferior to the external occipital protuberance). The authors used the relationship between the gon and the occipital artery for detecting the gon . At first, the authors identified the occipital artery pulsation about 2.5 cm from the midline, and the gon was located medial to the occipital artery . The needle (22 g) is introduced until there is bone contact or paresthesia is elicited . Sensory and motor nerve stimulations are then performed and we can easily find the target nerves . First, although the vas and tpi are validated tools for the quantification of pain, they are subjective outcome measures because they are dependent on personal interpretations and variation . Several limitations of our study are worth mentioning . First, although the vas and tpi are validated tools for the quantification of pain, they are subjective outcome measures because they are dependent on personal interpretations and variation . The results of this retrospective clinical study support the hypothesis that prf provides long - term reduction in headaches with minimal procedural risk in selected patients with medically intractable on . Prf should be considered an effective treatment modality for on, especially in medically intractable on patients . Future long - term prospective controlled clinical trials are warranted to establish more definitive conclusions.
Despite all the advances in the care of premature infants with respiratory distress syndrome (rds), including the use of antenatal steroids and early management with surfactant, bronchopulmonary dysplasia (bpd) continues to be a major cause of chronic morbidity among this population . There are large variations in the incidence and severity of this disease . According to the national institutes of health of usa (nichd) consensus, mild bpd is defined as a need for supplemental oxygen for 28 days at 36 weeks postmenstrual age (wpma) or discharge, moderate bpd as supplemental oxygen for 28 days plus treatment with <30% oxygen at 36 wpma, and severe bpd as supplemental oxygen for 28 days plus 30% oxygen and/or positive pressure at 36 wpma . Currently, the estimated incidence of bpd defined as need for supplemental oxygen at 36 wpma in the united states is approximately 30% for premature infants with a birth weight <1000 grams and <7% in infants with a birth weight> 1250 grams or who were at least 30 weeks of gestation at birth [1, 2]. Previous epidemiological studies have identified prematurity, oxygen toxicity, and mechanical ventilation as major risk factors associated with bpd [37]. Additional risk factors include perinatal or postnatal infection / inflammation [812], pulmonary edema resulting from patent ductus arteriosus (pda) [12, 13] or excess fluid administration [7, 14, 15], vitamin a deficiency, early adrenal insufficiency, and, more recently, genetic polymorphism . There is little information about trends in the epidemiology and pathogenesis of bpd in developing countries . The most recent report of the incidence of bpd in latin america comes from the neocosur neonatal group study in 1,825 very - low - birth - weight (vlbw) infants born in sixteen hospitals from argentina, chile, paraguay, peru, and uruguay . The authors found an incidence of bpd (oxygen requirement at 28 days of life with chronic radiographic changes) of 24.4% . A randomized controlled trial of early bubble nasal continuous positive airway pressure (ncpap) and surfactant in premature infants conducted in three different cities in colombia found an incidence of bpd (defined as supplemental oxygen requirement at 36 wpma) twice as large (44%) as the one observed in less mature premature infants in developed countries or in the neocosur study . The colombian trial also revealed significant variations in bpd rates among participating cities: bogot 50%, cali 18%, and bucaramanga 13% . These cities have different characteristics, the most important being altitude: bogot is 2600 meters above sea level (masl), bucaramanga 959 masl, and cali 956 masl . We conducted this study with the aim of identifying environmental, maternal, infant, and therapeutic risk factors associated with bpd in colombia . We also tested the hypothesis that differences in bpd rates were not explained by differences in city - of - birth - specific population characteristics or by differences in respiratory management practices during the first seven days of infants' life among cities . This is a nested case - control study based on data collected as part of a multicenter randomized controlled trial carried out by the colombian neonatal research network in eight neonatal intensive care units (nicus) located in three cities (bogot, bucaramanga, and cali) in colombia . Briefly, premature infants born between 27 and 31 weeks of gestation with clinical evidence of respiratory distress during the first hour of life, and who did not require intubation as part of their initial resuscitation and stabilization, were placed on bubble ncpap and then randomized to receive very early surfactant therapy through transient intubation followed by ncpap or to expectant management on ncpap alone . A total of 279 premature infants were enrolled in the trial from january 1, 2004 to december 31, 2006 . All study sites provided comprehensive continuous care for critically ill neonates and were staffed by trained nurses and specialized physicians with fully equipped and modern neonatal units . For the present study, all analyses were limited to infants who survived to 36 wpma . Bronchopulmonary dysplasia was defined as the need for supplemental oxygen for 28 days at 36 wpma . The target oxygen arterial saturation (sao2) was 92% for infants treated in bogota and 96% for those treated in other cities . Given the high altitude in bogot it was expected that sao2 would be lower than in infants at lower altitudes [21, 22]. Neonates who fell under this definition were considered as cases . Since we used an epidemiological definition for bpd and we did not have the radiographic findings to confirm bpd cases, to avoid misclassification bias controls were all infants who were not receiving supplemental oxygen at 36 wpma and had required <20 days of oxygen supplementation or had not required oxygen supplementation at all . Relevant exposure data included maternal and infant perinatal characteristics, infant postnatal diagnosis, and respiratory management practices . Preterm premature rupture of membranes (pprom) was defined as> 12 hours, use of antenatal steroids as the administration of a complete course (two 12 mg intramuscular doses of betamethasone within 24 hours) of maternal steroids at least 24 hours before delivery, confirmed chorioamnionitis as a positive amniotic fluid culture, and suspected chorioamnionitis as maternal fever during labor and fetal tachycardia . Gestational age (ga) at birth was estimated using the last menstrual period date; when ga was inconsistent with the physical examination, the ballard score was used . Small for gestational age (sga) was defined as a birth weight <10th percentile for age . Sepsis was defined as a clinical deterioration with temperature instability, recurrent apnea, and at least one of the following indications of altered organ function: lethargy, hypoxemia, and increased serum lactate level . Grades iii and iv ivh were identified by head ultrasound using the papile et al . Air leak syndrome included radiological evidence of pneumothorax, pneumomediastinum, or pulmonary interstitial emphysema . The score for neonatal acute physiology (snap ii) on the day of admission was calculated from clinical data collected prospectively during the first day of life . Respiratory management variables included type of ventilatory support received: (a) only ncpap when infants were placed on ncpap without subsequent mechanical ventilation (mv) during their nicu stay, (b) ncpap + mv when infants on ncpap met treatment failure criteria and required mv . Exposure to surfactant was divided into three categories: (a) no surfactant exposure included all infants who did not receive surfactant during the trial, (b) very early surfactant included infants administered surfactant within the first hour of life, and (c) rescue surfactant included infants administered surfactant after the first hour of life . Exposure to oxygen supplementation was measured by fraction of inspired oxygen (fio2) registered daily . Demographic and clinical characteristics of the study population were summarized . To identify differences in the distribution of studied variables between cases and controls, the statistical analysis included hypothesis testing using the pearson chi - square test for categorical variables and the students t - test for continuous variables . To explore associations between studied variables and bpd, crude and adjusted odds ratios (ors) with 95% confidence intervals (95% cis) were estimated in a bivariate analysis, followed by a multivariate regression analysis using a log - binomial generalized linear model that included all significant variables in a manual forward stepwise approach . The wilcoxon rank sum test was used to compare the median values between groups and the generalized cochrane mantel - haenszel test to assess the statistical significance of associations between categorical variables . To determine differences in population characteristics or in respiratory management variables during the first week of life that could explain the differences in bpd rates among cities, a descriptive analysis of selected variables was performed by city of birth, followed by a bivariate analysis to identify variables mainly associated with bpd in each city . To identify possible differences in the distribution of studied variables between three cities among cases of bdp, the statistical analysis included hypothesis testing using the pearson chi - square test for categorical variables and the analysis of variance or kruskall wallis test for continuous variables . Multivariate analyses were then conducted to explore associations between city of birth and bpd while controlling for differences in population demographic characteristics, postnatal diagnosis, and respiratory management variables during the first seven days of life . With the final sample of cases and controls, the power calculation for the research hypothesis tested was 82%, according to the following parameters: incidence of bpd 30%, type i error probability of 0.05, and expected size effect (odds ratio, or) of 2.0 . Results of all multivariate analyses are expressed as odds ratios (ors) with their corresponding 95% ci . All analyses were carried out using the sas program (sas institute, cary, nc). A total of 216 (77.42%) infants survived to 36 wpma, and four were excluded from the analysis due to missing data or because they did not meet the case or control definitions . The final analysis included 212 infants; 64 (30%) met the definition of bpd . Mean ga and birth weight were lower in the bpd group (table 1); no differences were observed in other perinatal characteristics between cases and controls . Tables 2 and 3 present the results of bivariate analysis controlling for ga at birth . Use of rescue surfactant, no surfactant exposure, diagnosis of pda, chorioamnionitis, and confirmed or suspected pprom were variables independently associated to bpd . Following a stepwise forward approach, the initial logistics model showed that pprom, ncpap + mv, no surfactant exposure, rescue surfactant, pda, median daily fio2 (sub index is not allowed in this website), and sepsis were associated with a higher risk of bpd . When city of birth was introduced into the model as a control variable, we observed that median daily fio2 and pda lost statistical significance, suggesting the presence of an interaction . We conducted a series of multivariate models testing for interactions between the variables: city of birth, median daily fio2; sepsis and pda . The only significant interaction observed was between the variables bogot as city of birth and median daily fio2 the results of the final logistic regression model controlling this interaction are showed in table 4 . Bogot as city of birth was the most significant independent variable associated with bpd; other variables associated with bpd in the initial model remained significantly associated (table 4). The distribution of maternal and infant perinatal characteristics, infant postnatal diagnosis, and respiratory management practices in the first seven days of the infants' life was similar in all cities (table 5); however, there are statistically significant differences between cases and controls among cities . The proportion of bpd cases in infants whose mothers received antenatal steroids was significantly higher in bogot than in any other city . In bucaramanga, infants with bpd had lower birth weights than infants in cali or bogot . Infants born in bogot that required mv after ncpap (ncpap+mv) and those treated with rescue surfactant or no surfactant exposure had a higher incidence of bpd compared to infants of similar characteristics born in cali or bucaramanga . Infants born in bogot had higher median values of daily fio2 compared to bucaramanga and cali, and infants diagnosed with bpd in bogot received higher concentrations of daily supplemental oxygen than controls, while bpd cases in cali and bucaramanga did not . In relation to postnatal diagnosis during the first 7 days of life, the diagnosis of pda was higher in bucaramanga, but the largest proportion of bpd cases in infants with pda was observed in bogot . The diagnosis of sepsis was also more frequent in bucaramanga and cali, but the largest proportion of bpd cases among infected infants was seen in bogot, followed by cali and bucaramanga (p <0.0001). Bivariate analysis stratified by city of birth (table 5) showed the following: for infants born in bogot, the diagnosis of sepsis in the first seven days of life and the incremental levels of daily median fio2 as variables mainly associated with a higher risk of bpd; for infants born in cali, air leak syndrome, pprom, and pda in the first seven days of life were the variables associated with bpd, while in bucaramanga pprom was the only significant risk factor associated with bpd . To explore for associations between city of birth and bpd multivariate results are shown in table 6; bogot as city of birth was strongly associated with an increased risk of developing bpd (or 1.82 95%ci 1.312.53). Other variables in the model associated with a higher risk of bpd were rescue surfactant, no surfactant exposure, ncpap + mv, median daily fio2, pda, and sepsis . This rate is higher than the average rates reported in populations with similar gestational ages in developed countries [1, 2, 57]. Our results showed that infants born in bogot had nearly twice the risk of developing bpd than infants born in bucaramanga or cali, independently of differences in maternal, infant, and therapeutic risk factors . Additionally, bogot showed the highest rates of bpd cases associated with the presence of air leak syndrome, exposure to mv, rescue surfactant or no surfactant exposure, pda, and sepsis, when compared to other cities . It also had the highest values of daily supplemental oxygen to reach the target sao2 among all the cities . Exposure to antenatal steroids did not appear to protect infants born in bogot from developing bpd . Because bogot is located a higher altitude (more than 2600 masl) than bucaramanga and cali, these results could suggest that altitude may play an important role in the pathogenesis of bpd in colombia . There are few publications on altitude - related disease and pulmonary hemodynamics in pediatric populations, and the altitude where an infant is born has not clearly been proven to be associated with the development of bpd . The effect of living at high altitudes (> 2500 masl) on lung diffusion capacity and pulmonary hemodynamics has been described in highland children . As a result of the low partial pressure of oxygen in the environment, oxygen uptake into the lungs . The decreased partial pressure of oxygen in the lungs of highland children has also been associated with higher pulmonary artery pressures [2831]. Several investigators have also found that functional closure of the ductus arteriosus in the newborn is delayed at high altitudes as a consequence of increased pulmonary vascular pressures [3235]. The transition from oxygenation via the placenta to oxygenation across the formerly fluid - filled lungs is especially precarious in a low - oxygen environment; with the onset of ventilation immediately after birth, the oxygen tension in the alveolus and pulmonary capillaries of neonates may not increase as expected, resulting in postnatal persistence of fetal circulation . In the absence of pulmonary disorders, normal neonates may experience frequent episodes of arterial oxygen desaturations and hypoxia during the first week of life . Studies comparing healthy infants born at high altitudes (> 3100 masl) with infants born at sea level have shown that a week after birth the sao2 of high - altitude infants declines, whereas sao2 gradually rises after birth or remains constant over time in infants born at sea level . As a result of these events, it is possible that premature infants born at high altitudes have a prolonged transition period and early dependency on high concentrations of oxygen and ventilatory support compared to their counterparts born at lower altitudes . The presence of rds would enhance ventilation perfusion mismatch leading to more hypoxia, oxygen dependency, oxidative stress, and higher levels of ventilatory support, increasing the risk and severity of bpd . It is also possible that the observed dependency on supplemental oxygen in our population of premature infants is the result of physiological oxygen dependency and not bpd . This may be due to the fact that the definition used for bpd does not take into account clinical or radiographic changes . The association between pda and bpd has previously been documented [4, 12, 37]. It is also possible that the prolonged closure of the pda may play a role in the pathogenesis of bpd, especially as the pulmonary vascular resistance begins to drop, but we cannot answer this question because we did not assess the duration of the pda, the presence of pulmonary hypertension, or the presence of pulmonary edema or hemorrhage in this population of infants . Likewise, we did not assess the effect of fluid intake on the development of pda and bpd because this information was not collected in the initial trial [7, 14, 37, 38]. Previous studies have demonstrated the relationship between the presence of late - onset sepsis, pda, and the development of bpd [12, 37]. Our study suggests that altitude enhances their negative effects and other traditional risk factors associated with bpd . In our population, pprom was found to be a significant risk factor for bpd while chorioamnionitis was not . This finding could be explained in part because amniocentesis and amniotic culture fluids were not taken routinely in all participating centers as part of the workup for suspected chorioamnionitis in mothers with preterm labor or premature rupture of membranes . The association of bpd with preterm labor and pprom has been well documented in the medical literature . Finally, our study emphasizes the need to minimize mv exposure and offer surfactant replacement therapy within the first hour of life to infants with rds on ncpap in order to decrease the incidence of bpd in this population [20, 40, 41]. To summarize, this study suggests that altitude may be an important risk factor associated with increased supplemental oxygen dependency and the development of bpd . Future studies need to determine whether altitude plays a role in the pathophysiology of bpd or if it is just a marker for physiologic oxygen demands . They also need to use a more accurate definition for bpd, measuring not only the need of supplemental oxygen but also the radiographic findings and clinical signs.
Anterior cervical fusion (acf) is a commonly performed surgical procedure to treat degenerative disorders of the cervical spine9). Although the use of tricortical iliac crest bone graft in acfs is associated with the excellent outcomes, the risk of donor site related complications have always incited the spine surgeon to use various contemporary graft substitutes . In 1965, urist14) discovered a subset of protein extract, which had a significant potential of inducing new bone formation even at the non - osseous tissues . Recombinant human bone morphogenetic protein-2 (rhbmp-2) is a member of this family, which has gained a widespread popularity for its application in spinal fusions . Although, their role in lumbar fusions is well established, the safety profile of rhbmp-2 in acfs is yet to be defined101113). Baskin et al.1) were the first authors who conducted a prospective randomized controlled trial and reported that the use rhbmp-2 in acfs is a safe and effective technique . Conversely several other authors reported a high incidence of neck swelling and dysphagia associated with the use of rhbmp-2 warranting either prolonged stay in the hospital or readmissions of the patients6101115). Despite the high fusions rates, some authors have even abandoned the use of rhbmp-2 for acfs due to its cost, side effects and availability of safer alternatives15). On the basis of results of earlier trials, food and drug administration (fda) issued a warning in 2008 against the use of rhbmp-2 in acfs until the sufficient evidence is available about its safety . Shields et al.10) reported a high rate of complications and attributed it to the use of greater amount of bmp dose (2.1 mg / level) placed both inside and outside the cage . Further emphasis on the influence of the dose and delivery method of rhbmp-2 in acfs was given by dickerman et al.4) and in their letter they proposed that the containment of low dose (1.05 mg) rhbmp-2 within the cage and placing demineralized bone matrix (dbm) putty surrounding the infuse ensures a controlled delivery of rhbmp-2 in the vicinity of desired fusion site, which therefore is associated with excellent fusion rates with no adverse effects related to rhbmp-2 . The purpose of our study is to share our clinical experience with low - dose of rhbmp-2 in acf . We also aim to elucidate the impact of dose and delivery techniques of rhbmp-2 on the safety profile of its use in acfs based on our clinical experience and review of the literature . We performed a retrospective analysis of 197 patients who underwent acf with the use of rhbmp-2 during 2007 - 2012 . Acfs included mainly anterior cervical discectomy and fusion (acdf, n=191), although few patients underwent anterior cervical corpectomy and fusion (accf, n=4). There were 80 males (40.6%) and 117 (59.4%) females included in our study . Surgeries were performed for herniated disc prolapsed in 91 patients, for spondylosis with or without instability in 104, and for pseudoarthrosis in 2 patients (revision acf was performed in both these patients). Most common presenting symptom was neck pain (n=175), followed by arm pain (n=163), sensory deficit (n=102) and motor weakness (n=87). History of cigarette smoking was present in 53 patients (26.9%) and diabetes mellitus in 21 (10.65%). Thirty nine patients had a history of previous neck surgery, most of which had undergone discectomy or nerve decompression by foraminotomy (n=37) and 2 patients had anterior fusions performed at different levels . In the majority of cases (n=191) polyetheretherketone (peek) cages were used as a spacer device and the construct was supplemented with anterior cervical plate . In 6 patients, peek prevail (medtronics sofamor danek) device was used, which has inbuilt slots for self drilling screws . Cages were filled with infuse bone graft substitute, which has 2 components: a reconstituted solution containing recombinant human bone morphogenetic protein-2 (rhbmp-2) and an absorbable collagen sponge (acs). We placed only 1/3 of sponge within the cage (measuring 1.72.540.5 cm), which would deliver 0.7 mg of rhbmp-2 per level considering a homogenous distribution of this protein within the sponge (fig . No additional acs was filled around the cage . In a large number of the patients (n=102), we also used demineralized bone matrix (dbm), which was placed from the top and the bottom over the centrally located infuse within the peek cage (fig . 1b). Unless contraindicated, intravenous dexamethasone (8 mg) was administered at the start of the procedure in all cases interbody spaces were prepared for cage placement, which were filled with infuse substitute in a proportion already discussed . Anterior plate fixation was supplemented in all patients except in those in which prevail peek device was used (n=6). In the patients with major anatomical deformities, the amount of infuse was the same even if a larger spacer was placed after accf . Closure was performed in layers without placing any drain . Unless it was contraindicated, all patients were given methylprednisolone orally (4 mg) in taper doses during the first week of postoperative period . Plain x - ray of the cervical spine was taken within few hours of surgery to document the appropriate placement of hardware . During hospitalization, any events such as dysphagia, neck hematoma, incisional swelling, and vocal cord palsy were recorded . The majority of the patients were discharged within 24 hours of surgery and the patients hospitalized beyond 48 hours were considered to have a prolonged stay . After discharge, the patients were evaluated at 2 weeks, 6 weeks, 3 months, 6 months, 12 months and 24 months after surgery . Incidence and severity of dysphagia was retrospectively determined by the 5 point swal - qol scale7). Score 1 indicates the patient almost always had difficulty; score 2 indicates the patient often had difficulty; score 3 indicates the patient sometimes had difficulty; score 4 indicates the patient hardly had any difficulty; score 5 indicates the patient never had difficulty . For the statistical analysis, patients with dysphagia were grouped into minimal (score 3 - 5) and substantial (score 1 - 2) dysphagia . All statistical analyses were performed using the spss software, version 21.0 (ibm corp . The comparison of incidence and severity of complications between dbm and non - dbm was performed by using fisher's exact test . In the majority of cases (n=191) polyetheretherketone (peek) cages were used as a spacer device and the construct was supplemented with anterior cervical plate . In 6 patients, peek prevail (medtronics sofamor danek) device was used, which has inbuilt slots for self drilling screws . Cages were filled with infuse bone graft substitute, which has 2 components: a reconstituted solution containing recombinant human bone morphogenetic protein-2 (rhbmp-2) and an absorbable collagen sponge (acs). We placed only 1/3 of sponge within the cage (measuring 1.72.540.5 cm), which would deliver 0.7 mg of rhbmp-2 per level considering a homogenous distribution of this protein within the sponge (fig . No additional acs was filled around the cage . In a large number of the patients (n=102), we also used demineralized bone matrix (dbm), which was placed from the top and the bottom over the centrally located infuse within the peek cage (fig . Unless contraindicated, intravenous dexamethasone (8 mg) was administered at the start of the procedure in all cases . Interbody spaces were prepared for cage placement, which were filled with infuse substitute in a proportion already discussed . Anterior plate fixation was supplemented in all patients except in those in which prevail peek device was used (n=6). In the patients with major anatomical deformities, the amount of infuse was the same even if a larger spacer was placed after accf . Closure was performed in layers without placing any drain . Unless it was contraindicated, all patients were given methylprednisolone orally (4 mg) in taper doses during the first week of postoperative period . Plain x - ray of the cervical spine was taken within few hours of surgery to document the appropriate placement of hardware . During hospitalization, any events such as dysphagia, neck hematoma, incisional swelling, and vocal cord palsy were recorded . The majority of the patients were discharged within 24 hours of surgery and the patients hospitalized beyond 48 hours were considered to have a prolonged stay . After discharge, the patients were evaluated at 2 weeks, 6 weeks, 3 months, 6 months, 12 months and 24 months after surgery . Incidence and severity of dysphagia was retrospectively determined by the 5 point swal - qol scale7). Score 1 indicates the patient almost always had difficulty; score 2 indicates the patient often had difficulty; score 3 indicates the patient sometimes had difficulty; score 4 indicates the patient hardly had any difficulty; score 5 indicates the patient never had difficulty . For the statistical analysis, patients with dysphagia were grouped into minimal (score 3 - 5) and substantial (score 1 - 2) dysphagia . All statistical analyses were performed using the spss software, version 21.0 (ibm corp ., armonk the comparison of incidence and severity of complications between dbm and non - dbm was performed by using fisher's exact test . A total of 197 patients with the mean age of 51.3 years and male to female ratio of 1.01 were included in the study . The mean duration of follow - up was 27.5 months (range 9 - 43 months). The majority of patients underwent single level acfs (n=110, 55.8%), while 2, 3, 4 levels acfs were performed in 72 (n=36.5%), 13 (6.6%), 2 (1%) patients respectively . Hospital stay for the majority of patients was uneventful except in 2 patients who had prolonged hospitalization . An 83 year old patient, who underwent 2 levels acdf, developed severe postoperative dysphagia on 2 day of surgery . The clinico - radiological assessment did not reveal any neck swelling or hematoma, but the patient failed swallow tests . The patient was inserted a percutaneous endoscopic gastrostomy (peg) tube until he started tolerating oral feeds over the next few weeks . The other patient was a 70 year old male with an existing pulmonary dysfunction, who underwent 3 levels acdf, developed acute respiratory failure on the day of surgery . The patient was reintubated; however, no hematoma or fluid collection in the neck was detected . A total of 26 (13.2%) patients complained of dysphagia and the mean duration of onset was 9.2 (range 2 - 21) days . Of these patients, 15 patients had minimal discomfort (score 3 - 5) and the remaining 11 patients complained of a substantial degree of discomfort (score 1 - 2). Neck swelling occurred on average postoperative day 5.4 (range 2 - 12 days). In 9 (34.6%) patients with dysphagia, there was also an associated visible neck swelling . On the other hand, more than half (n=9, 52.9%) of the patients with neck swelling had associated dysphagia . In a large number of the patients these adverse events resolved spontaneously (table 1). However, several other patients were given oral methylprednisolone as an outpatient; only few required either prolonged hospitalization or readmissions . As already discussed, one patient with severe dysphagia who was found to have swallowing dysfunctions received a peg tube and therefore, had an extended hospital stay . Two patients with neck swelling were readmitted for the management of associated dysphagia and respiratory difficulty (fig . Resolution of the symptoms could be achieved in both patients by administering intravenous dexamethasone, and the patients were also given oral methylprednisolone for 5 days following discharge . None of the patients necessitated surgical management for the aforementioned complications . In 102 patients dbm overall the incidence of dysphagia, substantial dysphagia and neck swelling was lower in the patients who were inserted dbm putty around infuse . On the contrary, the rate of spontaneous resolution was more in the non - dbm group; although, none of these observations reached statistically significance (table 2). There was no difference in the incidence of prolonged hospitalization and readmissions in two groups . Four patients (2%) developed vocal cord palsies, which were transient and subsided over the period of few weeks in all of these patients . Pseudoarthrosis was seen in 2 patients . In the first patient, who was 50 year female with a 3 level acdf patient deferred to any further intervention for the persistent symptoms arising from the involved segment . The second patient was a 66 year female, who underwent a single acdf for the traumatic subluxation of c5/6 region . This patient underwent posterior fusion and stabilization due the radiological presentation of implant loosening and impending loss of fusion at 1 month . One patient developed postoperative deltoid weakness, which subsided spontaneously over the next few months . The reports of high incidence of complication following its use in acfs invoked the debate on the safety profile of rhbmp-2 in these procedures . One of such reports was from schilds et al.10), who documented a significant rate of complications resulting after the use of a high dose rhbmp-2 in acfs . Interestingly, the incidence of the adverse events in the literature ranges from none to very high rates of complications (table 3)123561011121315). Lower incidence of the complications in some of the studies has been correlated with the lower dose of rhbmp-2 and methods of delivery241013). Boakye et al.2) produced the first report of rh - bmp-2 filled in a peek spacer, which is what we use at our institution . The authors studied 24 patients that underwent acdfs with peek implants and rh - bmp-2 . Three patients were found to have heterotopic bone formation, while the authors were using half of the amount of a small infuse kit (2.1 mg rhbmp-2/level). Subsequently, the authors decided to use one - fourth of the sponge (1.05 mg rhbmp-2/level) and found no heterotopic bone formation . The results of bmp in acfs were convincing until schilds et al.10) presented their study, which documented alarmingly high incidence of complications . However, one glaring difference was the amount of rh - bmp-2 used per level (2.1 mg). They also suspected that higher amount of infuse used in vertebrectomies led to a greater risk of the adverse events (7.9% vs 38.8% hematoma). The impact of bmp dose was also evident from the study by tumialan et al.13), who were initially using 2.1mg / level . The authors noticed excess interbody bone formation in the first 24 patients and subsequently reduced the dose to 1.05 mg of rh - bmp-2 per level in the next 93 patients . The authors went on to reduce the dose even more (0.7 mg / level) as more studies supported using a smaller dose . Recent attention has also been directed to the methods of delivery . While reviewing their results, schilds et al.10) pointed out that the placement of bmp sponge outside the cage could be related to the high incidence of complication in their series . Some surgeons have also used thrombin glue to control the diffusion of bmp into the surrounding tissues5). The influence of delivery techniques gained more ground when dickermann et al.4) shared their experience with using dbm around bmp sponge to minimize its leakage . In the present study, we used a low - dose rhbmp-2 (0.7mg / level) and placed it exclusively inside the cage . In approximately half of the patient the incidence of dysphagia and neck swelling in our series was 13.2% and 8.6% respectively . Only 2 patients necessitated readmission and both of them responded well to the intravenous dexamethasone . The incidence and severity of complications were lower in the patients, in whom dbm was used, though these observations were not statistically significant . Interestingly, in a large number of the patients, the aforementioned symptoms resolved spontaneously . A careful evaluation of previous studies also gives a clue to the effect of dose and delivery methods on the safety profile of rhbmp-2 in acfs (table 3). As already discussed, higher dose and placing bmp sponge outside the cage were probably responsible for the greater incidence of adverse events in the case series presented by schilds et al.10). Apparently, buttermann3) used a smaller dose of bmp as compared to boakye et al.2) (0.9 vs 1.05 mg); however, the incidence of complications was lower in the later study . Placement of infuse both within and outside the cage may be a possible explanation for higher incidence of adverse events observed by buttermann . Usage of rhbmp-2 in acfs has been described as a safe technique by several authors11213). It is also evident from the review of these studies that the authors either used low - dose bmp or placed it exclusively inside the cage or both (table 3). On the basis of our findings, it appears that low dose bmp placed only inside the cage has a lower complication rate than previously reported higher doses (table 3). Unlike higher readmission and reexploration rate in these reports, we had only 2 readmissions with bmp related complication and none of them required any surgical intervention . However, the risk of complications associated with the use of low - dose bmp seems to be greater than risk without its use . In a recent report from our institution, we documented relatively a lower risk of morbidity in a large group of patients who underwent acdf without bmp (overall 8.4%, dysphagia 3.3%, neck hematoma 0.1%, and recurrent laryngeal nerve palsy 0.1%)8). There are other factors, which may also influence the risk of complications in acfs using bmp . Tumilian et al.13) stated that irrigation should be avoided after the placement of bmp to minimize any displacement of the protein into surrounding structures that can lead to inflammatory reactions in the adjacent tissues or cause heterotopic bone formation . Perioperative steroid administration is also found to reduce the complaints of dysphagia and neck swelling3). Unless contraindicated the impact of type of spacer on the safety profile of bmp in acfs is unknown . The amount of rhbmp-2 in infuse sponge cannot be accurately measured and it is also difficult to determine that how precisely 1/3 portion was separated from the remaining sponge . The impact of selection bias related to the surgeon's choice of using dbm in approximately half of the cases also cannot be denied . We also believe that the cost - effectiveness of rhbmp-2 and dbm should also be evaluated, which is another limitation of the present study . The use of low - dose rhbmp-2 in anterior cervical fusion is not without risk . However, in comparison to previous reports with high dose bmp placed both inside and outside the cage, a low - dose of bmp placed exclusively within the spacer is associated with lower incidence and severity of complications . Placing dbm putty around the bmp sponge does not seem to affect the safety profile of bmp in acfs.
The aim of this study was to measure the level of oncostatin m (osm) a gp130 cytokine in the gingival crevicular fluid (gcf) and serum of chronic periodontitis patients and to find any correlation between them before and after periodontal therapy (scaling and root planing, srp). 60 subjects (age 2550 years) were enrolled into three groups (n=20 per group), group i (healthy), group ii (gingivitis) and group iii (chronic periodontitis). Group iii subjects were followed for 68 weeks after the initial periodontal therapy (srp) as the group iv (after periodontal therapy). Clinical parameters were assessed as gingival index (gi), probing depth (pd), clinical attachment level (cal), and radiographic evidence of bone loss . Gcf and serum levels of osm were measured by using enzyme linked immunosorbent assay (elisa). It was found that mean osm levels had been elevated in both the gcf and serum of chronic periodontitis subjects (726.65 283.56 and 65.59 12.37 pgml, respectively) and these levels were decreased proportionally after the periodontal therapy (95.50 38.85 and 39.98 16.69 pgml respectively). However, osm was detected in gcf of healthy subjects (66.15 28.10 pgml) and gingivitis - suffering subjects (128.33 22.96 pgml) and was found as below the detectable limit (0.0 pgml) in the serum of same subjects . Significant correlation has been found between clinical parameters and gcf - serum levels of osm . Increased osm level both in the gcf and serum, and the decreased levels after initial periodontal therapy (srp) may suggest a use as an inflammatory bio - marker in the periodontal disease.
Basal cell carcinoma (bcc) is a common skin tumor that constitutes 75% of non - melanocytic skin tumors . More than 80% of bcc cases are localized in areas exposed to the sun, such as the head and neck . The most important etiological factor is exposure to ultraviolet light, while other risk factors include advanced age, male gender, immunosuppression and arsenic exposure . Bcc can also develop on areas of chronic inflammation, burn scars, squamous cell cancer and ulcers [1, 2]. It is quite rare in areas with no ultraviolet exposure, especially the genital and perianal regions . A 34-year - old male presented with a bleeding perianal mass that had been growing for the last 6 months . There was nothing of significance in the personal or family history and especially no history of anogenital warts, sexually transmitted disease, malignancy, inflammatory dermatosis or arsenic exposure . Dermatology examination revealed an exophytic condyloma - like nodular lesion approximately 3 6 cm in size with surface ulceration in the left lateral perianal region and no other pathological findings on whole body examination (fig . Laboratory test values were normal and the hiv, hsv and syphilis serology results were negative . Histopathological evaluation of a punch biopsy obtained from the lesion showed an edematous stroma containing mononuclear inflammatory cells under a stratified squamous epithelium with epithelial cell islands containing pleomorphic cells and showing infiltrative growth and tumor cell islands with peripheral palisading (fig . The tumor cells had a pleomorphic character, with a large vesicular nucleus and narrow cytoplasm (fig . Histopathological examination of the total excision material was also found to be consistent with superficial bcc . Bcc is seen at a rate of 0.2% among all anorectal cancers and about 1% of all bcc cases are located in the perianal region [3, 4]. A review of the literature reveals that the disorder is more common in males, with a mean age of 73 years . The etiopathogenesis includes chronic trauma such as itching, chronic dermatitis, nevoid bcc syndrome, p53 mutation, immune deficiency, sexually transmitted disease and arsenic exposure [3, 5]. In contrast to squamous cell carcinoma, perianal bcc cases do not show hpv positivity . We did not find any other bcc lesions in our patient by whole body examination, and the trigger factors of disease development were not detected . The differences between the perianal bcc in our patient and those reported in the literature is the early age and lack of known risk factors, indicating that our patient's disease developed on a background of genetic predisposition via the sonic hedgehog pathway . The differential diagnosis of perianal bcc includes condyloma acuminatum, bowen's disease, paget's disease and squamous cell carcinoma . It is important to distinguish perianal basaloid (cloacogenic) squamous cell tumors because of their aggressive course and early metastatic potential . The typical palisading appearance and the presence of ber - ep4 monoclonal antibody expression indicate a diagnosis of perianal bcc [7, 8]. Patil et al . Emphasized that a lack of tumor retraction artifact and atypical mitotic figures on histopathology as well as the presence of strong ber - ep4 and bcl2 staining on immunohistochemical evaluation indicate bcc . Hpv positivity and p16ink4a upregulation can be present in anal squamous cell carcinomas, but hpv has been shown not to play a role in perianal bcc etiopathogenesis [9, 10]. The bcc diagnosis in our patient was made with the typical histopathological findings and hpv assessment was found to be negative . The most common perianal bcc type is nodular bcc [3, 8], whereas our patient was diagnosed as superficial bcc . A literature review revealed that gibson and ahmed had observed the superficial type as the second most common type at a rate of 18% among perianal and genital region bcc cases, and that patil et al . Had found one nodular / superficial and one superficial bcc case among the nine perianal bcc lesions that they had evaluated histopathologically . Bcc rarely metastasizes while perianal bcc can have an aggressive course and show more frequent metastasis . Local excision is frequently recommended for bcc treatment, but cryotherapy, radiotherapy, immunotherapy, electrodestruction, photodynamic therapy, immunotherapy, retinoids and chemotherapy are other treatment options when excision is not possible . We achieved cure with total excision of the lesion in our patient, and new lesion development was not observed during 8 months . Perianal bcc cases are diagnosed later than other types and can be mistakenly treated as inflammatory or infectious lesions . We also want to emphasize that local excision treatment at the early stage following an accurate diagnosis is effective in ensuring cure without advanced treatments.
A 74-year - old female patient underwent a roux - en - y cystjejunostomy for pancreatic pseudocyst developed several melena episodes and she was surgically reappraised . A 12 8.0 5.0 cm retro - gastric lesion was resected and pathology report indicated an unsuspected gastrointestinal stromal tumor (gist). The report aimed to describe an atypical presentation of gist medical literature has previously shown that a pancreatic cystic neoplasm could be mistaken for a pancreatic pseudocyst . Cystic tumors of the pancreas most commonly corresponds to serous microcystic adenoma, intraductal mucinous tumor, mucinous cystic tumor, and solid pseudopapillary tumor and, less commonly to cystic endocrine tumors, cystic metastasis, cystic teratomas, and lymphagiomas 1 . The case reported aimed to redefine the differential diagnosis of a presumed pancreatic pseudocyst, emphasizing the possibility of gist . A 74-year - old female patient was admitted to the internal medicine ward of so paulo hospital in april, 2012 . The main complaint was gastrointestinal bleeding identified as melena, occurring some weeks after a roux - en - y cystjejunostomy for a pancreatic pseudocyst . Bleeding was not, initially, thought to be related to the previously done cystjejunostomy, as it is an allegedly uncommon complication of that procedure . Several gastric erosions were observed but thought to be unrelated to the patients bleeding, and the urease test was positive for helicobacter pylori (hp) infection . Although there was no evidence of gastric bleeding, the initial approach consisted of a proton pump inhibitor and antibiotic therapy, for hp . Because the melena persisted, the patient underwent colonoscopy, which revealed diverticulosis in both the ascending and transverse colon . Other diagnostic procedures included an abdominal ct scan and technetium-99 m - labeled rbc scintigraphy, both of which had nonspecific findings and were not diagnostic . Repeat uge showed complete resolution of the gastric erosions which led to the search for a lesion in the small bowel . By this point in her clinical course, the patient had required 20 packed red cell transfusions . A small bowel barium study revealed findings compatible with a retro - gastric tumor causing extrinsic compression of the stomach . At that time, the medical team considered that the main diagnostic concern was a pancreatic cystic neoplasm masquerading as a pancreatic pseudocyst and the patient underwent a laparotomy (fig.1). At surgery, a retro - gastric lesion measuring 12 8.0 5.0 cm was resected and the pathology report indicated an unsuspected gastrointestinal stromal tumor (gist) (fig.2). Histologic findings were an epithelioid and fusiform mixed pattern tumor, with high mitotic grade (21 of 50 high - power fields) and central cystic degeneration . By immunohistochemistry, the tumor expressed c - kit, vimentin, dog 1, and ki 67 (figs.3 and 4). Gists are rare, with an estimated incidence of 1.5/100.000 per year 2 and encompass tumors primarily derived from cajal interstitial cells pacemaker cells which regulate gastrointestinal tract motility 3 . By immunohistochemistry, in fact, the kit mutation leads to kinase activation, and experimental studies have demonstrated that the mutation is capable of causing cajal cells hyperplasia and gist - like tumors in mice 3 . Any part of the gastrointestinal tract from esophagus to rectum may be involved, although stomach (60%) and small bowel (30%) are the predominant organs affected . The clinical expression of disease tends to be with gastrointestinal bleeding and nonspecific abdominal complaints . Some tumors from outside the gastrointestinal tract are called extragastrointestinal stromal tumors or egists; most of them are from the omentum, mesentery or retroperitoneum 3 . Gist pathology report encompasses three tumor subtypes: a fusiform cell (70%), an epithelioid cell (20%), and a mixed pattern . The fusiform cell subtype correlates with the kit mutation (cd117 positivity), the tumor cells present a spindled phenotype, and the clinical course of such tumors is usually less aggressive . The other histological patterns (epithelioid cells and mixed pattern) correlate with platelet - derived growth factor receptor alpha (pdgfra) expression (cd34 positivity), although such correlation does not consistently occur . Tumors with a predominantly epithelioid pattern and pdgfra mutation tend to have a favorable prognosis . On the other hand, an epithelioid or mixed pattern accompanied by the kit mutation not pdgfra mutation which occurs particularly in gists of the stomach, tends to have an unfavorable course . By immunohistochemistry, 95% of gists express cd117, and these kit mutated tumors respond to therapy with kit inhibitors (imatinib or sunitinib). Less than 5% of gists are kit immune - negative; many of them have the pdgfra mutation and respond to targeted therapy as well . A third type, called wild type and without either kit or pdgfra mutation, has been described too . Other histological markers are s-100 protein (5%), desmin (2%), a recently described discovered on gist protein (dog1, 95%) 3 and ki-67, the expression of which correlates with a high mitotic grade of tumor cells and with a worse prognosis 4 . By the time the patient had the second operation, the medical staff had already estimated gross probabilities of different diagnostic possibilities . First, bleeding should be regarded as a potential surgical procedure complication . According to newell et al . (1990), bleeding after roux - en - y cystjejunostomy is definitely an uncommon complication, occurring in only 2% of the cases 5 . Second, a colonic source of bleeding should be considered despite a presentation as melena . Previous studies have focused on the diagnostic yield of colonoscopy in the investigation of bleeding after a negative uge; positivity rates of 23.5% to 30% were reported . The main limitations observed were overvaluing the role played by lesions with low implication for bleeding and small sample sizes 6,7 . A recently published study with better design has reported a 14.2% positivity rate for colonoscopy (4.8% if only lesions with a high implication for bleeding are included) 8 . Finally, melena should be considered to result from some previously neglected condition, for example, a pancreatic cystic neoplasm mistaken for a pancreatic pseudocyst 9 . Other possibilities would be small bowel carcinoid 11, dieulafoys lesion 12 and other rare diseases . Combining data from those reports, it could be estimated a 2% bleeding probability related to the cystjejunostomy previously carried out; a 4.8% melena probability from a colonic source; finally, a 12% bleeding probability from an unsuspected pancreatic cystic neoplasm . The upper gastrointestinal bleeding investigation points to the small bowel whenever uge and colonoscopy are unrevealing, and endoscopic capsule and small bowel study should be considered . In the case presented here, a small bowel barium examination was able to uncover a suspicious lesion . By means of probabilistic reasoning, to our knowledge, gist masquerading as pancreatic pseudocyst is rather unusual, and we found only one prior report of such an atypical presentation in the medical literature 13 . In fact, the case reported by aggarwal and colleagues was a metastatic gastric gist mistaken for pancreatic pseudocyst . On the other hand, the case reported here seems to be a primary retroperitoneal gist . This type of tumor, actually known as egist, seems to be a rare medical finding . A case series from the national cancer institute of brazil (inca) reported only nine egist cases over a period of 13 years from 1997 to 2009 14 . Primary egist of the retroperitoneum seems even rarer, with only 58 cases reported in the medical literature according to casella and colleagues 15 . In conclusion, it is not an overstatement to say that any presumed pancreatic pseudocyst merits double attention . It might be a pancreatic cystic neoplasm as the medical literature has previously shown or even a gist, as this report has emphasized.
Acute acalculous cholecystitis (aac) is an acute necroinflammatory disease of the gallbladder without gallstones that is typically seen in critically ill patients (1,2). The pathology includes endothelial injury, gall bladder ischemia, and stasis and these conditions may cause a concentration of bile salts and gallbladder distension, eventually leading to necrosis of the gallbladder tissue (1,2). Marked gallbladder wall thickening (3.5 mm) and pericholecystic fluid are the most common imaging features of the disease (2). Approximately 10% of all acute cholecystitis cases are aac, and the mortality is high in such cases without prompt treatment (1,2). Aac can be related to trauma, surgery, shock, burns, sepsis, total parenteral nutrition, and/or prolonged fasting (2), and it can also be accompanied by autoimmune diseases . Macrophage activation syndrome (mas) is an aggressive and potentially lethal syndrome of uncontrolled immune activation . In mas, macrophages become activated and overproduce cytokines, thereby causing severe tissue damage and eventually leading to organ failure (3). Mas is thought to be a form of hemophagocytic lymphohistiocytosis (hlh) associated with a rheumatic disease . There are some reports describing the association of aac with systemic lupus erythematosus (sle) and other autoimmune diseases (4 - 6), but the coexistence of aac and mas has so far only rarely been reported . We herein report a case of aac complicating mas, occurring in a patient with recurrent histiocytic necrotizing lymphadenitis (kikuchi disease) and sjgren syndrome . Rheumatologists should be aware of this possible association and keep in mind the fact that aac in rheumatic or autoimmune diseases can be successfully treated with early immunosuppressive therapy, although the risk of perforation and the mortality rate is high in aac . A 41-year - old female was admitted to our hospital because of a high fever with lymphadenopathy that had appeared 4 days before admission . Kikuchi disease had been diagnosed 5 years earlier by a left cervical lymph node biopsy . Sjgren syndrome had also been diagnosed 2 years earlier, but she did not meet the criteria for sle . Physical examination revealed a temperature of 38.2, cervical and axillary lymphadenopathy with tenderness, but no tenderness in the right upper quadrant of the abdomen . Laboratory tests showed thrombocytopenia and a coagulation disorder including hypofibrinogenemia (143.5 mg / dl), suggesting disseminated intravascular coagulation (dic). Elevation of hepatobiliary enzymes, c - reactive protein, lactate dehydrogenase, soluble interleukin-2 receptor (5,040 u / ml), and ferritin (9,176.1 ng / ml) were also noted (table). A histological examination of the lymph node was not performed because of the urgent need for treatment . Mas due to the recurrence of kikuchi disease was diagnosed according to the 2004 diagnostic guidelines for hlh (7). Cervical and axillary lymphadenopathy, hepatosplenomegaly, marked thickening of the gallbladder wall (> 15 mm), pericholecystic fluid, but no signs of intestinal obstruction were detected on computed tomography (ct) and ultrasonography (fig . A gallstone was also found in the fundus, but neither any impaction of the stone nor dilatation of the common bile duct was detected (fig . So - called acute acalculous cholecystitis with a gallstone secondary to mas was therefore diagnosed clinically and radiologically . Aptt: activated partial thromboplastin time, dsdna: double - stranded dna, il-2r: interleukin-2 receptor, mpo - anca: myeloperoxidase - antineutrophil cytoplasmic antibody, pr3-anca: proteinase 3 antineutrophil cytoplasmic antibody, pt / inr: prothrombin time / international normalized ratio imaging performed on admission (a - f) and the sixth day of admission (g, h). A, b: ultrasonography showed marked thickening of the gallbladder wall, a gallstone (a), and pericholecystic fluid (b; arrow), but neither impaction of the stone nor dilatation of the common bile duct was detected . C: cervical lymphadenopathy (arrows) and d: axial lymphadenopathy (arrows) were detected by contrast - enhanced computed tomography (ct). E, f: marked thickening of the gallbladder wall (> 15 mm) and pericholecystic fluid were detected by ct . G, h: follow - up ct performed on the sixth day of the admission showed a significant improvement of the gallbladder wall thickening and pericholecystic fluid . Intravenous pulse methylprednisolone (mpsl) 1 g / day for 3 days, followed by psl 55 mg / day, and meropenem hydrate (1 g / day) were immediately initiated in addition to recombinant human soluble thrombomodulin (19,200 u / day) for dic treatment . Both the fever and lymphadenopathy improved by the next day, as well as thrombocytopenia, coagulation disorder, and hyperferritinemia . On the sixth day of admission, follow - up ct revealed a dramatic improvement of the gallbladder wall thickening and pericholecystic fluid (fig . She has remained clinically well without any relapse of kikuchi disease or cholecystitis even after the psl was tapered to 6 mg / day . Crp: c - reactive protein, ldh: lactate dehydrogenase, mepm: meropenem hydrate, mpsl: methylprednisolone, ne: not examined, plt: platelet count, psl: prednisolone, rhtm: recombinant human soluble thrombomodulin, t - bil: total bilirubin, wbc: white blood cell count aac can develop in patients with mas, but this phenomenon has only rarely been reported . Although the association between aac and mas is still not fully understood, endothelial damage caused by the cytokine storm may be primarily involved in the pathogenesis of aac (2,3,8). The pathogenesis of mas is characterized by an impaired immune response with the hypersecretion of pro - inflammatory cytokines and the activation of cytotoxic cells (8). This excessive immune reaction results in endothelial injury and increased vascular permeability, possibly leading to local microvascular ischemia of gallbladder wall and bile stasis in aac . Endothelial dysfunction has been suggested to play a central role in some forms of drug or infection - associated aac cases (9,10). The incidence of aac in mas is not clear, but it may be relatively frequent in children with hlh . Schmidt et al . Reported that 6 of 9 children with hlh had gallbladder wall thickening on ultrasonographic examination within 1 week of presentation (11). Reported that all of the 6 children with hlh examined by abdominal ultrasonography revealed thickening of the gallbladder wall, as well as in 14 of 21 children with hlh reported by fitzgerald et al . Reported that of 67 children with aac 4 of these children had hlh (14). There is only one case report of an adult patient with mas complicated with aac . Park et al . Reported the case of a 49-year - old female with a 7-year history of adult onset still's disease (aosd) who developed mas and aac (15). Unlike in children, there is no report evaluating the presence of gallbladder thickening in adult patients with mas . These facts suggest that aac could be more frequently associated with mas in children than that in adults, although the pathogenesis of aac is thought to be similar between children and adults (16). The precise mechanisms accounting for this difference are unknown, but one possible explanation is that children in critically ill and febrile conditions are more likely to develop hypovolemia, one of the risk factors for aac (2). Another explanation could be that the treatment for mas, using high - dose corticosteroids and broad - spectrum antibiotics, is normally initiated before abdominal imaging, which would thus improve aac before it can be identified . Aac associated with rheumatic or autoimmune diseases may possibly have a better response to immunosuppressive therapy, compared to that with other causes . In general, surgical intervention is recommended for patients with aac because of the high risk of perforation . It is reported that perforation occurs in 10% and the mortality rate is 30% overall (17,18). A delay in treatment could raise the mortality rate up to as high as 75% (19). However, in the case reported by park et al ., the patient who developed mas and aac was successfully treated with psl and cyclosporine a without surgical intervention (15). It has also been reported that some patients with sle complicated by aac were successfully treated with immunosuppressive therapy (4 - 6). Our patient recovered immediately after the initiation of intravenous pulse mpsl and the follow - up ct showed significant improvement in the gallbladder wall, resulting in no need for surgical intervention . Therefore, early immunosuppressive therapy under careful observation should be carried out for aac cases with rheumatic or autoimmune diseases.
Four randomized trials have been reported in three specific clinical settings that clearly document both the biological and clinical impact of bevacizumab, a human recombinant monoclonal antibody directed against vascular endothelial growth factor (table 1) [1 - 4]. However, while there has been a documented improvement in progression - free survival (pfs) in each of the studies, they have failed to reveal superior overall survival for patients managed on the bevacizumab - containing treatment arm . Bev, bevacizumab; gog, gynecologic oncology group; icon 7, international collaborative ovarian neoplasm group; oceans, ovarian cancer study comparing the efficacy and safety of chemotherapy and anti - angiogenic therapy in platinum - sensitive recurrent disease; pfs, progression - free survival . While it is recognized that not all agree on the interpretation of these findings, it is the opinion of this commentator that it is highly likely the failure to convert a statistically significant improvement in pfs to superior overall survival is a direct result of the fact that the large majority of ovarian cancer patients receive multiple anti - neoplastic agents (including possibly bevacizumab) previously demonstrated to have both biological and clinical activity in ovarian cancer after they complete treatment on the study, which may favorably impact their ultimate survival . Despite the solid evidence - based results of these multiple phase 3 randomized trials (and perhaps at least partly because of them), there remain many quite relevant unanswered questions regarding the optimal utilization of bevacizumab in the management of ovarian cancer (table 2). Hopefully, future clinical trials will help resolve the uncertainties surrounding the use of this important anti - neoplastic agent . The urgency in providing these answers is heightened when one recognizes the major financial impact associated with the routine administration of bevacizumab . Specific treatment setting (e.g., primary chemotherapy; recurrent platinum - sensitive disease; platinum - resistant disease) administered as a single agent (e.g., maintenance therapy) or in combination with cytotoxic chemotherapy appropriate dose (e.g., 7.5 mg / kg [employed in icon7 trial] or 15 mg / kg [employed in gog218 and oceans trials]) establishment of a reliable and clinically useful biomarker to predict the patient population who will benefit or (conversely) who will not benefit from bevacizumab administration treatment with bevacizumab during more than a single treatment episode (e.g., recurrent platinum - sensitive and platinum - resistant disease) overall safety of abdominal surgery performed shortly before or after drug delivery (e.g., risks of delayed wound healing, bowel perforation, vascular events) overall safety of drug delivery in the presence of extensive intra - abdominal cancer in the setting of chemoresistant disease development of longer - term toxicities (e.g., vascular, cardiac, renal) associated with prolonged delivery in patients who experience extended survival cost, economics, etc . Gog, gynecologic oncology group; icon7, international collaborative ovarian neoplasm group; oceans, ovarian cancer study comparing the efficacy and safety of chemotherapy and anti - angiogenic therapy in platinum - sensitive recurrent disease . A preliminary report of the results of a phase 3 trial examining a potential role for pazopanib employed as a maintenance strategy following the completion of primary cytotoxic chemotherapy has revealed that the administration of this agent (compared to placebo) improves pfs (median 17.9 months versus 12.3 months; p = 0.0021; hazard ratio [hr] 0.766). Although data on overall survival remain immature, the initial reported data do not suggest this strategy improves overall survival . Similar to the previously discussed situation with bevacizumab, this outcome is highly likely to be a result of the multiple anti - neoplastic agents delivered to patients once they have completed treatment on this trial . Data from phase 3 trials examining several additional anti - angiogenic drugs (nintedanib; cediranib; trebananib) in ovarian cancer management are eagerly awaited . Phase 2 trials have previously demonstrated the clinical activity of single - agent olaparib, a poly(adp - ribose) polymerase (parp) inhibitor, in ovarian cancer patients with a germline brca 1 or brca 2 mutation . As pre - clinical data revealed similarities in the molecular profiles of brca mutation - positive ovarian cancers and non - mutant - positive high - grade serous ovarian tumors, investigators conducted a phase 2 randomized trial (n = 265 patients) comparing olaparib to placebo employed as a single - agent maintenance strategy in women with high - grade serious ovarian cancers who had achieved a response or exhibited stable disease following a second - line platinum - based chemotherapy regimen . The study revealed a highly statistically significant improvement in pfs (median 8.4 months versus 4.8 months; p <0.001; hr 0.35) in favor of the olaparib strategy . Again, there was no difference in overall survival between the study groups . In a preliminary report of a re - evaluation of this study population, the investigators were able to examine 218 (of the original 265) patients for the presence of germline brca mutations . This analysis revealed an even greater impact of olaparib (compared to placebo) on pfs (median 11.2 months versus 4.1 months; p <0.001; hr 0.17) in patients with a germline brca mutation . It was also noted that of the 37 patients with a known germline brca mutation who were treated with placebo in this trial, 13 subsequently received a parp inhibitor (in addition to other biologically active agents this group of patients may have also received following completion of the trial), potentially seriously compromising an analysis of an overall survival endpoint in this study . Despite the enthusiasm for the development of novel anti - neoplastic agents in ovarian cancer, it must be remembered that classical anti - neoplastic agents (e.g. The platinums, the taxanes) remain the cornerstone in disease management . Furthermore, clinical research designed to optimize the utilization of such agents remains active in the gynecologic oncology investigative community . For example, a phase 3 randomized trial revealed the superiority (pfs endpoint) of a second - line regimen of carboplatin plus pegylated liposomal doxorubicin compared to carboplatin plus paclitaxel in recurrent, potentially platinum - sensitive, ovarian cancer, possibly due to a provocative, essentially unexplained, and striking reduction in carboplatin - associated hypersensitivity reactions when this agent is delivered with pegylated liposomal doxorubicin in the second - line setting . There has been a particular focus on strategies designed to optimize the method of paclitaxel administration in ovarian cancer . The japanese gynecologic oncology group reported the results of a phase 3 trial demonstrating the superiority (pfs and overall survival) associated with administering paclitaxel on a weekly (80 mg / m per week) schedule compared to the standard every-3-week regimen . In this program, the carboplatin was delivered every 3 weeks . In a preliminary report of a multi - institutional phase 3 randomized trial, investigators explored a quite different weekly strategy with both the paclitaxel (60 mg / m per week) and carboplatin (auc 2/week) administered by this novel approach . Compared to standard every-3-week carboplatin plus paclitaxel, the weekly (paclitaxel and carboplatin) schedule was revealed to produce equivalent efficacy but with substantially reduced side effects . Whether the differences in efficacy and toxicity outcomes between these two studies relate to the weekly dose of paclitaxel delivered, the approaches to the administration of carboplatin, unique genetic or environmental backgrounds of the patient populations, or a combination of these factors remains unknown and worthy of future investigative efforts . I would list potential conflicts of interest to include participation in medical advisory boards for: genentech, glaxo - smith kline, amgen, boehringer - ingelheim, celgene, novartis.
Injection lipolysis is a rapidly growing technique for dissolving fat for non - surgical body contouring . A case of solitary lipoma, treated with phosphatidylcholine / sodium deoxycholate without any recurrence even after 9 months is hereby presented . A review of literature regarding this controversial technique is also included . A 35-year - old healthy male with complaints of a gradually increasing growth of 9 months duration on right wrist presented to dermatology opd . Local area examination revealed a 3 3 cm, soft to firm, hemispherical, non - tender, mobile lesion on the medial aspect of right wrist [figure 1]. The patient was not open to any surgical intervention and was given the option of injection lipolysis . After discussing the possible side effects like hyperpigmentation, skin loss, prolonged pain, swelling, or tenderness, late onset hives or itching and skin contour irregularities in detail, an informed written consent for three sessions at 6 - 8 weeks gap was taken . A firm, mobile, 3 3 cm growth on right wrist the lesion was cleaned and draped . The area was divided into small grids (1.5 cm apart) [figure 2]. Solution of phosphatidyl choline and deoxycholate (50 mg / ml) was filled in insulin syringes (4 injections of 16 units of formula). The lipoma was pulled away from the underlying structures and four units were injected in each of four grids (total of 16 units). Care was taken not to inject any material while withdrawing the needle to avoid any intradermal deposition of the drug . The injections were not perceived as painful by the patient . Immediately after the injections there was swelling, redness and burning sensation which persisted for around half an hour [figure 3]. No external compression was used and gradually the swelling settled . In the next 48 h, the lesion shrunk to half of its original size and disappeared completely within next 3 weeks, after the first injection itself . The patient is under follow up for the last 9 months with no evidence of any recurrence [figure 4]. Swelling and redness immediately after the injection injection lipolysis or lipodissolve is the practice of injecting phosphatidyl choline / sodium deoxycholate (pdc / dc) compounds in the subcutaneous fat . Though the use of this technique for non surgical body contouring (e.g., reduction of areas of localized fat like double chin, love handles and abdominal fat) has been increasing rapidly over the past few years, very few studies have been carried out to determine its efficacy for dissolution of commonly encountered lipomas . This practice is growing rapidly but there are still controversies attached with this technique. [46] various injection techniques have been described including use of a syringe (as was done in our case), use of multi - injector device such as mesorelle, and use of mesogun . Indications for injection lipolysis include small, soft areas of localized fat, lipomas, post - liposuction deformities, skin contour irregularities due to traumatic fat necrosis, cellulite, post - fat grafting deformities, and depressed scar with adjoining areas of protruding fat . Larger fat deposits (> 500 ml), fat pad more than 3 cm . Thick, fibrous fat or thinner fat deposits spread over a broad surface area should not be treated with this technique . The exact role of pdc is not clear as dc is supposed to lyse the adipocytes by detergent action . Additives such as vasodilators (procaine, lidocaine, pentoxyphilline), local anaesthetics (bupivacaine) and vitamins such as alpha tocopherol or vitamin b complex are used in various formulas besides the pdc / dc . But none of these have been scientifically proven to improve the results . Currently the standard maximum dose of pdc per injection session is considered as 2500 mg . Average number of treatments required is 3 at a gap of 6 - 8 weeks . However, in the present case only a single session was required . In the past years many physicians worldwide have decried the use of injection lipolysis, claiming a lack of both scientific data and longevity of treatment experience . Source of controversy worldwide is the lack of safety and efficacy recognition by food and drug administration (fda), medicines and healthcare products regulatory agency (mhra), or any other health regulatory agency . A ban on the use of lipostabil in brazil was a source of major controversy . The side effects that can be encountered include poor outcome, atypical mycobacterial infection or an uneven contour . These injections could also cause skin loss, hematoma, muscle injury, or nerve damage if the injector is not aware of the underlying anatomy of the injection area . There have been reports of skin necrosis or multiple areas of skin ulceration and blistering after self injections . Majority of these side effects have been reported when the procedure was done by untrained individuals or in cases of self injections . Bechara et al, studied the effect of lipolysis in cases of multiple familial lipomatosis in 2006 . They achieved a reduction up to 45.8% after four injections at 6 - 8 weeks intervals . In the current study, we have seen a complete dissolution with a single injection and no recurrence in 9 months . There is no scarring and can be used to effectively treat multiple lipomas where surgery may not be a viable option . Hence, it would turn out to be a very cost - effective treatment modality . Absolute contraindications include age <18 years, pregnancy, breast feeding, patient, on anti - coagulant therapy, current serious, or significant illness or active infection, known allergy to soy products, injections for breast reduction, unstable diabetic control, or impaired circulation, body mass index bmi>30, previous adverse reaction to this treatment, severe needle phobia, immunocompromised patients such as transplant recipients and those undergoing chemotherapy . Vascular insufficiency should be ruled out . If performed by a trained doctor in the right patient, injection lipolysis could prove to be a safe and cost effective treatment modality for lipomas . In the current case a study with larger number of patients is needed to establish this technique as a safe and effective treatment modality for lipomas . Also, a comparative study between pdc plus dc versus dc alone for lipolysis would help in clarifying the effect of pdc in adipocyte destruction.
Resin cements are now widely used in clinical dentistry and improved or new versions are constantly being introduced which are claimed to offer advantages over their predecessors.1,2 the ability of luting multiple structures together, high resistance, less solubility in mouth liquids and colour options makes resin cements an alternative in cementation of esthetical restorastions.3 adhesive cementation systems have been considered the best option for luting ceramic restorations and the application of resin cement on prepared teeth has demonstrated good biomechanical behavior, particularly with ceramic restorations.1 due to their chemical structures, resin cements adhere to tooth structure . The bonding of resin to dentin is complex; it's achieved through penetration of hydrophilic monomers to partially demineralized apatite structure of etched dentin . Hence, adhesion is created via micromechanical interlocking of resin to hybrid layer or resin diffusion zone.3 the mechanism of modern adhesion is currently believed to be based on micromechanical interlocking rather than on primary chemical adhesion . Resin infiltration into demineralized dentin permits formation of hybrid layers and resin tags thus producing micromechanical retention.4 resin tags are formed in the opened dentinal tubules which are intended to contribute to the final dentin bond strength.5 the contribution of resin tags to bond strength relative to the role of intertubular dentin, depends on the orientation of dentinal tubules and dentin depth of tested materials . While the penetration of resin tags into dentinal tubules is believed to contribute little to final bond strength, the adaptation to inner tubule walls probably contributes significantly much more to bonding efficiency . Hybridization by resin interdiffusion into the exposed dentin collagen layer, combined with attachment of resin tags into dentin tubules, appeared to be essential for a reliable dentin bonding.4,5 current literature showed several contradictory interpretations regarding the formation of tags: some researchers found no correlation between bond strength and formation of resin tags,6,7 while others appreciated that the resin tags may contribute about 30% to the total strength of adhesive - dentin bonding8,9 or at least that the resin tags are a major factor influencing the bond strength.10 also, it was reported that micromechanical retention of the dentin surface is not adequate without resin tags.11 from that point, it may be thought that resin tags are essential for improving microshear bond strength . Residual cement and debris might impair etching quality of tooth surface, infiltration of adhesive system, or may even inhibit the polymerization of resinous monomers and thus the fit and final bonding of restoration.12 if proper cleaning of the dentin surface of abutment teeth is not carried out, the interaction between resin cement and dentin may be weakened or lost, resulting in bond failure . Although several investigators have studied methods for the removal of remnants of provisional cement in vitro,12 there is no research related to the techniques in cleaning of permanent resin cement residuals on dentin in dental literature . A great number of cleaning agents for dentinal surface, which are based either mechanical or chemical methods have been reported.13,14,15,16 the most common techniques for chemical cleansing include the use of clorhexidine digluconate, sodium hypochlorite, hydrogen peroxide and ethylene diamine tetra acetic acid (edta).13 in addition, endosolv r has been improved for cleaning of resinous materials and resinous remnants from dentin canals by its softening / dissolving ability on resins.17 so, it may be effective on eliminating resin remnants from dentinal tubules in repetitive cementations . Laser etching of dentin has been reported to yield an anfractuous surface and open dentin tubules, both apparently ideal for adhesion.18 the member of erbium laser family, the erbium, chromium: yttrium - scandium - gallium - garnet (er, cr: ysgg) hydrokinetic laser system has been useful for preparing tooth surfaces for adhesion.11,19,20 although its efficiency was reported, bond strength of composite resin cement to tooth substrate prepared by erbium laser are often confusing and contradictory.18 some researchers noted that lasers may be used to increase shear bond strength (sbs) by treating dentin surface in prosthodontic practice19,21,22 while others found similar bond strength values with or without the application of er, cr: ysgg laser.20,23 considering the previous experiments with erbium lasers in composite resin removal and roughening of the surface,24,25,26,27 the use of these systems may be applicable for removing adhesive cement remnants from dentinal tubules in repetitive cementation . The purpose of this study was to examine the effect of chemical agents and er, cr: ysgg laser operated at different outputs on microshear bond strength between dentin and resin cement in repetitive cementation via eliminating or decreasing the amount of adhesive cement residuals from dentin tubules . This study was conducted after approval of research ethical committee of near east university (approval no: 79/2013). A total of 90 non - carious, intact human molar teeth which were extracted within 3 month period, were selected and stored in distilled water at 4. prior to use, teeth were washed under running water to eliminate storage solution residues . The teeth were embedded in an acrylic resin (heraeus kulzer ltd, newbury, london) with the occlusal surface of the crown upwards and parallel to the base of resin block . Each sample was sectioned at a level below occlusal pit and fissure level perpendicular to the long axis of the tooth with a diamond blade saw (precision sectioning saw, isomet 1000, buehler, il, usa) on an automated sectioning device under water irrigation, to have superficial dentin cross sections in 1 mm . Then, exposed superficial dentin surfaces were polished with a 600 grit silicone carbide paper (zibo sisho mt coated abrasive co, ltd, shandong, china) to create a flat surface with standardized smear layer formation . Afterwards, all dentin cross sections with mesio - distal width facing upwards were fixed on a chemically cured acrylic resin blocks with 2 cm diameter and 1.5 cm height, with cyanoacrylate adhesive (zapit base, dental ventures of america, corona, usa). The dual cure resin luting cement (variolink n professional set, ivoclar vivadent, schaan, liechtenstein) was applied to the surface of sliced teeth with the use of cylindrical tubes with dimension of 4 mm 4 mm, according to the manufacturer's instructions . The cement was light cured for 40 seconds with halogen light source which has 450 - 500 nm wave length and 500 mw / cm power (hilux dental curing light unit 250, benliolu dental inc ., two parallel resin cylinders were located on each sample at least 1 mm far from dento - enamel junction (fig . Bonding areas of resins were defined with a permanent marker to help performing cleaning techniques and placing repetitive resin cylinders in appropriate area . Initial debonding was performed, after 24 hours which is required duration for complete polymerization, by removing of resin cement mechanically with a carving instrument without harming any teeth tissue until dentin surface appeared macroscopically clean (fig . Samples were randomly assigned to 6 groups of 30 specimens each, according to cleansing procedures on removal of adhesive remnants which is listed in table 1 . Any surface treatment was not applied to samples in group 1 (control group)., ny, usa) or endosolv r (septodont, cedex, france) which is consist of 66.5 g formamide and 33.5 g phenylethyl alcohol, was applied all over the marked areas of the samples . Following step for either group 2 or group 3 was rinsing of organic solvents and drying teeth with absorbent paper to avoid dentin dehydration . An er, cr: ysgg laser (waterlase md, biolase technology inc, ca, usa) with wide output power range between 0.1 - 8 w, operating at a wavelength of 2,780 nm was used for group 4, 5 and 6 . The laser energy was delivered through a fiber - optic system to sapphire tip terminal 600 m in diameter (mmg6, biolase md technology inc ., ca, usa). Samples were lased at 1.25 w (group 4), 2 w (group 5) and 3.5 w (group 6) output power for 15 seconds in non - contact mode with the angle of 85-95 to the flat specimen surface with a 1 mm fixed distance from the laser tip . A sweeping motion was used to achieve an even coverage of surface by overlapping the laser impact . To standardize the distance, holes, 4 mm 4 mm in size were constructed on the surface of 1 mm acrylic disc . The acrylic disc was placed on the teeth surfaces for all specimens which were irradiated, to avoid unnecessary laser irradiation . Investigation of treated dentin surfaces was performed by scanning electron microscopy (sem) (quanta 400f field emmision sem, tokyo, japan). The specimens had their surfaces desiccated and coated with au - pd alloy for sem examination at 1,500 - 2,000 magnification . A tube with an inner diameter 1 mm and height of 1 mm was attached to each of the treated or untreated (control group) dentin surfaces . Resin cement (variolink n, professional set, ivoclar vivadent, schaan, liechtenstein) was applied and polymerized for 40 seconds with halogen light source (hilux dental curing light unit 250, benliolu dental inc ., prior to microshear bond strength (sbs) test, the specimens were stored in distilled water at 37 in an incubator for 48 hours . Sbs tests were performed by using an universal testing machine (ez - test 500 n shimadzu, kyoto, japan) and a computer program (nexygen software, lloyd inc . 2) head at a cross head speed 1 mm / min until the composite resin cylinder was dislodged from tooth surface . Samples were oriented in a holding vise so that the direction of shear testing was parallel to the flat surface and the loading head is in contact to the bonded tooth surface . The maximum load to failure sbs was calculated according to the following formula and expressed in mpa: stress = failure load (n) / surface area (mm). After sbs testing, failure modes were assessed for each specimen under a light microscope (olympus sz 40, sz - pt, tokyo, japan) at 100 magnification and classified as: adhesive, cohesive and mixed (both adhesive and cohesive) failure . All statistical analysis were performed with statistical package for special science spss 18.0 software (spss inc ., descriptive statistics, including the mean, median, standard deviation, minimum, and maximum, were calculated for each of the six groups . After analysis of the normal distribution of sbss of samples with shapiro - wilk (sig) test and test of homogeneity of variances, comparisons of the medians of sbs in six groups were carried out with kruskal wallis h test with bonferroni correction . The control group showed scratches, fundamental smear layer and plugs in the sem observations of untreated dentin (fig . Sem revealed, diffuse cement layer particularly removed in small insular areas in group 2 . 3b). In group 3, there were sporadically closed dentin tubules (fig . When the circles were examined, some areas were found to be perforated up to enamel border and also dentin tubules were open . In addition, even dentin and opened dentinal tubules without the presence of cement remnants were appeared . Laser almost exposed even dentin and few open dentinal tubules in group 6 (fig . Mean microshear bond strength standard deviation (mpa) for each group was 34.9 17.7, 32.1 15.8, 37.8 19.3, 31.3 12.7, 44.4 13.6, 40.2 13.2 respectively . When the two by two comparison had been done among 6 groups (p<.05), group 5 revealed statistically significant difference from group 1, group 2 and group 4 . Also, comparison indicated that group 6 has statistically different microshear bond values from group 4 . The mode of failure of specimens after sbs test was presented in percent (fig . 4). Also, the most common fracture pattern observed in the study was mixed and cohesive . It is important to better understand what to expect when a tooth is debonded more than once . In a study, findings indicated that shear bond strength during second bonding / debonding sequence significantly decreased approximately 61% due to cement residues.27 in present study, the effectiveness of er, cr: ysgg laser in repetitive cementation for cleaning of adhesive cement remnants from dentin tubules was compared with some other methods . Bond tests are used for examining resistant of material by imitating the possible tension forces which the restorations would be exposed in oral circumstances.28 shear bond strength testing with bonded cross - sectional areas of 1 mm or less is also referred to as microshear bond strength . This relatively simple test permits efficient screening of adhesive systems, regional and depth profiling of a variety substrates and conservation of teeth . In addition, testing of multiple specimens from a single tooth conserves teeth and allows research design which is not possible using conventional macro methods29 that is why microshear bond test was preferred rather than shear bond test in this study . Generally, there is variability among the dentin bond values reported by various researchers.19,20,22 this may be attributed to different testing methods and conditions, the varying nature of dentin substrate and the composite adhesive used . Morphological and structural variations in dentin may influence the bond strengths of the adhesive systems to dentin.30 researchers noted that most of the adhesive systems gave higher microshear bond strength to superficial dentin and they mentioned progressive decrease in bond strengths to deeper dentin.31,32 in addition, it was shown that age factor may affect bond strength.33,34,35 for this reason, superficial dentin layers from the teeth which were collected from the patients with the age group between 20 and 30 were used in present study for both standardization and higher bond strength . Also, teeth were cut with micro saw under water cooling with diamond burs to avoid sudden increase in temperature rate and samples were kept in distilled water at 4 until usage.36 various techniques involving organic solvent application were used for removing cement residues . In a study, edta was effective in removal of the remnants of provisional cement, so edta was considered to create appropriate physical and chemical interactions between the resin cement and dentin.15 with regard to this finding, edta was preferred as one of the dentin cleansing agents in this study . According to some researchers, 17% edta usage did not significantly alter or decrease the sbs values14,37 while others concluded that dentin treatment with edta is effective in improving bond strength.15,38 however, in the present study, results indicated that edta did not show significant difference in sbs from control group so it was found ineffective in dentin cleansing . Concurring with previous study14 samples in edta group showed more adhesive fracture pattern compared to the untreated (control) group in failure of mode analysis . On the other hand, endosolv r group showed higher sbs than control group and also comparable mean to 2 w and 3.5 w laser groups . Also in a study, it was conducted that endosolv r prevented deterioration of resin - dentin bond strength17 while others concluded it had no positive effect on the bond strength.39 therefore it may be conceivable to use this organic solvent rather than laser systems which have much incommensurable cost . Although endosolv r was found to have superior penetration40 and eradicate residual materials,41,42 manufacturer suggested two - visit application for better utilizition40 which is a time consuming . With respect to micromorphological changes in lased dentin surface, sem analysis figured out better findings than endosolv r group which 2 w and 3.5 w laser groups were completely lacking out of smear with opened dentinal tubule orifices without any widening . Moreover, it is prudent to point out that the formamide which is one of the co - solvent of endosolv r, is a potential teratogen . Thus, female workers / patients of child - bearing age must be made aware that a known teratogen is being used and appropriate' right - to - know' compliance policies be adhered to by providing them with data from appropriate animal studies.17,43 besides, there are limited numbers of clinical reports which conducted on usage of endosolv r, in the dental literature so further investigations are mandatory . Currently, many studies have been focused on the evaluation of the effectiveness of er: yag and er, cr: ysgg lasers on bond strength between dentin and cement.11,19,21,22,23,24 er: yag and er, cr: ysgg lasers can selectively effect on the surface of dentin, resulting in an irregular surface pattern that may potentially improve the micromechanical retention of adhesive systems to dentin, when used at appropriate doses . The laser system used in this study was er, cr: ysgg which is a hydrokinetic one . The main advantage of lasers was avoiding immediate increase in temperature which may result in an inflammatory pulpal response . With this system not only could the temperature be suppressed, but also cutting efficiency could be increased . Laser energy is delivered through a fiber optic system to a sapphire tip terminal.19,22,44 the average output can be varied from 0.1 w to 8 w. for dentin irradiation, several investigations have suggested irradiation outputs varying from 1.25 w to 5 w can be used.19,21,22,38,44 in a study it has even used the maximum output power of 5 w,19 providing increased value of energy density and as a consequence, a higher efficiency of laser ablation . However, use of higher values of energy density has also been related to a more harmful thermal effect on dental hard tissues, particularly when laser irradiation is nor accompanied by a continuous air water spray . The presence of micro cracks under partially attached dentin particles may provide a weakened substrate, which is more prone to the occurrence of fractures during bond strength testing.44 to avoid these negative effects, we used continuous air - water spray continuously at 65% and 55% percentages, respectively . In the study, in order to irradiate the dentin, 1.25 w, 2 w and 3.5 w outputs were used . Laser irradiation with high power outputs show higher sbs means while lower laser outputs demonstrated significantly lower sbs and closed dentinal tubules . In laser groups, kruskal wallis h test detected significant difference between the sbs means of 1.25 w lased dentin surfaces and other 2 groups, while there was no any significant difference between 2 w and 3.5 w laser application . Commercial dental laser manufacturers claim that enamel and dentin can be successfully etched at lower power settings with the erbium lasers . Apel et al.45 investigated the ablation threshold of er: yag and er, cr: ysgg lasers used when preparing tooth structures and noted that there is a possibility of micro cracks developing in dental structures below the ablation threshold . These cracks act as starting points for fracture and failure, which may reduce or eliminate the possible positive effect of erbium laser irradiation . What is more, researchers considered power setting of 1.5 w is the lowest power setting that is able to thoroughly treat the dentin surface, as assessed by light microscopy.19 the sbs values of 1.25 w laser applied group which significantly differ from the other 2 laser groups may be explained by this theory . The literature had shown that surfaces irradiated by 2 w er, cr: ysgg laser displayed rough surface.46 in the present study, it may be thought that laser act as an etching agent to make rough dentin surface and therefore cause an increase in the sbs values of group 5 . However, in previous studies, researchers claimed that laser irradiation with 2 w, 3.5 w and similar doses had no positive effect on bond strength between dentin and cement,11,22,47 in other words they pointed out that there was no increase in etching . Moreover, moretto et al.48 concluded that er, cr: ysgg laser irradiation interacts with the dental hard tissue resulting in a specific morphological pattern of a dentin and collagen fibrils that negatively affected the bond strength to resin . At this point, it may be concluded that, the procedure done in this research primarily was not etching with laser, it was cleaning of resin cement remnants from dentin tubules that led new resin tag formation in repetitious cementation which increased sbs . In the limitations of the study, it appears that laser application of 2 w and 3.5 w laser irradiation may be effective in increasing sbs and an attractive alternative for cleaning cement residues from dentinal tubules in repetitive bonding.
There is evidence that sufficient and restoring sleep is associated with a wide variety of increased cognitive performance1,2 and emotional processing,3,4 and also with personality traits5 such as optimism6 and mental toughness.7,8 in contrast, dysfunctional cognitive emotional processes such as symptoms of depression,9,10 anxiety,9 and perceived stress1113 are associated with poor sleep quality . As regards the personality traits of perfectionism, numerous studies indicate that perfectionism and poor sleep are associated both cross - sectionally14,15 and longitudinally.16,17 perfectionism is understood as a set of cognitive emotional attitudes such as unrealistically high and rigid standards for performance, fear of failure, excessive self - criticism, and an inability to derive satisfaction from achieved goals.1820 in a search for the developmental origins of perfectionism, hibbart and walton21 conducted semistructured interviews and observed that unlike nonperfectionist undergraduate students, perfectionists reported feeling pressure from their families to succeed . Moreover, perfectionists also reported that their parents were overly critical of their mistakes when they were growing up . Hibbart and walton21 concluded that perfectionism was a personality trait that seemed to develop over childhood and adolescence as a result of parents (dysfunctional) expectancies and pressure . As regards the definition of the personality trait of perfectionisms, there is agreement that perfectionism consists of a set of dysfunctional cognitive emotional processes.1821 this definition is important because numerous studies indicate that dysfunctional cognitive emotional processes cause sleep disturbances . To illustrate, riemann et al13 proposed a cognitive behavioral and neurobiological model of insomnia to explain this link: briefly, psychological stress and inadequate problem - solving, along with worrying and rumination, lead to sleep disruptions (ie, delayed sleep onset latency, more awakenings after sleep onset, early awakening in the morning). In parallel to these cognitive emotional processes and sleep disruptions, physiological processes such as an increased secretion of cortisol and orexin, and a decreased secretion of serotonin, lead in the longer term to acute cortical arousal and a deterioration in homeostasis . Riemann et al13 thus explain sleep disturbances as the result of cognitive emotional processes in conjunction with underlying neurophysiological processes . In addition, harvey22 claims that individuals who suffer from insomnia tend to be overly worried about their sleep and about the daytime consequences of not getting enough sleep . Put simply, people suffering from insomnia maintain sleep disturbances via further dysfunctional cognitive emotional processes . However, only a few studies have focused so far on the relationship between perfectionism and sleep, and this relationship deserves particular attention . First, de azevedo et al14 observed, among a sample of 1,163 undergraduate students aged between 17 and 25 years, that socially prescribed perfectionism was associated with sleep disturbance . Second, azevedo et al16 showed that socially prescribed perfectionism predicted poor sleep 1 and 2 years later, suggesting therefore that perfectionism traits causally predicted sleep disturbances among young adults . The authors observed that relative to non - insomniacs, insomniacs were more likely to report doubts about action, frequent parental criticism, and concerns about action . However, focusing on sleep continuity variables, only the perception of increased parental criticism was associated with delayed sleep onset, whereas all other perfectionism traits (concerns about action, doubts about action) were unrelated to sleep continuity variables . Additionally, fourth, spiegelhalder et al23 investigated punctuality behavior (here understood as a sign of perfectionist behavior) among patients admitted to the sleep lab and observed that arriving at the sleep lab earlier than arranged was not associated with scores for insomnia, but a strong relationship was observed between polysomnographic sleep parameters and punctuality; short sleep duration was significantly associated with early arrival times at the sleep laboratory, suggesting therefore, that dimensions of perfectionist traits (here, punctuality) may causally be linked to poor sleep . Whereas the pattern of results of the abovementioned studies suggest a bidirectional relationship between poor sleep and perfectionist traits, to our knowledge possible confounders and latent factors have not thus far been taken into account . This might be surprising given that, among dysfunctional cognitive emotional processes, perceived stress or poor emotion regulation, for example, might contribute to an unknown degree to the explained variance between perfectionism traits and sleep . Fifth, in these respects, and again to our knowledge, jansson - frjmark and linton17 were the first to investigate the predictive value of perfectionism traits for preexisting and future sleep disturbances, while introducing emotional distress as a control . Second, when introducing emotional distress as a confounder, none of the perfectionism subscales contributed significantly to the explanation of preexisting or future insomnia . Importantly, we note that the approach of jansson - frjmark and linton17 corresponds to a mediational model of coping, emotional regulation as well as perceived stress in the relationship of trait perfectionism on current sleep disturbance.2428 collectively, only five scientific studies focused on the relationship between perfectionism and sleep more thoroughly, and only one study17 introduced possible mediating or confounding factors . We took these observations into account and investigated the associations between perfectionism traits and sleep pattern, while concomitantly assessing perceived stress, coping strategies, and dimensions of emotion regulation . We believe that the results may be potentially of practical importance in that, by definition, cognitive behavioral therapies focus on modifying dysfunctional cognitive emotional processes such as perceived stress, coping, and emotion regulation, while, on the flip side, modifying personality traits, which are products of long - term developmental processes, requires more psychotherapeutic effort, and with uncertain results; accordingly, treating personality - related sleep disturbances would turn out much more difficult . Or simply put, if it turns out that perceived stress, coping, and dysfunctional emotion regulation are responsible for the association between personality traits of perfectionism and sleep, then a cognitive behavioral approach to treat these dysfunctional coping strategies and emotion regulation underlying perfectionism traits and sleep disturbances might be more successful . The following two hypotheses were formulated: first, following azevedo et al,16 de azevedo et al14 and vincent and walker15 we expected a direct association between perfectionism traits and poor sleep . Second, following jansson - frjmark and linton17 we expected that the significant association between perfectionism and sleep would disappear when introducing perceived stress, coping, and emotion regulation as mediating factors, that is, following others2428 we expected that the association between perfectionism and sleep disturbances would be mediated via coping, emotional regulation, and perceived stress . A total of 346 young adults (m=23.87 years; sd [standard deviation] = 1.93; 54.6% females) took part in the study . They were recruited from among students of the faculties of medicine and psychology at the university of basel (switzerland). All participants were informed of the purpose of the study and the voluntary basis of their participation . Afterwards, they completed a booklet consisting of questionnaires related to perfectionism, sleep, stress, emotion regulation, and mental toughness, as described in more detail in the tools section . After completion, participants received a voucher of chf15.00 (about 12/usd 15.00) for a lunch at the university canteen . The study was approved by the local ethical committee of basel, switzerland (ethic commission beider basel, basel, switzerland) and performed in accordance with the ethical standards laid down in the declaration of helsinki . A total of 346 young adults (m=23.87 years; sd [standard deviation] = 1.93; 54.6% females) took part in the study . They were recruited from among students of the faculties of medicine and psychology at the university of basel (switzerland). All participants were informed of the purpose of the study and the voluntary basis of their participation . Afterwards, they completed a booklet consisting of questionnaires related to perfectionism, sleep, stress, emotion regulation, and mental toughness, as described in more detail in the tools section . After completion, participants received a voucher of chf15.00 (about 12/usd 15.00) for a lunch at the university canteen . The study was approved by the local ethical committee of basel, switzerland (ethic commission beider basel, basel, switzerland) and performed in accordance with the ethical standards laid down in the declaration of helsinki . Perfectionism was assessed with the german version (fmps - d)19 of the frost multidimensional perfectionism scale (fmps).18 this self - report measure consists of 35 items which, following frost et al18 are divided between the following dimensions: concern over mistakes, doubts about actions; parental expectations; parental criticism, and personal standards . Here, however, for the german version, we followed stber19 and scored the questionnaire for the following dimensions: a composite combining items for concerns over mistakes (i should be upset if i make a mistake .) And doubts about action (it takes me a long time to do something right), labeled concerns and doubts.a composite combining items for parental expectations (eg, my parents have expected excellence from me .) And parental criticism (eg,), labeled parental expectations and criticisms.personal standards (eg, i set higher goals than most people . ). A composite combining items for concerns over mistakes (i should be upset if i make a mistake .) And doubts about action (it takes me a long time to do something right), labeled concerns and doubts . A composite combining items for parental expectations (eg, my parents have expected excellence from me .) And parental criticism (eg, answers were given on 5-point likert scales ranging from 0 (= not at all true) to 4 (= definitively true), with higher scores reflecting greater perfectionism (cronbach s =0.90). The seven items, answered on five - point rating scales (1= not at all, 5= very much), refer to difficulty in falling asleep, difficulties maintaining sleep, increased daytime fatigue, and worrying about sleep . The higher the overall score, the more the respondent is assumed to suffer from insomnia (cronbach s =0.89). To assess sleep schedules and sleep - related psychological functioning, we administered a brief questionnaire based on the pittsburgh sleep quality index (psqi).30 this asks about sleep duration (hours), sleep onset latency (minutes), and number of awakenings after sleep onset for the last five working days . Additionally, participants reported, on eight - point likert - scales, for the mornings: sleep quality (8= very good sleep quality), feeling of being restored (8= completely restored), mood in the morning (8= very good mood); during the day: tiredness during the day (8= very tired; higher scores reflect greater tiredness), and concentration during the day (8= very good concentration); for the evenings: mood in the evening (8= very good mood); (cronbach s =0.84). The perceived stress scale31 consists of ten items and was used to determine perceived overall stress that occurred over the previous month . Answers were given on five - point rating scales ranging from 1 (never) to 5 (very often), with higher scores reflecting greater perceived stress (cronbach s =0.89). The questionnaire consists of 18 items and assesses positive and negative coping strategies.32 positive coping strategies are those that reduce tension in both the short- and long - term, including minimizing the situation, controlling the situation, and self - instruction . Negative coping strategies are those that reduce tension in the short - term but increase stress in the long - term, including social withdrawal, rumination, and resignation . Answers were given on five - point rating scales ranging from 1 (very unlikely) to 5 (very likely). The higher the score, the more pronounced is the coping strategy (cronbach s =0.87). Two composite mean scores were computed reflecting positive and negative coping strategies, respectively . To further calculate an overall score for coping strategies, the ratio between positive and negative coping was calculated, with higher ratios reflecting a greater reliance on positive as opposed to negative coping strategies . To assess emotion regulation, we used the emotionale- kompetenz - fragebogen (emotional competencies inventory).33 the inventory consists of 62 items and assesses the following dimensions: perception and acknowledgement of own emotions (pae: sometimes i feel sad without knowing why); regulation and control of own emotions (rce: when i feel myself getting angry, i know how to cool down again); emotional expressivity (ee: i can express my feelings very well); perception of others emotions (poe: i can perceive and describe my friend s emotions very well). Answers are given on five - point - likert scales with the anchor points 1 (= never / not at all true) to 5 (= practically always / definitively true). Participants were asked to fill in the 18-item mental toughness questionnaire, the short form of the mtq48.34 the short form provides a general score for mental toughness . Answers on the mtq18 are given on five - point likert - type scales ranging from 1 (= strongly disagree) to 5 (= strongly agree). Items were summed, with higher scores reflecting greater mt (cronbach s =0.91). Next, to calculate the relation between perfectionism traits and sleep parameters (sleep disturbance, sleep duration, sleep onset latency, awakenings after sleep onset, daytime sleepiness), perceived stress, coping, mental toughness, and emotion regulation, a series of pearson s correlations was calculated . Finally, to calculate both the direct and indirect relations of perfectionism with sleep disturbance via perceived stress, coping, mental toughness, and emotion regulation, a structural equation model (sem) was computed, using amos (ibm corporation, armonk, ny, usa)., all statistical analyses were calculated with spss 20.0 (imb corporation, armonk, ny, usa) for apple mcintosh . Perfectionism was assessed with the german version (fmps - d)19 of the frost multidimensional perfectionism scale (fmps).18 this self - report measure consists of 35 items which, following frost et al18 are divided between the following dimensions: concern over mistakes, doubts about actions; parental expectations; parental criticism, and personal standards . Here, however, for the german version, we followed stber19 and scored the questionnaire for the following dimensions: a composite combining items for concerns over mistakes (i should be upset if i make a mistake .) And doubts about action (it takes me a long time to do something right), labeled concerns and doubts.a composite combining items for parental expectations (eg, my parents have expected excellence from me .) And parental criticism (eg,), labeled parental expectations and criticisms.personal standards (eg, i set higher goals than most people . ). A composite combining items for concerns over mistakes (i should be upset if i make a mistake .) And doubts about action (it takes me a long time to do something right), labeled concerns and doubts . A composite combining items for parental expectations (eg, my parents have expected excellence from me .) And parental criticism (eg, answers were given on 5-point likert scales ranging from 0 (= not at all true) to 4 (= definitively true), with higher scores reflecting greater perfectionism (cronbach s =0.90). The seven items, answered on five - point rating scales (1= not at all, 5= very much), refer to difficulty in falling asleep, difficulties maintaining sleep, increased daytime fatigue, and worrying about sleep . The higher the overall score, the more the respondent is assumed to suffer from insomnia (cronbach s =0.89). To assess sleep schedules and sleep - related psychological functioning, we administered a brief questionnaire based on the pittsburgh sleep quality index (psqi).30 this asks about sleep duration (hours), sleep onset latency (minutes), and number of awakenings after sleep onset for the last five working days . Additionally, participants reported, on eight - point likert - scales, for the mornings: sleep quality (8= very good sleep quality), feeling of being restored (8= completely restored), mood in the morning (8= very good mood); during the day: tiredness during the day (8= very tired; higher scores reflect greater tiredness), and concentration during the day (8= very good concentration); for the evenings: mood in the evening (8= very good mood); (cronbach s =0.84). The perceived stress scale31 consists of ten items and was used to determine perceived overall stress that occurred over the previous month . Answers were given on five - point rating scales ranging from 1 (never) to 5 (very often), with higher scores reflecting greater perceived stress (cronbach s =0.89). The questionnaire consists of 18 items and assesses positive and negative coping strategies.32 positive coping strategies are those that reduce tension in both the short- and long - term, including minimizing the situation, controlling the situation, and self - instruction . Negative coping strategies are those that reduce tension in the short - term but increase stress in the long - term, including social withdrawal, rumination, and resignation . Answers were given on five - point rating scales ranging from 1 (very unlikely) to 5 (very likely). The higher the score, the more pronounced is the coping strategy (cronbach s =0.87). Two composite mean scores were computed reflecting positive and negative coping strategies, respectively . To further calculate an overall score for coping strategies, the ratio between positive and negative coping was calculated, with higher ratios reflecting a greater reliance on positive as opposed to negative coping strategies . To assess emotion regulation, we used the emotionale- kompetenz - fragebogen (emotional competencies inventory).33 the inventory consists of 62 items and assesses the following dimensions: perception and acknowledgement of own emotions (pae: sometimes i feel sad without knowing why); regulation and control of own emotions (rce: when i feel myself getting angry, i know how to cool down again); emotional expressivity (ee: i can express my feelings very well); perception of others emotions (poe: i can perceive and describe my friend s emotions very well). Answers are given on five - point - likert scales with the anchor points 1 (= never / not at all true) to 5 (= practically always / definitively true). Participants were asked to fill in the 18-item mental toughness questionnaire, the short form of the mtq48.34 the short form provides a general score for mental toughness . Answers on the mtq18 are given on five - point likert - type scales ranging from 1 (= strongly disagree) to 5 (= strongly agree). Items were summed, with higher scores reflecting greater mt (cronbach s =0.91). Next, to calculate the relation between perfectionism traits and sleep parameters (sleep disturbance, sleep duration, sleep onset latency, awakenings after sleep onset, daytime sleepiness), perceived stress, coping, mental toughness, and emotion regulation, a series of pearson s correlations was calculated . Finally, to calculate both the direct and indirect relations of perfectionism with sleep disturbance via perceived stress, coping, mental toughness, and emotion regulation, a structural equation model (sem) was computed, using amos (ibm corporation, armonk, ny, usa)., all statistical analyses were calculated with spss 20.0 (imb corporation, armonk, ny, usa) for apple mcintosh . Perfectionism traits were associated with sleep disturbances (isi), more awakenings after sleep onset, lower mood in the morning and in the evening, feeling less restored in the morning, poorer concentration during the day, and greater tiredness during the day . No significant correlations were found for sleep quality, sleep duration, and for sleep onset latency (with one exception). Perfectionism traits were significantly associated with greater perceived stress, more reliance on negative coping strategies, and less reliance on positive coping strategies . Perfectionisms traits were significantly associated with lower scores for perception and acknowledgement of own emotion, and the regulation and control of own emotions . No significant associations were found for emotional expressivity (with one exception) or for the perception of others emotions . Two perfectionism traits (concerns and doubts; personal standards) were associated with lower mental toughness . To calculate both the direct and indirect effects of perfectionism on sleep disturbance via perceived stress, coping, mental toughness, and emotion regulation, a sem was computed . With respect to the goodness of fit criteria as proposed by hu and bentler35 and mcdonald and ho,36 the model represented a satisfying fit (goodness of fit in square brackets): /df=22.36, p<0.05 [<df; here, 4], agfi (adjusted goodness of fit index) = 0.91 [0.95], pclose = 0.78 [> 0.50], cfi (confirmatory fit index) = 0.1791 [> 0.90], rmr (root mean squared residual) = 0.018 [<0.08] and rmsea (root mean square error of approximation) = 0.025 [0.05]. The statistically significant and direct association between perfectionism and sleep disturbances (isi) was =0.187 (p<001). When perceived stress, coping, emotion regulation, and mental toughness were entered in the equation, the direct effect of perfectionism on sleep disturbance (isi) decreased to 0; sleep disturbance was predicted by greater perceived stress, poor coping, low emotion regulation, and low mental toughness (figure 1). (sobel z = 2.01, p<0.05; 95% asymmetric ci [confidence interval] = 0.010.05, p<0.05). To calculate both the direct and indirect effects of perfectionism on sleep disturbance via perceived stress, coping, mental toughness, and emotion regulation, a sem was computed . With respect to the goodness of fit criteria as proposed by hu and bentler35 and mcdonald and ho,36 the model represented a satisfying fit (goodness of fit in square brackets): /df=22.36, p<0.05 [<df; here, 4], agfi (adjusted goodness of fit index) = 0.91 [0.95], pclose = 0.78 [> 0.50], cfi (confirmatory fit index) = 0.1791 [> 0.90], rmr (root mean squared residual) = 0.018 [<0.08] and rmsea (root mean square error of approximation) = 0.025 [0.05]. The statistically significant and direct association between perfectionism and sleep disturbances (isi) was =0.187 (p<001). When perceived stress, coping, emotion regulation, and mental toughness were entered in the equation, the direct effect of perfectionism on sleep disturbance (isi) decreased to 0; sleep disturbance was predicted by greater perceived stress, poor coping, low emotion regulation, and low mental toughness (figure 1). (sobel z = 2.01, p<0.05; 95% asymmetric ci [confidence interval] = 0.010.05, p<0.05). The key findings of the present study are that perfectionism traits were associated with poorer sleep, though if perceived stress, coping, emotion regulation, and mental toughness were entered into the equation, the importance of perfectionism traits dramatically decreased . Therefore, the pattern of results suggests that the underlying psychological mechanisms of perfectionism seem to be greater perceived stress, poor coping, low emotion regulation, and low mental toughness, which are in turn associated with poorer sleep . Our first hypothesis was that there would be a significant association between perfectionism traits and poor sleep, and this hypothesis was supported . In this respect, the present pattern of results concurs with previous research,1416,23 showing that perfectionism, understood as a dysfunctional set of attitudes toward achieving high performance as a function of one s own standards, concerns about mistakes and doubts, but above all as a function of parents expectations and criticism, is associated with poor sleep . The present study expands upon previous work in that we showed that in a sample of young adults perfectionism traits were also negatively associated with a broad range of psychological processes such as greater perceived stress, poor coping, poor emotion regulation, and low mental toughness, along with increased tiredness and decreased concentration during the day, and lower mood in the morning and in the evening . Our second hypothesis, however, was that psychological factors such as stress and emotion regulation would mediate the relation between sleep and perfectionism . This hypothesis was derived from the findings of morin et al 24 leblanc et al25,26 and fernandez - mendoza et al27,28 who claimed the importance of coping and perceived stress as an important factor to explain the emergence and maintenance of sleep disturbances, and from the study by jansson - frjmark and linton.17 they reported two main findings: first, concerns over mistakes were significantly related to preexisting and future insomnia . Second, when introducing emotional distress as a control, none of the perfectionism subscales contributed significantly to explaining preexisting and future insomnia . We took this pattern of results into account and confirmed the second hypothesis, that is jansson - frjmark and lintons17 observation: when perceived stress, poor coping, low emotion regulation, and low mental toughness were entered in the equation, perfectionism traits were no longer associated with poor sleep, that is to say, they were mediated by coping and perceived stress . Therefore, we conclude that dysfunctional cognitive emotional processes such as stress perception and coping, and poor emotion regulation underlying perfectionist behavior explain most of the association between perfectionism traits as such and poor sleep . Most importantly, the present pattern of results fits well within the large body of research showing that it is not personality traits per se, but the underlying coping strategies and emotion regulation processes, which are predisposing and perpetuating factors of sleep disturbance.2428 the present data do not shed any further light on the reasons for the association between perfectionism and stress or poor emotion regulation, though three overlapping theoretical frameworks support the following possibility . Both riemann et al13 and harvey22 claim that sleep disturbances are caused and maintained by dysfunctional cognitive emotional processes; more specifically, riemann et al13 propose that psychological stress and inadequate problem - solving, along with worrying and rumination, lead to sleep disruptions (ie, delayed sleep onset latency, more awakenings after sleep onset, early awakening in the morning). Importantly, spiegelhalder et al23 showed that hyper - punctuality among patients admitted to the sleep lab arriving earlier than agreed at the lab (interpreted here as a form of perfectionist behavior) was significantly associated with poor polysomnographic sleep parameters . Short sleep duration was significantly associated with early arrival times at the sleep laboratory, suggesting therefore, that aspects of perfectionism (here, punctuality) may be causally linked to poor sleep . Likewise, harvey22 claims that individuals who suffer from insomnia tend to be overly worried about their sleep and about the daytime consequences of not getting enough sleep or, put simply, people suffering from insomnia maintain sleep disturbances via further dysfunctional cognitive emotional processes . Last, morin et al24 leblanc et al25,26 and fernandez- mendoza et al27,28 showed that it is not personality traits per se, but the underlying coping strategies and emotion regulation processes, which are predisposing and perpetuating factors of sleep disturbance . Taken together, the three theoretical frameworks help to explain why it is not perfectionism per se, but rather the underlying psychological mechanisms that best explain the association between perfectionism and poor sleep . As regards the association between mental toughness (a construct consisting of a high degree of control, commitment, challenge, and confidence; clough et al34) and sleep, we observed that the present pattern of results fits well within previous literature showing that mental toughness was associated with increased subjective7,37 and objective sleep8 among adolescents . In this respect, the present findings expand upon earlier results in that the positive association between higher mental toughness and favorable sleep also holds among young adults . Despite the intriguing results, several limitations warrant against overgeneralization of the results, and findings should be interpreted cautiously . First, participants were recruited from among university students and therefore the sample is not representative of young adults as a whole . More specifically, it remains unanswered to what extent the present sample is representative for other samples of adults such as nonacademic people, older adults, or people suffering from severe insomnia . However, we claim that the present pattern of results is a useful step in differentiating cognitive processes that may impact sleep, which could act as a catalyst for future research in clinical samples . Second, the data consists entirely of self - reports; experts ratings such as a brief psychiatric interview could have provided further insight into possible psychiatric or personality disorders . Third, and most importantly, sleep was not objectively assessed, leaving it uncertain to what extent participants with poor sleep were also inclined to report poor psychological functioning . Or simply put: there is the risk, that self - reported poor sleep quality was associated with inadequate and negative self - reported psychological functioning . Future studies should therefore also include objective sleep measurements (actigraphy, sleep - eeg [electroencephalogram]), though we also note that for large samples questionnaire - based measures remain the gold standard.38 fourth, no biological markers were assessed, though, specifically, there is evidence that, for instance, cortisol secretion is associated with sleep and stress regulation . Further, bdnf as indicator of neuronal plasticity plays a role in insomnia and coping with stress.39 fifth, the present pattern of results might have emerged due to further latent but unassessed variables . Sixth, no cognitive academic performance was assessed, which is a global concern for students; accordingly, a wealth of studies shows the association between cognitive abilities, academic performance, and sleep.1,40 last, the cross - sectional design of the study precludes any conclusion as to the direction of influence between perfectionism traits, perceived stress, coping, mental toughness, and sleep, and haario et al41 showed the bidirectional association between sleep and psychological functioning . Longitudinal studies such as the research of jansson - frjmark and linton17 and azevedo et al16 allow for a more detailed picture as to the cause the pattern of results suggests that among a sample of young adults, perfectionism traits are related to poor sleep, though cognitive emotional processes such as stress, coping, and emotion regulation underlying perfectionism traits better explain variance in sleep than perfectionism traits per se . In our opinion, these results should allow more focused and successful psychotherapeutic treatment of perfectionist patients suffering from poor sleep . However, to further prove these observations, the present pattern of results should be replicated among clinical samples.
Our understanding of the mechanisms that regulate the organization of developing tissues is based on the idea that cells gain information determining their fates by monitoring the levels of morphogens released by discrete signaling centers, or organizers, in their vicinity (rogers and schier, 2011). One possibility is that feedback from transcription factors whose expression is regulated by morphogens contributes to the control of the organizers and their morphogen production . The identification of such mechanisms is particularly interesting because they are likely to play a major role in enhancing the precision, stability, and robustness of gene expression patterns in the developing embryo (sokolowski et al ., 2012). Here, we tested whether feedback via the transcription factor pax6 regulates the size and function of a forebrain organizer, the zona limitans intrathalamica (zli). The diencephalon is the caudalmost component of the forebrain and contains the thalamus . During development, interactions between genes expressed around and within the thalamic anlage establish regions with different identities and fates along the embryonic rostral - caudal axis . The transcription factors fezf1 and fezf2 specify a rostral diencephalic domain (the future prethalamus; hirata et al ., 2006; jeong et al ., 2007), whereas the transcription factors otx2 and irx1 specify a caudal diencephalic domain (the future thalamus; hirata et al ., 2006). The zli forms as a thin strip of tissue in the progenitor cell layer at the interface between these domains . It contributes to the organization of the regions around it mainly through its expression of the diffusible morphogen shh (hashimoto - torii et al ., 2003; jeong et al ., 2011; kiecker and lumsden, 2004; scholpp et al ., 2006; scholpp and lumsden, 2010; zhou et al ., 2004; robertshaw et al ., it appears as a thin spike of shh - expressing tissue extending from basal plate through alar plate toward roof plate (shimamura et al ., 1995). Immediately caudal to it, a small rostral area (called pth - r; figure 1b), which comprises thalamic progenitors exposed to relatively high levels of shh, expresses ascl1 and nkx2.2 and generates mostly gabaergic neurons that contribute to the ventral lateral geniculate (vlg) nucleus (inamura et al ., 2011; suzuki - hirano et al ., 2011; vue et al ., 2007; robertshaw et al ., . A larger region of thalamic progenitors caudal to pth - r, called pth - c (figure 1b), expresses ngn2 and olig3 rather than ascl1 and nkx2.2 and generates glutamatergic neurons that innervate cortex (vue et al ., 2007; it starts in the neural plate and is initially throughout the entire alar forebrain neuroepithelium (mastick et al . Pax6 is repressed in the zli by developing shh expression (ericson et al ., 1997; macdonald et al ., 1995; robertshaw et al ., pax6 is retained by prethalamic progenitors and postmitotic cells and by thalamic progenitors; the latter express it in a gradient, with pax6 levels increasing with distance from the zli . Mutant mice lacking pax6 show progressive defects of diencephalic size and patterning (grindley et al ., it has been considered that pax6 functions downstream of shh, which represses pax6 (ericson et al ., 1997; 2013), but it has also been reported that loss of pax6 increases the size of the shh - producing zli (grindley et al ., 1997; pratt et al ., 2000; chatterjee et al ., 2014), suggesting that pax6 might somehow regulate shh . We examined the influence of pax6 on diencephalic patterning and identified an important cell - autonomous action of pax6 on the expression of shh . Our findings indicate a crucial role for mutual repression between pax6 and shh in diencephalic cell specification . In normal e12.5 mice, pax6 expression is highest in the progenitor and postmitotic layers of the prethalamus, absent along the thalamic / prethalamic border, and distributed in a gradient through the progenitor layer of the thalamus (lowest near to the zli) (figure 1a). We examined the consequences of pax6 absence for patterning in each of these regions (figure 1b). A marker of prethalamic progenitors, gsx2, was undetectable in pax6 diencephalon (figures 1c and 1d), and numbers of islet1 + cells were greatly reduced in prethalamic postmitotic cells (asterisk in figure 1d). The absence of gsx2-expressing lineages was shown using a cre recombinase transgene controlled by the gsx2 promoter (kessaris et al ., 2006) with a floxed - stop - gfp reporter (miyoshi et al ., 2010). In controls, most prethalamic cells expressed gfp (figure 1e), consistent with their descent from gsx2 + prethalamic progenitors, but no gfp+ cells were detected in an equivalent region of pax6 mutants (asterisk in figure 1f). To test whether pax6 is required cell autonomously for expression of gsx2 by prethalamic cells the contributions of pax6 cells to each chimera varied (nine chimeras were analyzed), allowing us to analyze situations in which mutant cells were surrounded by much larger numbers of wild - type cells and those where the opposite was the case . In all, pax6 cells contributed to the prethalamus . In chimeras with a high contribution of mutant cells, none of these cells expressed gsx2, whereas even small clusters of wild - type prethalamic progenitors embedded among them retained gsx2 expression (figure 1h). Similarly, in chimeras with a low contribution of mutant cells, all pax6 progenitors were gsx2, even those in very small isolated groups (figures 1i1 m), whereas all wild - type progenitors, even individually isolated ones, were gsx2 + (figures 1l and 1 m). In low - contribution chimeras, islet1 was expressed by most pax6 cells (as it was by most wild - type cells) exiting the progenitor layer (figure 1 m), but in high - contribution chimeras, it was expressed by a smaller proportion (about 50%) of differentiating cells (figure 1 g). These results indicate that cells in the prethalamus have an absolute cell - autonomous requirement for pax6 in order to express gsx2 . Pax6 is not required for prethalamic islet1 expression, although the observation that proportions of islet1 + cells are reduced when large proportions of prethalamic cells are pax6 suggests a non - cell - autonomous effect of extensive pax6 loss on prethalamic islet1 expression . In normal diencephalic development, nkx2.2 expression marks a thin strip of cells around the prethalamic / thalamic border, including progenitors around the zli that coexpress olig3 (figure 2a) and postmitotic cells extending through the neuroepithelial wall to the developing vlg (figures 1e and 2a; kitamura et al ., 1997; scholpp and lumsden, 2010; vue et al ., 2007). In pax6 diencephalon, this domain was greatly expanded (double arrow in figure 1f; figure 2b); it still reached a collection of superficially located nkx2.2 + cells in the same relative position as the vlg of normal embryos (arrowheads in figure 2b). The domains of lhx1 and lhx5 expression in postmitotic cells around the prethalamic / thalamic border were also greatly enlarged in pax6 embryos (figures 2c2f, s1a, and s1b). We tested whether the mechanism of expansion of these domains of expression involved incorrect cellular specification, causing an abnormally broad swathe of cells, many of which would normally have adopted distinct thalamic or prethalamic identities, to take on the identities of cells normally located very close to the prethalamic / thalamic border . Ngn2 + lineage cells, which populate the thalamus of wild - type and pax6 embryos (figures 2i and 2j), were labeled using a transgene expressing a tamoxifen - inducible cre recombinase, creer, under the control of the ngn2 promoter (zirlinger et al ., 2002) and a floxed - stop - gfp reporter allele (miyoshi et al ., 2010). Pregnant females received tamoxifen at e10.5 so as to induce reporter activation by e12.5 (kessaris et al ., 2006), and embryos were collected at e14.5 . In e14.5 controls, gfp+ cells were present throughout the body of the thalamus, and their axons could be seen exiting through the prethalamus; ngn2 lineage cells close to the thalamic / prethalamic border did not express lhx1/lhx5 (figures 2i and 2k). In e14.5 pax6 embryos, about 75% of the gfp+ cells located in the rostral part of the thalamus were double labeled for lhx1/lhx5 (figures 2j and 2l; cells were counted in three sections from each of three embryos), providing evidence for the misspecification of many postmitotic cells in this region . Examination of the thalamus in pax6 embryos showed an overall reduction in its size (as marked by sox2, gbx2, and ngn2 expression: figures 1e, 1f, 2c, 2d, and s1c s1f) but an expansion of the part of its progenitor domain closest to the zli at e12.3e13.5 (figures 2e2h, 2 m, 2n, s1 g, s1h, and s2). This region, known as pth - r (inamura et al ., 2011; suzuki - hirano et al ., 2011; 2011), expresses ascl1, gsx1, and nkx2.2 but only low levels of ngn2, and its expansion in pax6 embryos coincides with a reduction in the size of the pth - c, the caudal region of thalamic progenitors, which expresses ngn2 . Previous studies showed that pth - r contributes gabaergic neurons (vue et al ., 2007). Our analysis of postmitotic gabaergic neurons (vgat and gad67) and glutamatergic neurons (mglur1) confirmed shrinkage of the glutamatergic population in caudal thalamus and expansion of the gabaergic population in rostral thalamus (figures s3a s3i). Expression of six3 in a small group of very rostral postmitotic thalamic cells was also expanded in pax6 mutants (figures s3j s3l, arrows). These findings indicate that the absence of pax6 results in expansion of both pth - r and the gabaergic population of cells that it generates at the expense of the glutamatergic pth - c - derived population . The fact that pax6 is expressed more strongly in pth - c than pth - r progenitors and that rostral territory expands if pax6 is lost suggests that pax6 in pth - c normally suppresses the molecular phenotype associated with the rostral thalamus . To begin addressing how pax6 suppresses the rostral thalamic phenotype, we tested whether its requirement is cell autonomous by examining the expression of rostral thalamic markers in pax6 pax6 chimeras . Figure 3 shows examples of chimeras containing high or low proportions of pax6 (gfp+) cells . Regardless of the balance of wild - type and mutant cells, the boundary region between the prethalamus and thalamus was always identifiable . Its progenitor layer contained cells that were both olig3 + and nkx2.2 + (figures 3a, 3b, 3d, and 3e), and the overlying mantle zone contained nkx2.2 + cells (figures 3a and 3d) and lhx1/lhx5 + cells (figure 3f), whose expression overlapped slightly with that of thalamic sox2 expression as occurs in wild - type embryos (figures 3 g and 3h). Both wild - type and pax6 cells contributed to this boundary region, which was broader in those chimeras that contained a high proportion of mutant cells (marked by double - headed arrows in figure 3a). Nkx2.2, lhx1/lhx5, and ascl1 are not normally expressed through the body of the thalamus, but they were expressed by some thalamic pax6 cells in chimeras . Examples are shown in figures 3a, 3c, 3f, 3i, 3l, and 3n3q . Interestingly, the mutant thalamic cells that showed abnormal expression of nkx2.2, lhx1/lhx5, and ascl1, which their surrounding wild - type neighbors did not, were all located relatively close to the thalamic / prethalamic border (within about 100200 m) in clusters of 100200 cells or more (figures 3a, 3c, 3f, 3i, 3l, and 3n3q). Even large clusters of pax6 cells more distant from the border expressed the same markers as their wild - type neighbors (figures 3a, 3f, 3i, 3l, and 3n). Smaller clusters of pax6 cells always showed the same expression as their wild - type neighbors (figures 3d, 3j, 3k, and 3r3v). These findings indicate that absence of pax6 does not cause a straightforward cell - autonomous upregulation of the rostral thalamic phenotype (nkx2.2 +, lhx1/lhx5 +, and ascl1 +). Although mutant cells do upregulate rostral thalamic markers in some circumstances, they need to be in a critical mass relatively close to the thalamic / prethalamic border . Another revealing result was that in chimeras containing a high contribution of mutant cells, we observed expanded ascl1 domains immediately caudal to the expanded pax6 zli (i.e., corresponding to expanded pth - r) that comprised wild - type cells, providing clear evidence for a non - cell - autonomous upregulation of ascl1 and loss of ngn2 around the expanded mutant zli (figures 3l and 3 m). In summary, our findings strongly suggest that a major underlying cause of abnormal thalamic patterning in the absence of pax6 is a position - dependent defect of local intercellular signaling . This contrasts with the absolute cell - autonomous requirement for pax6 for gsx2 expression in prethalamus described above . Position - dependent defects of signaling among cells around the thalamic / prethalamic border might be caused by defects of the zli . We compared the expression of markers of the zli in control and pax6 embryos using in situ hybridization and immunofluorescence at e12.5e13.5 . The wild - type e12.5 zli is a thin shh - expressing wedge - shaped structure in the alar plate (figure 4a). Close to its tip, it appears as a single line of cells expressing both shh and ngn2 (vue et al ., 2007). We found, however, that as it widens toward its base (e.g., at the level of the broken line in figure 4a), only its caudal part expresses both ngn2 and shh (figure 4c; solid double - headed arrow in figure 4e). Its rostral part expresses dbx1 (broken double - headed arrow in figure 4 g), and a central domain is both dbx1 + and ngn2 + (arrow in figure 4i; summarized in figure 4k). In e12.5 pax6 mutants, there was a large rostrocaudal expansion of shh expression in the alar plate in a location equivalent to that of the zli (figures 4b, 4d, 4f, 4h, 4j, and 4l). This was divisible into an enlarged caudal domain expressing both shh and ngn2 (solid double - headed arrow in figure 4f) and an enlarged rostral domain expressing both shh and dbx1 (broken double - headed arrow in figure 4h) with an enlarged central domain expressing dbx1 and ngn2 + (double - headed arrow in figure 4j). These results indicate that, in the absence of pax6, the zli and its subdomains, identified by expression / coexpression of shh, ngn2, and dbx1, are enlarged, as summarized in figures 4k and 4l . The pitx2-expressing mantle zone immediately superficial to the zli is also expanded in pax6 mutants (figures s3m s3o). We then tested whether the expansion of the zli in pax6 mutants results from the misspecification of cells flanking the normal position of the zli (e.g., rather than an overproliferation of correctly specified zli progenitors). The normal zli develops at about e9.5e10.5 in the mouse at the interface between a rostral diencephalic fezf1 + domain (figure 4 m) and a caudal diencephalic irx1 + domain (figure 4o) (hirata et al ., 2006). It comprises shh+ cells sandwiched between pax6 + domains rostral and caudal to it (figures 4q and 4u). We tested whether expansion of the zli in pax6 embryos involves the erroneous activation of shh by flanking cells expressing the pax6 gene . We used two approaches to identify the pax6 + cells in pax6 mutants . In the first, they were labeled by gfp produced from a pax6-yeast artificial chromosome - reporter transgene that expresses gfp in pax6-expressing cells regardless of whether they are pax6 or not (pax6-gfp; tyas et al ., in e10.5 pax6 embryos, the boundary between fezf1 and irx1 expression was as clear as in controls (arrows in figures 4m4p), indicating that pax6 is not required for this early patterning and allowing us to identify the position of zli formation . In complete contrast to controls (figures 4q and 4u), we observed a relatively large block of tissue whose cells coexpressed high levels of pax6-gfp and shh (figures 4r and 4v). This tissue was largely caudal to the position marked for zli development by the interface of fezf1 + and irx1 + territories (compare figures 4n and 4p with figures 4r and 4v). Normally, the difference in pax6 expression between prethalamus (high) and thalamus (low) becomes greater by e12.5, as shown by expression of pax6-gfp in figures 4s and 4w . As at e10.5, in e12.5 pax6 embryos (figures 4 t and 4x), the caudal part of the abnormally broad shh+ domain extended abnormally far into the low pax6-gfp - expressing thalamic progenitor layer (double - headed arrow in figure 4x). The rostral part of the abnormally broad shh+ domain overlapped the region of high pax6-gfp prethalamic expression (broken double - headed arrow in figure 4x), with cells in this region being double positive for shh and pax6-gfp expression . At e12.5, we also observed abnormal coexpression of pax6 and ngn2 mrnas in this rostrally extended part of the expanded pax6 zli (figures 4y4bb). Whereas ngn2 and pax6 expression is normally complementary at e12.5 (with ngn2 in the zli and high pax6 rostral to it; figures 4y and 4aa), in pax6 embryos, many ngn2 + cells in the expanded zli coexpress high levels of pax6 (figures 4z and 4bb). These findings indicate that pax6 progenitor cells rostral and caudal to the position of normal zli formation are misspecified . The failure of cells around the thalamic / prethalamic border to repress shh expression in pax6 embryos might provide an explanation for aspects of the mis - patterning of this region in terms of its abnormal marker - gene expression in pax6 embryos and chimeras . To test this, we first examined chimeras to discover whether the abnormal upregulation of shh in pax6 cells around the thalamic / prethalamic border is cell autonomous . We focused on chimeric embryos in which small minorities of pax6 cells were surrounded by vast majorities of wild - type cells, in which influences from outside pax6 cell groups should be relatively normal . In chimeras, small clusters of pax6 (gfp+) cells throughout the diencephalon all expressed shh regardless of their size and whether they were in the zli, thalamus, or prethalamus (figures 5a5e). Cells embedded in the zli expressed shh at levels that were indistinguishable from those of their shh - expressing wild - type neighbors (figures 5a5c). Outside the zli, pax6 cells achieved levels of shh expression that, although raised above those of their wild - type neighbors, were lower than those of cells in the zli (e.g., horizontal arrow in figure 5b, and double - headed arrows in figures 5d and 5e). We measured the relative intensities of shh labeling across the diencephalon of wild - type and pax6 embryos and in the clusters of pax6 cells in chimeras . The relationship between intensity and distance from the zli was very similar in pax6 mutants and in pax6 cells in chimeras (figure 5f). In control chimeras comprising a mixture of pax6 and pax6 gfp - expressing cells, the gfp+ cells were not clustered but were scattered throughout the diencephalon, the in situ hybridization staining for shh appeared normal, and there was no evidence that levels of shh depended on whether cells were gfp+ or not (figures s4a s4c). These findings indicate that pax6 diencephalic cells activate shh cell autonomously because activation occurred even when mutant cells were embedded in an environment comprising a large majority of wild - type cells . Moreover, the results suggest that the mechanism that sets the level of activation with reference to distance from the zli in the absence of pax6 acts cell autonomously because the relationship between the locations of the pax6 patches and their levels of shh expression reflected the relationship in pax6 embryos . These results have the potential to explain the observed mis - patterning of those pax6 cells close to the thalamic / prethalamic border in pax6 mutants and chimeras . They might also explain the expanded ascl1 domain affecting wild - type cells close to the border (figures 3l and 3 m) and the lack of repatterning among wild - type cells further from the border . We do not exclude the possibility that factors other than levels of shh expression might also be important in region - specific repatterning . Previous studies have shown that expression of ptch1 and gli1 can be upregulated by shh signaling and is normally high in cells adjacent to the zli (figures 5 g, 5i, and 5k; bai et al ., 2002, 2004;, 1997; goodrich et al ., 1996; kiecker and lumsden, 2004; lee et al ., 1997; marigo and tabin, 1996; wijgerde et al ., 2002; vue et al ., we tested the prediction that if the abnormal shh expression of pax6 cells has functional consequences for those cells, then they should upregulate ptch1 and gli1 . In situ hybridizations on e10.5 pax6 whole mounts showed that staining for ptch1 spread more broadly than normal through surrounding diencephalic tissue (arrowheads in figures 5 g and 5h). Coronal sections of e12.5e13.5 pax6 embryos showed expansion of the ptch1 + areas and abnormally strong gli1 expression in the thalamus and prethalamus (figures 5i5l). Outside the zli, clusters of pax6 (gfp+) cells were ptch1 + (figures 5m5o), indicating a response to the shh that they produce . Wild - type cells surrounding the zli were ptch1 +, as in normal embryos (figures 5q and 5r). Interestingly, ptch1 and gli1 are normally downregulated within areas of high shh expression in the zli and basal plate (figures 5 g, 5i, and 5k; marigo and tabin, 1996). Ptch1 and gli1 are also downregulated in the expanded zli of pax6 embryos (figures 5j and 5l) and by pax6 cells embedded in the zli in chimeras (figures 5p5r). To test whether this might also be caused by exposure to high levels of shh, we administered vismodegib (gdc-0449), a selective inhibitor of the shh receptor smo (expressed throughout control and pax6 diencephalon; figures s5a and s5b), to pax6 embryos . This treatment caused a dose - dependent overall reduction of the area of ptch1 expression but an upregulation of ptch1 at the expanded shh+ pax6 zli (figures s5c s5f), in agreement with the hypothesis above . In the forebrain, pax6 is also strongly expressed in the cerebral cortex, and a previous microarray - based analysis found small but significant upregulation of both shh and ptch1 in cortical cells from pax6 embryos (shh: + 1.38-fold, p = 0.038; ptch1: + 1.36-fold, p = 0.043; mi et al ., 2013). In situ hybridization on pax6 embryos did not show obvious upregulation of shh or ptch1 (figures s4d s4 g), but in chimeras, where mutant and wild - type cells are together in the same sections, we did detect slightly stronger staining for shh and ptch1 in pax6 cells (figures s4h s4k). These findings indicate that pax6 also has a cell - autonomous repressive effect on shh in cortex, although its magnitude appears less than in diencephalon . We then tested whether abnormalities of diencephalic patterning in pax6 embryos result from abnormally high shh signaling around the zli, by administering vismodegib (gdc-0449) to inhibit shh signaling in pax6 embryos . We examined the effects of shh blockade on markers whose expression is altered at or around the zli and the thalamic / prethalamic border in pax6 mutants (dbx1, ngn2, lhx5, nkx2.2, and ascl1). In wild - types, dbx1 is expressed in the zli and in a gradient through the thalamus (figures 4 g, 4i, 4k, and 6a), whereas in pax6 mutants, its expression is expanded at the zli and greatly reduced in the thalamus (figures 4h, 4j, 4l, and 6d). Its expression at the zli was abolished by administration of vismodegib, both in wild - types and pax6 embryos (figures 6b, 6c, 6e, and 6f), indicating that its expression / expanded expression in this region is dependent on shh . In wild - types, the intensity of staining for lhx5 expression around the thalamic / prethalamic border declined with increasing doses of vismodegib, but otherwise, the pattern of expression appeared unaffected (figures 6g6i). In pax6 mutants, the expanded domain of intense lhx5 expression around the thalamic / prethalamic border (whose position was recognized by shh expression in adjacent sections) was reduced by increasing doses of vismodegib (figures 6j6n), indicating the importance of shh signaling for this aspect of the mutant phenotype . The pattern of ngn2 expression in wild - types appeared unaffected by treatment with vismodegib, showing high expression in the thalamus and zli with reduced expression in pth - r (figures 6o and 6p). In pax6 mutants, however, the region of low ngn2 expression at the pth - r was no longer visible after vismodegib treatment (figures 6q6s). Ascl1 has a complementary expression pattern to that of ngn2: wild - types show high expression in pth - r and prethalamus and low expression in the zli and thalamus (figures 2 g and s6a), whereas pax6 mutants show expanded expression at the pth - r (figures 2h and s6b). Vismodegib had no obvious effect on the ascl1 expression pattern in wild - types (figure s6c) but caused a marked loss of expression at the pth - r of pax6 mutants (figure s6d), reducing both the area containing ascl1 + cells and their density (from 20.5 1.9 sem, n = 3, to 9.78 1.8, n = 3, cells per 0.005 mm; p <0.05, student s t test). Vismodegib had little effect on nkx2.2 expression in wild - types (figures 6 t, 6u, and 2a), but it reduced substantially the expanded nkx2.2 expression around the zli in pax6 mutants (figures 2b and 6v6x). These findings indicate that altered patterns of ngn2, ascl1, and nkx2.2 expression around the thalamic / prethalamic border of pax6 mutants are dependent on shh signaling . Our finding that the normal expression of these three genes is not shh dependent indicates that other factors can maintain their normal patterns . They suggest that enhanced shh signaling is a major contributor to many diencephalic patterning defects in pax6 mutants . We examined the potential functional consequence of the slight increase in shh signaling in the pax6 cortex but could not detect any effects of even high doses of vismodegib on the abnormal patterning in this region . For example, loss of pax6 causes upregulation of ascl1 expression in the cortex (figures s6e and s6f), but vismodegib did not reverse this defect (figure s6 g). This indicates that the small upregulation of shh expression in pax6 cortex is not a significant cause of patterning defects in this region, highlighting regional differences in pax6 s mode of action . We carried out luciferase assays using a 775 bp sequence corresponding to the shh promoter (table s1; mutoh et al ., 2010) inclusion of the shh promoter into the firefly luciferase reporter construct caused a large increase in firefly luciferase over renilla luciferase activity; this increase was significantly reduced when cells were cotransfected with a pax6-expressing construct (mi et al . Chromatin immunoprecipitation (chip) experiments (figure 7b) showed significant enrichment of fragments containing shh promoter sequence (table s1) compared to that of fragments containing syt8 promoter (a pax6 nonbound control; mi et al ., 2013), demonstrating that pax6 binds to the shh promoter in vivo . Interestingly, bioinformatics using position weight matrices based on previously reported pax6 binding sites did not identify the likely positions of pax6 binding within the shh promoter, indicating that pax6 binds this region through one or more previously unrecognized, noncanonical sites . We also tested whether an indirect mechanism might contribute to the upregulation of shh in pax6 diencephalic cells . It was possible that pax6 loss might increase wnt signaling around the zli, and this might cause upregulation of shh because early wnt expression in the zli permits the induction of shh (martinez - ferre et al ., 2013). We used a -galactosidase transgenic reporter (bat - gal) (maretto et al ., 2003) to show strong wnt/-catenin signaling around the zli whose domain expands in pax6 mutants (figures s7a s7f). In chimeras, however, we found no evidence that clusters of pax6 cells expressed increased levels of axin2 and lef1 (figures s7g s7l), which are readouts of levels of wnt signaling (hsu et al ., 1998; jho et al ., 2002), indicating that increased wnt signaling is unlikely to be a cause of changes in shh expression by pax6 diencephalic cells . Previous studies showed that loss of pax6 results in diencephalic patterning defects including expansion of the zli (grindley et al ., 1997; pratt et al ., 2000; chatterjee et al ., we find that a combination of cell - autonomous and non - cell - autonomous effects is responsible . Pax6 is required cell autonomously to repress diencephalic shh expression . Some of the major patterning defects that occur around the zli when pax6 is absent are caused by enhanced shh activity . We show that expansion of the zli and territories around the thalamic / prethalamic border is caused by cells acquiring abnormal molecular identities . Theoretical studies by others have indicated that the involvement in control systems of elements that mutually repress each other, such as shh and pax6, can enhance the system s robustness by buffering it against stochastic, interindividual fluctuations or temporal changes in morphogen levels and can generate bistability (sokolowski et al ., 2012). These properties are exactly those required within the diencephalon, which is a relatively small but intricately patterned structure, to maintain its cells in either a zli or a non - zli state with a sharp, reproducibly positioned transition between them . We propose that direct repression of shh by pax6 creates a feedback loop that is critical for the precision of normal diencephalic patterning by ensuring extremely tight, robust control of the size and influence of the zli . Several aspects of our findings highlight the importance of the context within which interactions between regulatory molecules occur for their outcomes . Although previous studies have shown that shh can repress pax6 expression, including at the zli (ericson et al ., 1997; kiecker and lumsden, 2004; macdonald et al ., 1995), we found no evidence for repression of the pax6 gene expression in diencephalic regions surrounding the normal position of the zli in pax6 mutants . In these areas, expanded shh expression caused an anticipated increase in the expression of its targets ptch1 and gli1, but it did not prevent pax6 transcription, as shown by the abnormal double labeling of cells around the zli for both shh and the pax6 reporter or pax6 mrna . This suggests that, whereas shh may normally contribute to the absence of pax6 at the zli, elsewhere, the potential repressive effects of shh on pax6 expression are likely moderated by other factors . Another example of context dependency is seen in the prethalamus, where pax6 is required for gsx2 expression, whereas in the telencephalon, loss of pax6 results in upregulation of gsx2 in cortex, where it would not normally be expressed (rallu et al ., 2002; toresson et al ., 2000; yun et al ., the way in which a transcription factor affects its target genes probably depends mainly on the nature of the cofactors that are available for it to interact with . Many studies have shown that shh can drive expression of ptch1 (bai et al ., 2002, 2004; balaskas et al ., 2012; goodrich et al ., 1996; this explains the expression of ptch1 by cells adjacent to the zli in normal embryos and by pax6 cells more widely throughout the diencephalon in mutants and chimeras, but it leaves unexplained the paradoxical observation that ptch1 is not expressed within the zli itself . Ptch1 and gli1 are downregulated in regions expressing the highest levels of shh not only at the zli but also along the neural tube (marigo and tabin, 1996), where cells become progressively refractory to shh (ribes et al ., this suggests a biphasic response of ptch1 and gli1 to shh, with very high levels of shh repressing their expression (figure 7c). They indicate that administration of shh inhibitor to mutant embryos caused ptch1 upregulation at the expanded zli, where shh levels were blocking ptch1 production, while simultaneously reducing ptch1 expression in cells outside the expanded zli, where lower levels of shh were activating ptch1 . In our model, shh activates dbx1 in the zli s ventricular zone and enhances lhx5 expression in the overlying differentiating zone . Because the shh receptor ptch1 is not expressed at the zli, it is likely that this activation and enhancement involve double repression, i.e., ptch1 receptor activation likely represses dbx1 and lhx5, and so loss of ptch1 in the ventricular zone at the zli would allow expression of dbx1 by progenitors and lhx5 by their progeny . Other factors must also be involved in fine - tuning the expression of dbx1 because dbx1 is not expressed throughout all of the shh - rich zli, but only in its rostral part . Lineage analysis has shown that progeny from the zli are incorporated into the rostral vlg, a nucleus that comprises gabaergic neurons whose axons do not project to the cortex (suzuki - hirano et al ., 2011; vue et al . It is possible that there are subtle differences in the vlg neurons derived from the different types of zli progenitor . In summary, we found that mutual antagonism between pax6 and shh, involving cell - autonomous repression of shh by pax6, constrains the development of the zli, its production of shh, and its influence on surrounding diencephalon . Pax6 is likely to have similar effects on shh in other regions of the cns, including the cortex (present findings and mi et al ., 2013) and the spinal cord, where its misexpression has been shown to reduce shh expression (lek et al ., 2010). The functional importance of this repression outside the diencephalon is currently unclear; we show here that inhibition of shh signaling in pax6 cortex does not prevent abnormal cortical patterning . In the diencephalon, however, we conclude that pax6 plays many of its essential roles in patterning by cell autonomously regulating shh expression at the zli . Animals were bred in - house following home office (uk) regulations . The pax6 null allele used was pax6 . Pax6 and wild - type embryonic stem (es) cells were stably transfected with tau - gfp expression construct ptp6 (pratt et al ., 2000). Chimeric embryos were produced by injection of pax6 tau - gfp or pax6 tau - gfp es cells into blastocysts . Antibodies used were mouse anti - islet1 (39.4d5), mouse anti - lhx1/lhx5 (4f2), and mouse anti - nkx2.2 (74.5a5) obtained from the developmental studies hybridoma bank (university of iowa), mouse anti - ascl1 (bd biosciences), mouse anti - ngn2 (clone 7g4; lo et al ., 2002), rabbit anti - olig3 (chemicon), rabbit anti - sox2 (ab5603; chemicon), goat and rabbit anti - gfp (abcam), and mouse anti - gfp (roche). Probes were labeled with digoxigenin, fluorescein, or dinitrophenol (dnp). For fluorescence double in situ hybridization, the probes were detected sequentially, and the slides were incubated in 10 mm hcl before detection of the second probe . Suspensions of gdc-0449 powder were prepared in methylcellulose - tween vehicle (mct) (lipinski et al ., 2010). Dna - protein complexes were precipitated with anti - pax6 antibody (covance) or with anti - immunoglobulin g (igg) antibody (abcam) (sansom et al ., 2009). The amount of qpcr product obtained with anti - pax6 antibody was expressed relative to that obtained with anti - igg antibody . Shh promoter sequence (table s1) was cloned into pgla4.10 promoterless firefly luciferase reporter vector (promega). Pax6 was expressed using the pcmv - pax6 construct (mi et al ., 2013). Human embryonic kidney 293 cells were transfected using lipofectamine 2000 (invitrogen), harvested 48 hr after transfection, and analyzed with the dual - luciferase reporter assay system using the glomax luminometer (both promega). Did most of the experiments in collaboration with m.m . And helped write the manuscript.
We performed the first comprehensive, population - based brachytherapy (bt) patterns of care study (pocs) in the australian setting by performing a bt pocs for new south wales (nsw). In comparison with our model of optimal bt utilization, gynaecological bt was underutilized in nsw, as well as in the usa and western europe . The other aim of the pocs was to assess the technical quality of nsw bt practice by comparing with known quality benchmarks, and to thereby determine if a caseload effect exists . Herein we focus on these results as they pertain to cervical bt quality . Although uncommon in australia, worldwide cervical carcinoma is the fourth largest cause of cancer death in females . Brachytherapy is an integral part of cervical cancer treatment, and the technical quality of cervical bt has been shown to correlate with outcome . Previous cervical cancer pocs have shown that institution size / type affects cervical treatment quality and outcome [5, 6], but as yet there is no data directly comparing institutional cervical bt caseload and cervical bt quality . In 2005, a retrospective nsw bt pocs was performed for patients treated in 2003 . The population for the current study was all nsw resident cervical cancer patients treated with bt in nsw in 2003 . Site visits and data collection by the principal investigator were made to all 9 radiation oncology departments in nsw that deliver bt . Quality of bt treatment was assessed using published benchmarks: point a dose 80 gy [3, 7], icru (international commission on radiation units and measurements) rectal point dose <75 gy, icru bladder point dose <80 gy, and total treatment time 8 weeks [3, 7]. Quality was compared between the four departments that performed more cervical bt (higher bt caseload) compared to the five departments that performed fewer (lower bt caseload). Doses were compared using the linear quadratic equation to convert high dose rate (hdr) bt doses to equivalent low dose rate (ldr) doses, which are approximately equivalent to standard fractionation at 2 gy per fraction (eqd). For late effects, means of two variables were compared using the to - sample t - test (two - tailed). Pearson correlation test was performed to assess relationships between bladder / rectal doses and point a doses . Permission for the study was obtained from all relevant radiation oncology departmental directors and human research ethics committees . In nsw in 2003, there were 15 radiation oncology departments, of which 9 (60%) treated patients with bt, all administering gynaecological bt . After exclusion of 14 cervical cancer patients resident interstate, 76 nsw cervical cancer patients were treated with bt in 2003 . Four departments treated 52 (68%) patients, median 13 (range 11 - 15) per department, and are categorized in further analysis as higher cervical bt caseload departments . The other five departments treated the remaining 24 (32%) patients, median 4 (range 1 - 8) per department, and are categorized as fifty - seven (75%) patients were stage iib - iva, with technically inoperable disease . Seventy - five patients had known histology: with most (87%) having squamous cell carcinomas, the remainder being adenocarcinomas (9%) or adenosquamous carcinomas (4%). One patient was treated for locally recurrent disease, and five patients were treated adjuvantly . Definitive radiotherapy with ebrt and bt for 70 patients with non - recurrent disease forms the subject of the remainder of the analysis . Tandem and ovoid bt was the commonest applicator type, used for 61 (87%) patients, with 6 of 9 departments using only this equipment . Ldr cs bt was used by three departments to treat 40 (57%) patients, pdr ir was used by two departments (treating as ldr) to treat 5 (7%) patients, and hdr ir bt was used by four departments treating 25 (36%) patients . Seven departments used individualized planning, while two (both lower caseload departments) applied a best - fit library plan to their nine anaesthetised hdr patients . All departments dosed to point a. there was no difference in type of applicator used by caseload, with almost all centres using tandem and ovoids . Lower caseload departments were more likely to treat using a best - fit library (rather than individualised) plan, treating 38% versus 2% of patients this way (p = 0.002, or: 0.034, 95% ci: 0.004 - 0.296). No consistent dosing schedule was used, with a wide range of doses and fractionations prescribed across the state . Median ebrt dose to the cervix was 50 gy (range: 39 - 50.4 gy) in 1.8 gy or 2 gy fractions for all except two patients who were treated with 1.67 gy fractions . Median and modal hdr dose was 18 gy (range: 6 - 24 gy), delivered in one (8% of patients), two (8%), or usually three fractions (84%). Dose was known for 44 of the 45 patients treated in an ldr fashion (with ldr or pdr equipment): median and modal dose was 30 gy (range: 20 - 43 gy), in one (69% of patients), two (29%), or rarely three fractions (2%). Median total eqd2 dose to point a (/ = 10) was 76 gy (range: 47 - 88 gy). Higher caseload departments treated to higher mean doses (78 gy vs. 71 gy, p <0.001, 95% ci for difference: 3.5 - 10.2 gy). Benchmark dose 80 gy was delivered in only 31 (45%) patients with higher caseload departments more likely to achieve this benchmark: 28 of 48 (58%) compared with 3 of 21 (14%) (p = 0.001, or: 8.40, 95% ci: 2.18 - 32.4) (fig . Adequacy of point a dose, by institutional cervical brachytherapy caseload (p = 0.001). Adequate dose is eqd2 (/ = 10) 80 gy hdr patients were treated to lower doses than ldr patients (mean 72 gy vs. 79 gy, p <0.001, 95% ci for difference: 3.5 - 9.8 gy), and only 5 of 25 (20%) hdr patients were treated to the benchmark point a dose, compared to 26 of 44 (59%) ldr patients (p = 0.002, or: 0.17, 95% ci: 0.05 - 0.55). When using hdr bt, higher caseload departments were more likely to treat to an adequate dose (4 of 9 patients, 44%) compared to lower caseload departments (1 of 16 patients, 6%) (p = 0.02, or: 12.0, 95% ci: 1.07 - 134). This distinction was not necessarily true in the case of ldr bt: adequate dose was achieved for 24 of 39 (62%) patients treated in higher caseload departments compared to 2 of 5 (40%) patients treated in lower caseload departments, but this difference was not statistically significant . Icru rectal point data were routinely recorded by all departments and were available for 66 of the 70 (94%) patients . Median total eqd2 dose to the icru rectal point (/ = 3) was 64 gy (range: 43 - 77 gy). There was no statistically significant difference in mean doses by departmental caseload (p = 0.11). The recommended dose of <75 gy was achieved in 61 of 66 (92%) patients with no differences by caseload (p = 0.6). Although hdr patients averaged lower rectal doses (hdr 60 gy vs. ldr 66 gy, p <0.001, 95% ci for difference: 3.8 - 9.8 gy), there was no significant difference in the likelihood of the <75 gy benchmark being achieved by bt dose rate (hdr 96% of cases vs. ldr 91%, p = 0.47). Icru bladder point dose data were routinely recorded by all departments except one, and were available for 56 of the 70 (80%) patients . Median total eqd2 dose to the icru bladder point (/ = 3) was 80 gy (range: 53 - 175 gy). There was no significant difference in mean doses by departmental caseload (p = 0.23). The benchmark dose <80 gy was achieved in 28 of 56 (50%) patients with no differences by caseload (p = 0.34). Hdr patients averaged higher bladder doses (hdr 94 gy vs. ldr 77 gy, p = 0.04, 95% ci for difference: 1.3 - 32 gy) and hdr bt was significantly less likely to achieve a bladder point eqd2 dose less than the recommended 80 gy maximum (hdr 29% of cases vs. ldr 59%, p = 0.042, or: 0.29, 95% ci: 0.09 - 0.98). A maximum of 8 weeks is recommended after which accelerated repopulation may negatively affect survival [3, 7]. This benchmark was achieved in the treatment of 38 of 67 (57%) patients, more commonly in higher caseload departments (31 of 47 patients, 66%), compared to lower caseload (7 of 20 patients, 35%) (p = 0.02, or: 3.60, 95% ci: 1.20 - 10.8) (fig . There was no statistically significant difference between hdr or ldr treatments (p = 0.27). In nsw in 2003, 76 nsw residents with cervical cancer underwent bt, in 70 of whom treatment was part of definitive radiotherapy . At this time, no nsw radiotherapy departments were using 3-dimensional bt treatment planning, the majority of patients were treated with ldr bt, and many patients were treated to inadequate total xrt doses over prolonged treatment time, particularly in the departments that treated fewer patients with cervical bt . Comparing the results of our nsw pocs with shows that there appears to be a move towards incorporation of more advanced bt technology . Only two years later, 6 of 21 (29%) australasian departments had incorporated some form of 3-dimensional treatment planning . There is also a move towards hdr bt, which was used in nsw in 2003 by 4 of the 9 (44%) departments for 25 of the 70 (36%) definitively treated patients . Two years later, according to van dyk's survey, 12 of 21 (57%) australasian departments were using hdr to treat cervical cancer, reflecting the gradual replacement of ldr by hdr for the treatment of cervical cancer as manufacture of this equipment wound down . Given this move to hdr bt, a concern is that nsw benchmark point a doses for hdr bt compared to ldr were less likely to be reached (20% vs. 59%, p = 0.002), and benchmark bladder doses were also less likely to be achieved (29% vs. 59%, p = 0.042). A reason for this poor therapeutic ratio may be the use of insufficient fractionation in nsw, with hdr bt usually delivered in three fractions of 8 gy . This compares unfavourably with the japanese and canadian medians of four hdr fractions [11, 12], and the us median of 5 - 6 . Fewer fraction numbers implies increased hdr bt fraction size for a given total dose and is correlated with increased late toxicity . For optimal local control, guidelines recommended total point a doses greater than eqd2 80 - 85 gy [3, 7]. In nsw, this benchmark was achieved in only 45% of patients . This compares unfavourably with the 1996 - 1999 pocs in the usa, showing that 69% of patients were treated to a point a dose of 80 gy . In nsw, median total eqd2 dose to point a was 76 gy for the entire cohort (72 gy for hdr bt and 79 gy for ldr bt). Median prescribed eqd2 for hdr bt was 83 gy, and in the 1996 - 1999 usa pocs, median total eqd2 for hdr bt was 86 gy and 83 gy for ldr bt . It is likely that local control may be sub - optimal in nsw patients as a result of inadequate point a doses . Bowel and bladder complications of definitive radiotherapy for cervical cancer are not infrequent, can be severe, and severity and frequency correlate with dose; guidelines therefore recommend routine calculation of doses to organs at risk . New south wales departments demonstrated a high rate of compliance with the documentation required, with all departments recording rectal doses and 8/9 (88%) recording bladder doses, similar to canada (89% rectal doses and 86% bladder doses) and comparing favourably to japan (25% and 18%) and usa (25% overall for hdr and 93% for ldr) [1113]. The new south wales pocs showed that 92% of patients were treated to a rectal point dose of less than the recommended maximum of 75 gy, but only 50% of patients were treated to a bladder point dose of less than the recommended maximum of 80 gy . The greater likelihood of patients being treated within rectal tolerance rather than bladder tolerance probably reflects the known greater risk of gastrointestinal toxicity compared to bladder toxicity after definitive radiotherapy for cervical cancer . Given accelerated repopulation of tumour clonogens in cervical cancer, this benchmark was achieved in only 57% of cases, with local control likely to be adversely impacted as a result . This compares favourably with the 47% rate in the 1996 - 1999 usa pocs, but unfavourably with the 72% rate reported by the japanese 1999 - 2001 pocs . Technical quality of brachytherapy insertion for cervical cancer has been correlated with local control and toxicity with a strong trend towards improved survival [4, 16]. Given this, the american brachytherapy society (abs) has recommended that radiation oncologists with low cervix cancer caseloads (less than four cases per year) refer to larger centres or more experienced physicians . In the uk in 2007 it has been recommended that a minimum of 10 cases be treated per centre per year, although no data were referenced to support this recommendation . Our results support these abs and uk assertions by showing a direct correlation between departmental cervical bt caseload and bt quality . The higher caseload departments were more likely than lower caseload departments to complete treatment within the benchmark 8 weeks (66% vs. 35% of cases, p = 0.02, fig . 2), to treat to the recommended point a dose 80 gy (58% vs. 14% of cases, p = 0.001, fig . 1), and to treat using an individualised rather than library plan . As there were no significant differences between higher and lower caseload departments in mean rectal and bladder point doses (or in the proportions of patients with rectal or bladder doses greater than recommended), the higher prescribed point a doses of the higher caseload departments imply that these higher doses were achieved by technically superior bt applicator placement, given that there was no cost in terms of higher doses to organs at risk . The nsw results are consistent with the international literature, generally (but not always) showing significant correlations between departmental caseload or facility size / type and treatment quality / outcomes [5, 6, 1921]. However, our nsw pocs data are the first data to directly compare cervical bt quality benchmarks by institutional cervical bt caseload . All the higher cervical bt workload nsw departments treated more than 10 cases per year, lending support to the uk royal college of radiologists assertion that the minimum number of cases per centre per year should be ten . Considering that in nsw 5 of 9 departments did not meet this benchmark, that patients treated in these departments were significantly more likely to be treated to lower doses over a longer treatment time with (probably) sub - optimal implants, and that the median number of cervical bt cases per centre in nsw is less than half that of the uk (see table 1), there is an implication that cervical cancer outcomes in nsw may be improved if cervical bt were to be centralised . The same conclusions might be drawn from the data showing low caseload per centre in the usa (table 1). Given that we have already shown that gynaecological bt is underutilized in nsw and the usa, this would have significant implications for health resource planning and patterns of patient care and referral . New south wales cervical brachytherapy caseload per centre and comparison with other jurisdictions nsw new south wales, abs american brachytherapy society, us pocs united states patterns of care study, uk united kingdom, not available; abs 1995 data per radiation oncologist this study did have a number of limitations . It was retrospective, involved small patient numbers, and represents state - of - play in 2003, and therefore not necessarily reflecting current nsw practice . Nevertheless, our results remain applicable: in the uk in 2012, dose was prescribed to a volume in only 29% of centres . In the developing world, many centres, treating the majority of the world's cervical cancer patients, are probably using 2-dimensional treatment planning and dosing to point a. as cervical bt becomes more conformal with the adoption of mri - bzased volumetric planning, although target definition is safe in expert centres, there may be a greater risk of geographical miss and poorer tumour control in inexperienced hands . Importantly, this study reports technical factors, but not outcome data . The lower point a doses and longer treatment times in patients treated at lower caseload departments are likely to have resulted in poorer disease control [24, 25]. Assuming, the present results are valid, it is not known whether or not further improvements in bt quality might be achieved by increasing the recommended minimum number of cases per centre further beyond ten . Cervical bt in nsw in 2003 was dispersed amongst a large number of departments and was of sub - optimal quality in the lower caseload departments . Higher quality brachytherapy was achieved in departments treating at least 10 patients per year . Although previous studies have shown improved treatment quality in larger and/or academic radiation oncology departments, this is the first study showing an association between cervical bt caseload and treatment quality, and it is likely that improved outcomes will be achievable if at least 10 patients are treated per department per year.
Increasing prevalence of type 2 diabetes mellitus (t2 dm) has stimulated research focused on the pathogenesis and treatment of t2 dm and its complications . Initial studies examining fracture as a possible complication of t2 dm indicated that type 2 diabetics were not at risk of fracture based on bone mineral density (bmd), the clinical standard for screening [13]. However, data analyzed from clinical trials with fracture as an outcome variable instead of bmd revealed that both men and women with t2 dm experience an increase in fracture (i.e., 1.53-fold) beginning 510 years after diagnosis [48]. Collectively, the clinical evidence indicates that, independent of bmd, type 2 diabetics are at increased risk of fracture that is exacerbated over time . Rodent models have enabled investigators to study the molecular mechanisms involved in altering bone quality in t2 dm . One of the most commonly utilized models has been the c57bl/6 young growing mouse fed a diet high in total and saturated fat (hf), which exhibits an increase in adiposity, impaired glucose tolerance, and dyslipidemia, similar to prediabetes in humans [1012]. C57bl/6 mice have been reported to exhibit decreased trabecular bone and either increased, decreased, or no change in cortical bone in response to long - term intake of a hf diet [1317]. When alterations in bone microarchitecture occur, they are usually accompanied by impaired bone quality as evidenced by compromised biomechanical properties [15, 1721]. Factors that may contribute to some of the discrepancies in the literature describing the skeletal response to a hf diet could be due to the composition of the diets, the age of the mice, the duration of the study, and, importantly, differences in the c57bl/6 mouse substrain's response (e.g., c57bl/6j or c57bl/6n) [1316, 21]. A review of published reports revealed that studies utilizing different c57bl/6 substrains (e.g., c57bl/6j and c57bl/6n) are often treated interchangeably without mention of genetic variations that could have important implications on the results and their interpretation . For example, the c57bl/6j mouse has a missense mutation in the gene encoding nicotinamide nucleotide transhydrogenase (nnt) that alters rna splicing and leads to the deletion of exons 711 [2224]. When c57bl/6j mice are fed a high fat diet, they exhibit impaired glucose tolerance that appears to result from suppressed insulin secretion by pancreatic -cells . Thus, genetic differences in the c57bl/6j mouse could contribute to some of the discrepancies in the literature in regard to metabolic and skeletal responses reported in the diet - induced obesity model of t2 dm . In contrast to the c57bl/6 mouse, the c3h / hej mouse model has been used in mechanistic studies because of its blunted metabolic response to a high fat diet [26, 27]. C3h / hej mice have a nonfunctional toll - like receptor (tlr) 4 due to a point mutation in the toll - interleukin 1 receptor domain [2628]. Tlr-4 is expressed on bone cells (i.e., both the osteoblasts and osteoclasts) [29, 30]. Our lab and others [3134] have shown that tlr-4 ligands (e.g., lipopolysaccharide or lps and saturated free fatty acids or sffas) as well as downstream inflammatory mediators have the potential to uncouple bone turnover . Because of interest in sffas and gut - derived lps in the pathophysiology of t2 dm and its complications, the c3h / hej strain has become an important research tool to examine the role of tlr-4 in these metabolic responses . To date, a direct comparison of the long - term metabolic and skeletal responses of the c57bl/6j and c57bl/6n substrains to a hf diet has not been reported in the literature . If, as we hypothesized, the metabolic response to a hf diet in these two substrains differs due to genetic variations, this may alter the inflammatory response, hormones, and adipokines, subsequently affecting the bone . Such differences would be important relative to the interpretation of results and could assist investigators in selecting the most appropriate model . Furthermore, because of our laboratory's interest in tlr-4 and bone, in this study c3h / hej mice were used as a negative control for comparative purposes [26, 27]. Eight - week - old male mice, c57bl/6n from charles river (wilmington, ma) and c57bl/6j and c3h / hej mice from jackson labs (bar harbor, me), were obtained (n = 30 mice / strain) for these studies . Animals were acclimated for 7 days and then randomly assigned to a control ain-93 m (10% kcals from fat) or a hf (45% kcals from fat; harlan teklad, td.06415) diet for 24 wk . Body weight and food intake were recorded throughout the study . Total feed efficiency was calculated by determining the gain in body weight (mg) per energy unit consumed (kcal). Venous tail blood was collected following a 6 hr fast for evaluation of glucose and insulin at 4 wk intervals . After 24 wk, mice were anesthetized (ketamine / xylazine cocktail 70 and 30 mg / kg body weight, resp .) As previously reported and whole body dxa (lunar pixi, ge medical systems, madison, wi) scans were performed . An aliquot of blood was collected for total white blood cell (wbc) counts and the remainder processed for plasma in edta coated tubes and stored at 80c . All procedures were approved by the institutional animal care and use committee of oklahoma state university . One week prior to the end of the study (23rd wk), mice were fasted for 6 hrs and an intraperitoneal (ip) glucose tolerance test (igtt) was performed . An ip glucose solution (2 g glucose / kg bodyweight) was administered, followed by blood glucose monitoring at 15, 30, 60, 90, and 120 min . Area under the curve (auc) was determined by calculating the sum of rectangular area between each time point . Plasma insulin was assessed at 4 wk intervals, whereas plasma leptin, adiponectin, and osteocalcin (ocn), both total ocn (gla - ocn) and undercarboxylated ocn (glu - ocn), were determined only at the final time point . All assays were performed using commercially available elisa kits including insulin (crystal chem, downers grove, il), leptin and adiponectin (emd millipore, billerica, ma), and gla - ocn and glu - ocn (clontech takara bio, mountain view, ca), following the manufacturer's protocol . Gla - ocn is reported as an indicator of bone turnover and given the importance of the carboxylation status of ocn relative to total ocn on glucose metabolism, the ratio of [glu - ocn]/[gla - ocn] was calculated to provide insight into the relationship between the skeletal and metabolic response to treatment . Whole body dxa scans were performed to determine body composition, bone mineral area (bma), content (bmc), and bmd . All scans were analyzed using piximus series software version 1.4x (ge lunar pixi, madison, wi). Micro - ct (micro - ct 40, scanco medical, switzerland) was used to evaluate bone microarchitecture at the proximal tibia metaphysis, tibia middiaphysis, and 4th lumbar vertebral body . Analysis of trabecular bone was performed at the proximal tibia metaphysis on high resolution scans (2048 2048 pixels) and the volume of interest (voi) included 750 m of secondary spongiosa . The voi was analyzed using a threshold of 300, a sigma of 0.7, and support of 1.0 . Trabecular bone of the vertebra was assessed on images 80 m from the dorsal and caudal growth plates at medium resolution (1024 1024 pixels) and included only secondary spongiosa . Images generated from the scans of the vertebrae were analyzed at a threshold of 340 and a sigma and support of 1.2 and 2.0, respectively . Trabecular parameters evaluated included trabecular bone volume expressed as a percentage of total volume (bv / tv), trabecular number (tb.n . ), trabecular thickness (tb.th . ), trabecular separation (tb.sp .) Cortical bone was evaluated by analyzing a 120 m section at the mid - diaphysis of the tibia . Assessment of cortical bone parameters included cortical porosity, thickness, area, and medullary area of the tibial middiaphysis . The acquired images were analyzed at a threshold of 300, a sigma of 0.7, and support of 1.0 . Tibiae were cleaned of soft - adhering tissue and stored in phosphate buffered saline (pbs) at 4c until analyses were performed . Reference point indentation (rpi) was applied laterally at the tibia - fibula junction using a biodent (active life scientific, inc ., santa barbara, ca), and the first cycle indentation distance and touchdown distance were recorded . Each tibia was subjected to a testing protocol of 2 n force, 2 hz, and 10 cycles . Fixed (10% neutral buffered formalin) liver samples were processed and sectioned (5 m) for staining with hematoxylin and eosin to assess histological changes associated with nonalcoholic fatty liver disease (nafld) that occurs in obesity and/or diabetes . Steatosis and fibrosis were scored on a scale from 0 to 4, with 0 indicating the absence of hepatic lipid droplets or fibrosis, whereas 4 indicated pronounced steatosis or fibrosis . Lobular and portal inflammation was scored using a range of 03, with 0 indicating the absence of macrophage infiltration and 3 corresponding to severe inflammation . Balloon degeneration was scored using a 02 system, with 0 defined as the lack of degeneration and 2 indicating modest presence of parenchymal cell death . Total rna was isolated from the liver and bone marrow using trizol reagent (invitrogen, grand island, ny) as previously described [36, 37]. Cdna was synthesized following a standardized laboratory protocol and qpcr was performed using sybr green chemistry (7900ht fast real - time, applied biosystems, foster city, ca). Hepatic genes of interest included fatty acid synthase (fasn), sterol regulatory element - binding protein (srebp1c), glucose transporter 2 or solute carrier family (slc2a2), peroxisome proliferative - activator (ppara), and glutathione peroxidase (gpx1) and in the bone marrow fasn, ppara, and gpx1 (table s1 available online at http://dx.doi.org/10.1155/2015/758080). All qpcr results were evaluated by the comparative cycle number at threshold (cq) method (user manual #2, applied biosystems) using peptidylprolyl isomerase b or cyclophilin b (ppib) as the invariant control . Statistical analyses were performed using statistical analysis software version 9.3 (sas institute, nc). The primary objective was to determine the difference in response to a hf diet of a given strain and, therefore, student's paired t - test was used unless stated otherwise . However, to further assess differences in the responsiveness between the two c57bl/6 substrains, if a statistical difference (p <0.05) was observed for a given parameter between con and hf within a given strain, the magnitude of response (i.e., percent change of hf compared to con) was compared between strains using one - way anova . When the f value was <0.05, post hoc analyses were performed with fischer's least square means separation test . All data are presented as mean standard error (se) and a p <0.05 was considered statistically significant . At baseline, body weight between strains differed (c3h / hej> c57bl/6n> c57bl/6j); however, no differences existed within a given strain between the two dietary treatment groups (i.e., con versus hf; data not shown). After 5 wk on the hf diet, the c57bl/6j exhibited a significant increase in body weight compared to the c57bl/6j con, whereas the c57bl/6n on the hf diet had a higher (p <0.05) body weight after only 3 wk (figure 1). The c3h / hej mice on the hf diet also exhibited a more rapid increase in body weight after only 1 wk compared to their respective controls (figure 1). Analysis of body composition revealed the increase in body weight was due to a significant increase in both lean and fat mass for the two c57bl/6 substrains as well as the c3h / hej mice (table 1). The amount of food consumed was less for the mice on the hf diet in each strain (table 1). However, on a kcal basis the c57bl/6n mice on the hf diet consumed + 2.1 kcal / day and the c57bl/6j and c3h / hej on the hf diet consumed + 1.2 kcal / day compared to their respective controls (data not shown). Overall, feed efficiency was higher in the c57bl/6n mice than in the c57bl/6j mice on the hf diet (table 1), further demonstrating the differences in metabolic responsiveness between these two substrains . After 24 wk on a hf diet the c57bl/6n mice exhibited splenomegaly, thymic hypertrophy, and decreased wbc, but the c57bl/6j mice failed to demonstrate these immunological changes (table 1). C3h / hej mice had a similar response to the hf diet in terms of tissue weights (i.e., spleen and thymus) and total wbcs compared to the c57bl/6n mice (table 1). C57bl/6n mice on the hf diet were the only strain that had elevated fasting blood glucose (figure 2(a)) and plasma insulin (figure 2(b)) after 4, 8, 12, 16, 20, and 24 wk of treatment compared to their con counterparts . The c57bl/6j substrain on the hf diet was hyperglycemic at 16 and 20 wk (figure 2(a)) and hyperinsulinemic at 24 wk (figure 2(b)). Importantly, neither substrain achieved a fasting blood glucose consistent with frank diabetes (i.e.,> 250 mg / dl) that is associated with polyuria and polydipsia . Similar to the c57bl/6j mice, the c3h / hej strain on the hf diet exhibited delayed - onset of hyperglycemia (figure 2(a)), while their plasma insulin was increased at 12, 20, and 24 wk (figure 2(b)). At the end of the study, igtt showed that the c57bl/6j and c57bl/6n as well as the c3h / hej mice on the hf diet exhibited impaired glucose intolerance (figures 3(a) and 3(b)). The percent change in auc to the hf diet demonstrated that the magnitude of response of the two c57bl/6 substrains was similar (data not shown). The c3h / hej mice also exhibited impaired glucose intolerance after 24 wk on a hf diet (figure 3). It should be noted that, despite elevated auc, the c3h / hej mice on the hf diet maintained the ability to restore blood glucose by the final igtt time point . Both the c57bl/6j and c57bl/6n substrains had elevated plasma leptin after 24 wk on a hf diet (table 1), but there was no significant difference in the magnitude of the response between the two substrains . Similarly, the c3h / hej mice on the hf diet also had higher plasma leptin (table 1). Interestingly, at 24 wk the c57bl/6j mice, but not the c57bl/6n substrain, exhibited a decrease in plasma adiponectin in response to a hf diet (table 1). After 24 wk on a hf diet, there were no differences in gla - ocn as an indicator of bone turnover due to diet in the c57bl/6 substrains or c3h / hej mice (data not show). The carboxylation status of ocn (i.e., glu / gla - ocn ratio), which has been shown to influence insulin sensitivity and systemic energy metabolism, was reduced only in the c57bl/6n mice after 24 wk on a hf diet (figure 4). Representative micrographs of liver sections from each group show that the c57bl/6j and c57bl/6n strains as well as the c3h / hej strain experienced some degree of hepatic steatosis in response to the hf diet (figure 5). The c57bl/6n mice on the hf diet had a significantly higher lobular and portal inflammation mean score compared to the con (table 2). Although the c57bl/6j mice on the hf diet had more lobular inflammation than their respective controls (p = 0.0038), the frequency of the inflammatory response was markedly lower in this substrain compared to the c57bl/6n (i.e., lobular inflammation in 92% c57bl/6n versus 54% c57bl/6j and portal inflammation in 77% c57bl/6n versus 23% c57bl/6j) (table 2). While none of the c57bl/6j mice on the hf diet exhibited liver fibrosis, 23% of the treated c57bl/6n mice had fibrotic changes (table 2). Balloon degeneration was also more severe in the c57bl/6n mice on the hf diet compared to the c57bl/6j (table 2). Despite a lack of lobular and portal inflammation and fibrosis in the c3h / hej mice, balloon degeneration was severe in this strain (table 2). Both the c57bl/6j and c57bl/6n mice demonstrated a decrease in whole body bmc and bma, but no change in whole body bmd in response to the hf diet after 24 wk (table 3). When bmd was expressed relative to body weight, differences due to diet were observed suggesting that the bone density did not increase relative to the increase in body weight (table 3). Micro - ct analyses of the lumbar vertebra revealed significant loss of trabecular bone or bv / tv with the hf diet in c57bl/6n, while the skeletal response of the c57bl/6j mice did not reach the level of statistical significance (p <0.0579) (figure 6(a)). As expected, the c3h / hej mice were protected from vertebral bone loss (figure 6) or nonmorphometric parameters with hf diet (table 3). Both the c57bl/6j and c57bl/6n mice on the hf diet had a higher smi indicative of a weaker, more rod - like trabecular bone in the vertebra (table 3). In contrast to the vertebra, no changes were observed in trabecular or cortical parameters analyzed at the proximal tibial metaphysis or the tibial middiaphysis in the c57bl/6j or the c57bl/6n mice . The c3h / hej mice failed to demonstrate alterations in trabecular bone of the proximal tibia but did exhibit an increase in the medullary area at the middiaphysis (table 3). Based on reference point indentation testing on cortical bone at the tibia - fibula junction, no changes were observed in first cycle indentation distance or touchdown distance in any strain following 24 wk on a hf diet when compared to their respective con (table 3). Determination of genes involved in hepatic metabolism and inflammation revealed that the c57bl/6n mice on the hf diet had altered metabolic processes, including the upregulation of glucose uptake (slc2a2), triglyceride storage (fasn and srebp1c) and adipogenesis (ppara), as well as antioxidant capacity (gpx1) (table 4). Interestingly, none of these alterations in gene expression were observed in the c57bl/6j mice after 24 wk on the hf diet . To determine the degree to which oxidative stress and adipogenesis contributed to bone loss with the hf diet model, gpx1 and pparg mrna abundance similar to the hepatic tissue, the abundance of gpx1 mrna was increased in the c57bl/6n mice on the hf diet, suggesting an increase in antioxidant capacity (table 4). In contrast, the c57bl/6j mice on the hf diet demonstrated a decrease in the relative abundance of gpx1 (table 4). Additionally, no alterations were observed in the transcriptional regulator of adipogenesis, pparg, in any strain after 24 wk (table 4). The findings of this study show that the c57bl/6j and the c57bl/6n mouse differ in their metabolic response to a hf diet over a 24 wk study period . Discrepancies in the metabolic response between the two strains may be attributed in part to the missense mutation (m35 t) in exon 1 and a multiexon deletion of nnt in the c57bl/6j mice [39, 40]. This mutation in nnt has been reported to uncouple -cell mitochondrial metabolism leading to less atp production in pancreatic islets, enhanced katp channel activity, and, consequently, impaired glucose - stimulated insulin secretion [23, 39, 41]. Only fasting insulin was assessed in the current study; however, early onset of hyperinsulinemia with hf diet was only observed in the c57bl/6n mice with an intact, functional nnt . The coincident lower feed efficiency in the c57bl/6j compared to the c57bl/6n mice resulted in a delay in the development of hyperglycemia and shorter duration of exposure to conditions associated with impaired glucose tolerance . The c57bl/6j and c57bl/6n mice had a markedly different hepatic response to the hf diet after 24 wk . Increased mrna abundance of fasn and a modest increase in srebpc1 in the presence of severe liver steatosis in the c57bl/6n mice on the hf diet suggest an increase in hepatic triglyceride synthesis and storage . Conversely, the c57bl/6j substrain, which has lower glucokinase activity and thus impaired glucose sensing, may explain the lack of transcriptional regulation of fasn and srebp1c . Furthermore, c57bl/6n mice on the hf diet demonstrated an increase in slc2a2 gene expression, which encodes the non - insulin - sensitive glucose transporter 2 and has been reported to be upregulated in response to a hf diet . Histological evaluation suggests that the c57bl/6n mice on the hf diet also experienced the most pronounced hepatic inflammation, compared to the c57bl/6j mice . Interestingly, the hf - induced nafld that develops in the c57bl/6j was previously attributed, in part, to the spontaneous mutation in the nnt [39, 43]; however, the data demonstrate that the c57bl/6n mice, with a functional nnt, develop more severe nafld in response to hf diet feeding . Given the more pronounced metabolic phenotype in the c57bl/6n mice on the hf diet, it was counterintuitive that only the c57bl/6j mice demonstrated the anticipated decrease in plasma adiponectin following a hf diet . Although the mechanism for reduced adiponectin during obesity and t2 dm has been attributed, in part, to an increase in local tnf- in adipose tissue, strain - related variability of adiponectin gene expression and plasma has been previously documented . As mice continue to be used for models of impaired glucose homeostasis, further investigation is warranted to fully understand these strain differences relative to metabolic handling . The findings of this study also demonstrate that the c3h / hej mice may not be completely resistant to diet - induced obesity and the subsequent metabolic changes . Differences in the c3h / hej strain's response to a high fat diet compared to previous reports may be attributed to the difference in the control strain used [26, 28]. Specifically, previous studies have compared the c3h / h3j response to hf diet to c3h / heouj or c3h / hen, both of which have a functional tlr-4 [26, 28]. However, in these studies, no comparisons were made with c3h / hej mice on a control diet . Therefore, it is not possible to determine if the differences in metabolic response to a hf diet are a result of tlr-4 or genetic variability in the control substrain background . Furthermore, the c3h / hej strain has recently been shown to have a genetic variation in the leptin receptor gene (lepr). This mutation could account for the impaired metabolic response observed in the c3h / hej strain on a hf diet due to the central role leptin has on regulating energy intake and expenditure . However, in the present study, food intake in the c3h / hej strain was not significantly altered and the absence of a leptin - mediated effect on food or energy intake does not rule out implications of the lepr defect on the skeletal response [18, 19, 47]. In conjunction with the metabolic comparisons, the other primary objective of this study was to compare the skeletal response to a hf diet in two commonly used c57bl/6 substrains . The c57bl/6j mice on the hf diet experienced a 13.6% reduction in trabecular bone of the vertebra, although not statistically significant . C57bl/6n was the only strain that exhibited significant trabecular bone loss which occurred only in the vertebra . In the absence of alterations in tibia trabecular and cortical bone microarchitecture, it is conceivable that the absence of alterations in the tibia could result from site - specific changes associated with increased adiposity and greater weight - bearing, which could offset some of the negative effects of glucose intolerance on bone [48, 49]. Based on reports in the literature that bone biomechanical properties are changed in t2 dm independent of alterations in bone mass [8, 50, 51], the tibia was subjected to rpi testing . No detectable alterations in cortical bone strength were observed after 6 months in the absence of structural changes . The c57bl/6n mice had more prolonged exposure to hyperglycemia and hyperinsulinemia in response to the hf diet compared to the c57bl/6j substrain, and c57bl/6n were the only substrain to lose significant trabecular bone in the spine . While there have been conflicting reports on how a hf diet impacts bone in c57bl/6 mice [12, 1417], the results of this study indicate the skeletal response may be linked to the duration of disrupted insulin signaling and glucose intolerance . This idea is further supported by the response of the c3h / hej mice in which case an attenuated glucose, leptin, and insulin response to the hf diet failed to induce bone loss . Several reports have shown that a high fat diet uncouples bone turnover by increasing bone resorption and decreasing bone formation in various rodent models [16, 17, 21]. In this study, osteocalcin which is considered a marker of bone turnover was the only bone marker assessed and it was not altered at the end of the study . Although this does not rule out alterations in bone metabolism occurring earlier, future studies are needed to investigate the mechanism involved in the site - specific loss of bone observed in this animal model . Based on recent literature describing the hormone ocn as a regulator of systemic energy metabolism [5254], the role of ocn on both metabolic and skeletal changes induced by hf was investigated . After 24 wk, the c57bl/6n mice on the hf diet had a lower ratio of plasma glu - ocn / gla - ocn . Because circulating undercarboxylated (glu - ocn) can act directly on pancreatic -cells to stimulate insulin secretion [20, 5557], it would be expected that a reduction in glu - ocn would lead to a decrease in insulin secretion . Instead, fasting plasma insulin was elevated in the c57bl/6n mice on the hf diet compared to their respective controls . Alternatively, these results could indicate the direct effects impaired glucose tolerance has on osteoclastogenesis and bone resorption . In this regard, the acidic milieu of the osteoclast - resorption lacunae is capable of decarboxylating ocn, resulting in its release from the bone matrix and its subsequent circulation in the blood . The complexity of ocn's role on bone and energy metabolism during glucose intolerance, as well as these implications in various mouse strains, warrants further investigation . Additional studies are also needed to determine how genes involved in the gamma carboxylation of ocn (i.e., ggcx and esp1) by osteoblasts as well as the role of decarboxylation of ocn by osteoclast are regulated in response to changes energy homeostasis [55, 59]. To date, this is the first study to directly compare the c57bl/6j and c57bl/6n substrains' response to a hf diet from a metabolic and skeletal perspective . Although neither substrain developed frank t2 dm, the data presented here show that c57bl/6n mice exhibit an earlier metabolic response consistent with impaired glucose tolerance or prediabetes to the hf diet compared to the c57bl/6j mice . Moreover, the skeletal response followed that of the metabolic changes; this was demonstrated by the fact that significant trabecular bone loss occurred in the c57bl/6n mice, which demonstrated the robust metabolic alterations associated with clinical t2 dm . Given the observed differences in feed efficiency between the c57bl/6 substrains, further research is also warranted to identify the mechanisms underlying altered energy utilization . In contrast to the c57bl/6 mice, the c3h / hej strain was protected from the metabolic and skeletal changes induced by a hf diet . While a number of questions remain including how bone metabolism is being altered in response to a high fat diet on a molecular level, this study highlights the need to consider not only the most appropriate strain but also the most appropriate substrain of mouse when designing experiments . Other important factors to consider when studying the relationship between glucose intolerance and bone include the site - specific skeletal response and the study duration . These decisions could significantly impact data interpretation and the translational implications as they relate to understanding how bone metabolism is altered in the context of t2 dm.
Appropriate management of farm waste such as manure is critical in controlling the spread of pathogens such as e. coli o157:h7 to vegetable crops . In most management schemes, fumigants are used for the control of plant pathogens, nematodes, and weeds before high - value cash crops such as strawberry and tomato are planted . Outbreaks of e. coli o157:h7 infections historically have been associated with consumption of undercooked ground beef; however, many recent outbreaks have resulted from consumption of contaminated raw vegetables, including lettuce [13]. Although many pathogens have been associated with fresh produce, e. coli o157:h7 is of particular concern because ingestion of relatively few cells can cause illness . E. coli o157:h7 can survive for 60 to 120 days in water and in soil, and under dry and acidic conditions . The steps in the production chain that have the greatest potential for pathogen contamination are soil preparation (use of uncomposted manures) and planting and growing (use of contaminated irrigation water and animal manures and manure from animals grazing locally or nearby) [68]. Prevention of preharvest contamination of fresh produce is an essential part of systems approach focused on interventions designed to achieve delivery of microbiological safe produce to consumers . Suppression of human pathogens in agricultural soils and the subsequent prevention of spread into the food chain by contamination of produce must be realized by the adoption of best management practices . In the absence of known phytopathogens, many crops have exhibited an increased growth response when planted into soil that had been fumigated with mebr at the recommended application rate . One of the likely reasons for this observation may be that fumigation altered the microbial composition of the soil, either enhancing beneficial colonizers or reducing populations of deleterious rhizosphere colonizers . Fumigation is to control plant pathogens such as nematodes, soil - borne diseases, and weeds . The immediate impact of fumigation may be the reduction of certain bacterial species in the soil and the development of new communities after a few weeks [11, 12]. Some fumigants may be toxic to some microbes, and this may enhance the selection of microbes that may be beneficial to plants [11, 12]. Due to the increased focus on food safety related to fresh produce, there are more studies using real - time pcr to quantify pathogens such as e. coli o157:h7 in the environment [13, 14]. The main objectives of this study were to determine the effects of soil microbial diversity and fumigation on the survival of e. coli o157:h7 in soils contaminated with the pathogen . To accomplish these objectives, a preliminary study was conducted to determine the survival of the pathogen in both autoclaved and nonautoclaved soils at different concentrations of the pathogen . For our main objectives, both plate count and real - time pcr approaches were used to determine the survival of e. coli o157:h7 in the two soils . This strain produces shiga - like toxin stx1 and stx2 and the pgfp expressing green fluorescent protein (gfp) and ampicilin resistance . E. coli o157:h7 was cultured at 37c overnight in modified tryptic soy broth (mtsb)(difco laboratories inc ., cockeysville, md) supplemented with 100 g of ampicillin ml (sigma, st louis, mo). Cells were harvested by centrifugation at 3500 x g for 10 min and resuspended in phosphate buffered saline (pbs) (fisher scientific, pittsburgh, pa) to a concentration of ~10 cfu ml . Bacterial strains (except strain 72) used to determine the specificity and sensitivity of pcr assays were obtained from the national animal disease center (ames, ia) and were cultured on luria - bertani (lb) broth agar and sorbitol macconkey (smac) agar plates at 37c . Clay soil (willows silty clay, saline - alkaline) and sandy soil (dello loamy sand) were collected from mystic lake dry bed and the santa ana river bed, respectively, and treated as described by ibekwe and grieve . The clay soil had a bulk density of 1.51 mg m with 3.7% sand, 49.1% silt, and 47.2% clay . The sandy soil has a bulk density of 1.67 mg m with 99.1% sand, 0.20% silt, and 0.70% clay . The moisture content of the clay soil was 4.02% and that of sandy soil was 5.32%, before both were increased to about 12% at the start of the experiment . The ph of sand was 6.85 and that of clay was 7.45 . Each soil (100 g dry wt) was placed in 150 ml beakers (18 beakers of each soil type) and the soil was autoclaved for 1 h, cooled for 24 h, and autoclaved again before use for the study . Inoculums were made in pbs and added to the appropriate soil beaker by spraying and mixing 10 ml of the culture mixture with a spray bottle (sprayco, detroit, mich . ; sanitized by soaking in 70% ethanol) on the surface of 100 g of soil to obtain the following inoculums concentrations in triplicate: 10, 10, 10, 10, 10, and 0 for both autoclaved and unautoclaved soils.serial dilution was made with 10 g portion of each soil for the enumeration of bacteria . Soils were mixed for 5 min with sterile specula to homogeneously distribute the e. coli o157:h7, and covered with foil and incubated at 20 c in the growth chamber for the duration of the experiment . E. coli o157:h7 population was determined by plating on tryptic soy agar (tsa; becton dickinson) plates containing 100 g of ampicillin ml (tsa - a) at days 0 (inoculation), 1, 3, 5, 10, 20, 30, 40, 50, and 60 . The fumigant methyl iodide (mei, iodomethane,> 99% purity) was purchased from chem service (west chester, pa) and methyl bromide (mebr> 99% purity) was obtained from great lakes chemical company (west lafayette, in). Plastic trays (58.2 43.2 18.5 cm) were filled with approximately 40 kg of soil . Bacteria were inoculated into the irrigation lines with a cole - parmer hplc pump (cole - parmer, chicago, illinois) and delivered through pvc pipes to each tray with five surface drip lines . Soil samples were collected on the day of inoculation for community analysis, e. coli o157:h7(pgfp) concentration and heterotrophic plate counts . After the initial sample collection, trays were manually covered with a virtually impermeable plastic film; 0.038 mm hytibar film (klerk plastics, belgium) and fumigants were applied . Fumigant rates and application methods were selected according to the recommended field application rate for each chemical by california department of pesticide regulation (http://www.cdpr.ca.gov/). To avoid the emission of fumigants to the growth chamber, syringes were used to inject fumigant (mebr - gas and mei - liquid) into the trays; injection ports covered immediately with duct tape and left in the growth chamber for 10 days . Trays remained outside in an area covered with barb wires, opened and aerated for 2 days before they were moved back to the growth chamber for the continuation of the experiment . At this point, a total of 14 days has elapsed since fumigation, and soil samples were collected for e. coli o157:h7(pgfp) concentration, bacterial diversity, and heterotrophic plate counts . The sandy soil received the above nutrient solution twice daily, because this was a very poor river bank soil with poor nutrients for plant growth . Soil samples were collected weekly for 5 weeks for e. coli o157:h7, heterotrophic plate counts and for total bacterial dna extraction . Soil was transferred to ziplock bags and 10 g sample was used for serial dilution . E. coli / pgfp colonies were enumerated under a hand - held spectroline ultraviolet lamp (spectronics corporation, westbury, n.y). Community dna was extracted from 0.5 g soil with the ultra clean soil dna kit (mobio laboratories, solana beach, ca) according to the manufacturer's protocol and stored at 20 c. a 236-bp dna fragment in the v3 region of the small subunit ribosomal rna genes of eubacteria was amplified by using primer set prba338f and prun518r . Ready - to - go pcr beads (amersham pharmacia biotech, piscataway, nj) and 5 pmol of primers in a total volume of 25 ml were used in the pcr reaction . Pcr amplifications were done under the following conditions: 92c for 2 min; 30 cycles of 92c for 1 min, 55c for 30 s, 77c for 1 min followed by a final extension at 72c for 6 min . Dgge was performed with 8% (wt / vol) acrylamide gels containing a linear chemical gradient ranging from 30% to 70% denaturant with 100% defined as 7 m urea and 40% formamide . Gels were run for 3 h at 200 v with the dcode universal mutation system (bio - rad laboratories, hercules, ca). Dna was visualized after ethidium bromide staining by uv transillumination and photographed with a polaroid camera . Dna fingerprints obtained from the 16s rrna banding patterns on the dgge gels were photographed and digitized using imagemaster labscan (amersham - pharmacia biotech, uppsala, sweden) and analyzed . The comparison of diversity was done by using a one - way analysis of variance, and tukey hsd test for post hoc analysis . Diversity was calculated by using the shannon index of diversity (h) to compare changes in diversity of microbial communities within all treatments at each time by using the following function: (1)h = pi log pi, when pi = ni / n, ni is the height of peak, and n is the sum of all peak heights in the curve . Genomic dna was isolated from pure culture of e. coli o157:h7, grown for 12 h at 37c and extracted with the qiagen tissue kit (qiaamp dna mini kit; valencia, ca). Dna extracted from o157:h7 was used for the construction of standard curve and for the determination of detection limits of the e. coli by real - time pcr . Total bacterial dna was extracted from soil with the ultra clean soil dna kit (mobio laboratories, solana beach, ca) as stated above and stored at 20c . Primers and probes used for the detection and quantification of the stx1, stx2, and the eae gene in e. coli o157:h7 were as described [21, 22]. Real - time, quantitative pcr was performed with the icycler iq (bio - rad, hercules, ca) as described by ibekwe et al . . Briefly, pcr was performed in a total volume of 50 l volume containing 200 m of dntps, 2 l of genomic dna from each concentration, 2.5 u of amplitaq gold polymerase, 5 l of 10x taqman buffer (pe applied biosystems, foster city, ca), 0.3 m of each primer, 0.1 m of probe, and 3.5 mm of mgcl2 . Genomic dna purified from e. coli o157:h7 was used as a template for the positive control and no template for negative control . Pcr was performed using the following cycle conditions: denaturation at 95c for 10 min, 50 cycles of 94c for 20 s, 55c for 30 s, 72c for 40 s, followed by a 5 min extension at 72c and a hold at 4c . Standard curves generated from plotting the threshold cycle (ct) versus log10 of starting dna quantities (pg) were used for determining the detection limit of the assay . Optimization of the multiplex assay was done as previously discussed [21, 22]. Amplification efficiency (e) was estimated by using the slope of the standard curve and the formula: e = (10)1 . Reaction with 100% efficiency generated a slope of 3.32 . Statistical analyses were done with the general linear model (glm) procedure of the statistical analysis system . The population data were log transformed to obtain a normal distribution of the data . Comparisons between pairs of treatment means at any date were accomplished with the tukey's test . The log - transform data of e. coli o157:h7 population size of all individual samples were plotted over time after inoculation, and analyzed by regression analysis . Plate counts and real time pcr data were transformed to log10 values and survival curves were obtained by plotting the logarithm of survivors against the treatment time . The survival data were fitted to a biphasic model as proposed by coroller et al . [23, 24] with the geeraerd and van impe inactivation model - fitting tool (ginafit) as shown in (2) and (3) and as described by franz et al . : (2)n(t)=n01 + 10[10(t/1)p++10(t/2)p],(3)=log 10 (f1f), where n is the number of survivors, n0 is the inoculums size; t is the time; p is the shape parameter, when p> 1 a convex curve is observed; when p <1 a concave curve is observed, when p = 1 a linear curve is observed . F, varying from 0 to 1, is the fraction of subpopulation 1 in the population . Another parameter,, varying from negative infinity to positive infinity, is obtained by logit transformation of f as shown in equation 2 . The strong correlation between the scale () and the shape (p) parameters makes it possible for the double weibull model to fit most of the shapes of deactivation curves . Additionally, when 1 = 2, the double weibull model can be simplified into a single weibull model, and the survival curve can be described by only three parameters . A very important and useful parameter, time to detection limit (td) can also be calculated when using ginafit to fit the experimental survival data . The effects of inoculum density on the survival of e. coli o157:h7 in sandy and clay soils was first determined in the two soils used for this study . This was done to determine the influence of indigenous microorganism on the survival of e. coli o157:h7 in autoclaved and unautoclaved soils . Data from the survival study showed that within the first 7 days e. coli o157:h7 populations decreased by ca . 0.24 log10cfu g in the 10 dilution and by 0.67 log10cfu g in the 10 dilution for the unautoclaved soil (figures 1(a) and 1(b)). The reverse was the case with autoclaved soil where there was an increased in population by 2.13 log10 cfu g in the 10 dilution and an increased of 1.68 log10 cfu g in the 10 dilution . Survival curves showed a concave curvature in autoclave sandy soil and a convex curvature in the unautoclaved sandy soil (figure 1(a)). In the clay soil, the shape parameter was different from sandy soil with soils with inoculums density of 10 showing the convex shape whereas soils with cells at 10 showed either concave or linear shape . Modeling parameters (alpha (), delta (), and the shape parameter - p) were calculated from equation (2) and (3) used to explain the inactivation kinetics . More variations in values were observed from different soils (figures 1(c) and 1(d)). When the strain was characterized in sandy and clay soils, distinct 1 and 2 were observed indicating that the two subpopulations behaved differently in both soils, thus the survival data in both soils might not be simplified into the single weibull model that can be described by only three parameters,, and p. the initial sharp decrease in cell numbers in sandy soil (concave shape) might largely be attributed to the faster decline of subpopulation as shown with smaller 1 (figure 1(c)). However, with the time going, the subpopulation with greater 2 (i.e., the more resistant) dominated the cell population, leading to a slower and steadily decline of the cell concentration as the curves showed little or no decline . After the first 10 days, e. coli o157:h7 populations in unautoclaved soil declined considerably more rapidly than in autoclaved soil below the detection limits of 10 cfug soil . After 60 days, the concentration of e. coli o157:h7 in the 10 dilution was undetectable by plate count, and there was a 6.18 log10cfu g reduction for the 10 dilution . Survival of pathogen was greater in the sandy soil (p = .05) than clay unautoclaved soil within the first 7 days (figure 1). E. coli o157:h7 at 10 cfu g dilutions survived for more than 60 days in both unautoclaved and autoclaved soils used in this study . Before the enumeration of e. coli o157:h7 in the different matrices, background concentrations of heterotrophic bacterial the initial heterotrophic plate count in soil was 2.1 10 cfu g. after storage at 20c in the growth chamber, the total aerobic plate counts decreased steadily from ca . There were no differences in the levels of heterotrophic plate count in the two soils during the study period (data not shown). Mean comparison by days and methods were used to determine the impact of fumigants on the survival of e. coli o157:h7 in the two soils after fumigation (table 1). Since one of our objectives was to determine the effects of fumigants on e. coli o157:h7 on a weekly basis, direct comparison of the two fumigants and the control was done using plate count and real - time pcr to quantify the concentrations of e. coli o157:h7 . In the growth chamber soil, ten days after fumigation, e. coli o157:h7 was significantly lower (p = .0001) in fumigated soils than the control clay soil at the recommended application rate . During the rest of the study, there were no significant differences on the effect of the two fumigants on the pathogen, except on day 36 (p = .046) where the effects varied . Real - time pcr analysis showed that 10 days after fumigation, e. coli o157:h7 concentration in non - fumigated soils was significantly higher (p = .002) in sandy soil than clay soil . There were no significant differences (p = .56) in pathogen concentration during day 23 when real - time pcr was used for the analysis . The same effect was observed during day 36 and 50 (data not shown). Direct comparison between mebr and mei within each soil showed that neither had significant greater impact on e. coli o157:h7 . The majority of the survival curves (figure 2) showed a concave shape, with a relatively fast initial decline followed by a slower decline phase . Survival reached the detection limit faster in clay soil than in sandy soil without fumigation using plate counts (figures 2(a) and 2(b)). When the pathogen was exposed to fumigants (mei), inactivation was faster than in control, especially with plate count (figures 2(c) and 2(d)). The same pattern was observed with mebr (figures 2(e) and 2(f)). However, for both control treatments the population size did not reach the detection limit (ttd) of 10 cfu g during the experiment due to earlier onset of tailing at about 35 days using real - time pcr . Also, both soils showed that it took less than a day to inactivate the first log10 of microbial population in most of the fumigated samples . Effects of soil types on the survival e. coli o157:h7 in clay and sandy soils after fumigation was model by fitting the experimental data into the survival functions (figure 3). Similar modeling parameters (,, and p) were calculated when they were inoculated into the same soil (figures 3(a)3(f)). However, there more little variations in these parameters from the two soils, except the values . When the pathogen was characterized in sandy and clay soils, distinct 1 and 2 were observed indicating that the two subpopulations behave differently in both soils . The goodness - of - fit statistics (r) did not differ significantly between survival curves in sandy soil irrespective of fumigants or no fumigant . The same effect was observed in clay soil, indicating that the model is suitable to fit survival curves of e. coli o157:h7 in an array of different soils . Dgge analysis of 16s rrna fragments was used to examine the effects of mebr and mei on soil microbial communities during week 1 to7 after fumigation . The most drastic effect occurred on the first week of the experiment where there was a significant effect (p .05) of fumigants as determined by the shannon - weaver index of diversity between clay and sandy soil (table 2). During this period, microbial diversity in the mebr fumigated treatments was significantly lower (p = .0003) in sandy soil than in clay soil at the recommended application rate . The same effect was observed with mei fumigated soil . Bacterial communities were not different at week three (p = .13), week four (p = .06), and week five (p = .11). However, at week seven there was a significant (p = .0001) shift in microbial community structure as determined by diversity index with all the treatments (table 2), and the effect was greater in sandy soil than clay soil . This resulted in the initial higher survival rate of e. coli o157:h7 in sandy soil compared to clay soil . Microbial diversity (expressed as shannon weaver index of diversity h) was positively correlated with survival of e. coli o157:h7 in sandy soils (figure 4(a); r = 0.56, p = .015) and in clay soil using the plate count method (figure 4b; r = 0.47; p = .019). Using data from real - time pcr analysis, survival of e. coli o157:h7 were positively correlated with microbial diversity in both clay and sandy soils (data not shown). Before the start of this experiment, a preliminary study was conducted in autoclaved and unautoclaved soil to determine the influence of indigenous soil microorganisms on the survival and growth of e. coli o157:h7 (figures 1(a) and 1(b)). The antagonistic effect of indigenous soil microorganisms was likely a factor in killing e. coli o157:h7 cells in unautoclaved soils used in this study . E. coli o157:h7 was inactivated more rapidly in unautoclaved soil than in autoclaved soil in all the different dilutions of e. coli o157:h7 used as inoculums (figures 1(a) and 1(b) for 10 and 10 cfu g). Our study is in agreement with jiang et al . Who showed that small numbers of e. coli o157:h7 from an unautoclaved soil were only detected by enrichment culture, and survived for longer period of time at 15c than 21c . This suggests that there was a small population of cells that have survived in the soil under different environmental stress . The long - term survival of this pathogen in the environment has been reported by many authors [6, 2731], but very little has been done on the survival in fumigated soil . We have shown from this study that e. coli o157:h7 can survive in fumigated soils for over 60 days due to long - term persistence of a small percent of the population . Our study showed that e. coli o157:h7 can survive longer in sandy soil than in clay soil during a short term experiment . However, populations persisted longer in clay than in sandy soil during a long - term study . Our results showed that e. coli o157:h7 survived longer than 60 days in both soils . Others have reported survival of more than 54 days in manure amended soil [28, 3234] and 34 days or more in sandy loam soil amended with cow manure [26, 35], and over 90 days in clay soils (8, 40). Others reported longer e. coli o157:h7 survival times of between 154 and 217 d in soils amended with inoculated compost . This study with longer survival period agrees with our study because both studies relied on inoculating the substrate with relatively high densities of the pathogen (> 10 cfu g). Also, during our preliminary experiment with e. coli o157:h7 with population of lesser than 10 cfu g, survival of the pathogen was less than seven days this is in agreement with franz et al . That monitored the fate of the pathogens in manure - amended soil after they declined to below detection limit within a short period with low and more realistic levels of pathogens inoculated into manure (approximately 10 cfu g). Therefore, the survival of e. coli o157:h7 in the environment may depend on the initial concentration at the beginning of the experiment . In this study, we used the double weibull model, which is the cumulative form of the underlying distribution of individual inactivation kinetics, and it was a suitable model for describing the decline of e. coli o157:h7 . The survival curves generated from our study in most cases showed a convex fitting, indicating changes in biological stress over time . The model is sufficiently flexible to account for different survival patterns and has been previously used to model thermal inactivation of listeria monocytogenes in sucrose solutions of various water activities and the survival of e. coli o157:h7 in manure amended soil . Franz et al . ; van boekel,; peleg, discussed the different processes and mechnisms responsible for the different shape parameters during inactivation of e. coli o157:h7 in soil . They pointed out that even though the weibull model is an empirical model, it can be linked to physiological properties at population level and that the population is heterogeneous with respect to the stress encountered in the soil . These authors noted that a convex curve would mean that the remaining cells become increasingly susceptible to stress, and the cells are therefore subjected to more damages with time . A linear survival curve means that inactivation does not depend on time or other biological activities and the concave survival curve means that sensitive members of the population are rapidly eliminated and that the sturdier survivors remain . Longer persistence of e. coli o157:h7 in clay soil may be influenced by interaction between soil particles in the clay particle sizes that provided niches for the pathogen and moisture / nutrients within the niches . Other factors that contributed to the survival of pathogen in soil were soil microbial diversity . Recently, the effects of e. coli o157:h7 was assessed in a loamy sand soil obtained from species - rich grassland, in which the microbial community composition had been modified by progressively enhanced fumigation depths . The authors showed that e. coli o157:h7 in the soils with modified community structures due to fumigation was clearly consistent with the hypothesis that within the single selected habitat (soil), which was relatively unaffected with respect to abiotic conditions like ph, moisture and soil chemical conditions, microbial community structure was the main determinant of the survival of the pathogen . With the present study we found that the values of the log reduction time and the shape parameter of the double weibull model were higher for clay soils compared to sandy soils . This means that with sandy soils the initial rate of decline of e. coli o157:h7 was faster than in clay soil . E. coli o157:h7 was more vulnerable to mortality during the first few weeks in the sandy soil than in clay soil . Finer - textured (clayey) soils result in prolonged survival of introduced bacteria compared with coarser - textured (sandy) soils because of higher availability of protective pore spaces against feeding by soil fauna like protozoa . This could explain the faster initial decrease in e. coli o157:h7 numbers in the sandy soils compared with the loamy soils, but survivors are increasingly more sturdy compared with survival in the clay soils . The implication of long - term survival of this pathogen in the environment may involve the recontamination of the environment after the initial contamination event from few surviving strains . E. coli o157:h7 was significantly lower in fumigated soils than the control at the normal application rate during the first 2 weeks of the experiment . However, survival was significantly affected by soil type, with survival greater in sandy soil in the short run than clay soil . However, long - term persistence occurred in clay soil than sandy soil due to properties of clay soil . None of the fumigants showed significant higher toxicity effects on the pathogen at the normal application on the long run, and toxicity was higher in fumigated soil than nonfumigated soils during the first two weeks of the study . Therefore, mebr and mei have identical toxicity effects on e. coli o157:h7 at the normal application rate.
Whole blood samples collected during the 20082009 dengue epidemic were tested for denv igm by elisa (dengue igm capture elisa; panbio, brisbane, qld, australia). All reactive samples were tested with a second elisa (anti - dengue igm elisa; standard diagnostics inc ., giheung - gu, south korea) and by the public health virology laboratory at queensland health forensic and scientific services (qhfss) (11). Serologic evidence of recent exposure (presence of denv igm) was observed in 12 (0.22%) donors (table 1). Positive samples that were examined for type specificity, 7 (88%) were denv-3 specific, which was the dominant type during the epidemic (3). We used these igm seroprevalence rates to estimate the rate of subclinical dengue infection (technical appendix). We estimated 168921 subclinical cases (clinical: subclinical ratio 1.0:0.59; range 0.181.0) in cairns, the city where the epidemic was centered . Our estimate was toward the lower end of that observed in dengue - endemic areas (12,13) but higher than that estimated during a denv-2 outbreak in charters towers (14), which probably reflects the different methods used in the respective studies . Selected samples were tested for denv igg by elisa (dengue igg indirect elisa; panbio). Serologic evidence of previous exposure (presence of denv igg) was influenced overall by donor location (p<0.05) and age (p<0.05). The proportion of the northern queensland donor population with denv igg was 9.43% (95% ci 7.98%10.89%), and this proportion increased with age (table 2), which indicates cumulative previous exposure . The proportion of melbourne (control area with no dengue activity) donors with denv igg was 6.78% (95% ci 4.48%9.09%); however, no change was observed with age (table 2), demonstrating no cumulative exposure . Previous exposure in melbourne was surprisingly high; these persons may have been exposed during travel to dengue - endemic areas (subsequent follow - up demonstrated 94% reported travel to countries to which dengue may be endemic). The proportion of donors with denv igg did not change from the beginning to the end of the outbreaks in cairns and townsville, nor in northern queensland as a whole (table 2), which suggests that the epidemic was not of a scale to result in a change in population seroprevalence . This study was powered to detect a change in incidence of at least 10%; small changes might have been missed, which would be difficult to detect through such studies . We used our seroprevalence data along with donation frequencies to estimate the risk of collecting a dengue - infectious donation, based on published models (9,15) (technical appendix). Using this model, the risk of collecting a dengue - infectious donation in cairns during the epidemic was 1 in 7,146 (range 2,21850,021) (figure). These estimates are similar to those obtained by using a published probabilistic model (9) revised to incorporate the outbreak specific subclinical infection rate reported herein, which predicts the risk to be 1 in 9,303 (range 3,09232,344) donations in cairns . Because both methods derive estimates within comparable ranges, it would appear valid to use the revised probabilistic model as a predictive risk estimator during future outbreaks . Risk of collecting a dengue - infectious blood donation, northern queensland, australia, 20082009 epidemic . The dengue management strategy used during the epidemic cost the blood service 13.8 million australian dollars (2009 terms). This estimate is publically available and was based on: the number of donations affected by the dengue management strategy, collection targets for 2009, costs associated with whole blood collections, additional costs to meet national targets, transportation costs to meet demand in affected regions, and additional waste costs . An offset for any plasma obtained through a whole blood donation (used for fractionation) was included in selected estimates . Subclinical dengue infection rates vary by population, specific outbreak, and area examined (12,14). We demonstrate that the clinical to subclinical infection rate during the 20082009 dengue epidemic in northern queensland, where dengue occurs seasonally, was toward the lower end of that observed in dengue - endemic countries (12,13). This observation, together with our data suggesting that the incidence of dengue in the northern queensland population did not change from the beginning to the end of the epidemic, suggests the control and clinical management of dengue during this epidemic was comprehensive . We also estimated that the risk of collecting a dengue - infectious blood donation in cairns during the epidemic was 1 in 7,146 (range 2,21850,021). Given these risks, the increasing need for plasma in australia, and the absence of a screening test for blood donations in australia, the continuation of the dengue management strategy during future outbreaks is warranted . However, this strategy may have added strain on the inventory available to meet clinical demand for fresh blood components and was associated with a cost to the blood service of> 1 million australian dollars . Although this strategy is a necessary precaution to maintain safety, alternative approaches may exist, such as implementation of a suitable screening test (were one available) or pathogen reduction technology (a process designed to inactivate pathogens in blood products), which may offer a similar level of safety but be more cost effective . With dengue becoming increasingly common in australia (3) and the world (1), use of igm seroprevalence rates to estimate the rate of subclinical dengue virus infection and associated transfusion - transmission risk.
Based on our rodent studies on mixed chimerism, we initially developed a clinically relevant non - myeloablative preparative regimen that permits the induction of mixed chimerism and renal allograft tolerance when combined with simultaneous donor bone marrow transplantation (dbmt) in mhc fully - mismatched cynomolgus monkeys . This approach has been successfully extended to hla matched or mismatched clinical kidney transplantation . In murine models, the primary mechanism of tolerance induction through mixed chimerism was shown to be via thymic deletion . That is, donor derived dendritic cells (dc) migrate to the recipient thymus, where they induce negative selection of donor reactive t cell clones . Therefore, induction of stable mixed chimerism appeared to be a prerequisite for stable allograft tolerance through this strategy . However, the mixed chimerism induced in primates with our non - myeloablative regimen has always been transient in nature, but nevertheless, essential to induce renal allograft tolerance in this model . This led us to conclude that the mechanisms associated with induction of tolerance in primates include peripheral as well as central thymic deletion pathways . Our original protocol requires treatment of subjects beginning six days prior to organ transplantation, which limits its applicability to living donor transplant recipients . Therefore, our next major goal has been to develop a strategy that is applicable to deceased donor organ transplantation . We initially evaluated regimens in which conditioning was begun within 24 hours of kidney transplantation (ktx). However, simple compression of the previously effective six - day therapeutic protocol into a 24-hour period failed to induce chimerism and also led to unacceptable toxicity (fig . Approach, with which the recipient initially undergoes organ transplantation with conventional immunosuppression, followed by conditioning and donor bone marrow transplantation (dbmt) at a later date . This approach would potentially extend the applicability of our regimen to not only current recipients of deceased donor transplantation but also to any recipient of a previously transplanted allograft from either a living or deceased donor, if dbm is available . Strategy has the theoretical disadvantage that donor - specific memory t cells (tmem) might have been elicited despite administration of potent immunosuppressive agents during the interval between transplantation and attempted tolerance induction . Therefore, we have extensively monitored tmem subsets and alloreactive tmem responses in these studies . Renal allograft survival in the delayed tolerance (deaths due to infectious complication censored). Simple compression of the previously effective 6-d therapeutic protocol into a 24-h period (d) failed to induce chimerism and also led to unacceptable toxicity . The conditioning regimen that was successful in simultaneous kidney and bone marrow transplantation (skbmt) failed to induce long - term allograft survival in the delayed tolerance protocol at 4 mo (b). When anti - cd8 mab was added to the original regimen, however, this modified regimen with anti - cd8 mab was not successful in recipients of the delayed tolerance protocol at 1 mo (c). Primates including monkeys subjected to these experiments typically exhibit rigorous heterologous tmem responses even before ktx . In addition to nave t cell responses, these preexisting tmem that heterologously respond to alloantigens may further impair induction of chimerism and allograft tolerance . Somewhat unexpectedly, the initially high alloreactive tmem responses appeared to decline after ktx in a time - dependent fashion . As shown in figure 2, ifn and il-2 tmem responses progressively fell after ktx and became almost undetectable by four months . Since third party tmem responses were relatively preserved, this was not simply due to the global effects of immunosuppression . Development of such donor - specific tmem hyporesponsiveness has also been reported in clinical ktx and is speculated to result from the interaction between recipient lymphocytes and tolerogenic graft parenchymal cells . The important point is that, if these elispot results truly reflect the in vivo status of tmem responses, induction of chimerism might be even easier when dbmt is delayed . Post ktx anti - donor responses were measured by elispot in various populations, bulk(pbmcs), tmem(cd16cd95), cd8 mem(cd16cd8cd95) and cd4 mem(cd16cd4cd95). In the delayed tolerance, recipients initially underwent ktx alone and were treated with conventional immunosuppression (tacrolimus, mmf and steroids). Four months later, the recipients received our standard conditioning regimen (low dose total body irradiation, local thymic irradiation, atg and anti - cd40l mab). With this regimen, recipients of simultaneous kidney and dbm transplantation (skbmt) consistently developed multilineage chimerism and most achieved long - term survival without immunosuppression . In contrast, no recipients conditioned at four months with the same therapeutic regimen developed multilineage chimerism (fig . 3c) and all rejected their previously well - functioning kidney allografts soon after discontinuation of immunosuppression (fig . This homeostatic recovery was faster than that observed in skbmt (data not shown) leading us to conclude that cd8 tmems had been insidiously activated by the kidney allograft but that this had not been detectable by elispot monitoring of ifn and il-2 . (a) in recipients treated with the conditioning regimen without anti - cd8 mab, rapid homeostatic recovery of cd8 tmem was observed after day 5 . (b) by adding humanized anti - cd8 mab to the conditioning regimen, cd8 tmems were effectively suppressed until day 30 . (c) all recipients who received the delayed protocol at 4 mo without anti - cd8 mab (4 m) failed to develop chimerism . By adding humanized anti - cd8 mab to the conditioning regimen since the faster homeostatic recovery of cd8 tmem seemed to prevent induction of chimerism, we added anti - cd8 mab to the conditioning regimen . 3b) and most recipients (11/13) successfully developed mixed chimerism (fig . If death from infectious complications is censored, approximately 70% of recipients survived long - term following withdrawal of all immunosuppression (fig . These observations suggest that, although not detected by elispot, cd8 tmem had been activated during the four months following ktx despite the administration of immunosuppression potent enough to prevent rejection of the kidney . More recently, we have evaluated replacing anti - cd8 mab in the conditioning regimen with lfa-3/igg1 (lfa3ig) anticipating that the agent will be more readily available for clinical use . Lfa3ig modulates the function of cd2 (+) and depletes efficiently primate cd95cd28 effector tmem in vivo . This molecule mediates cognate interactions between cells expressing human cd2 and cd16 to activate cells, increase extracellular signal - regulated kinase phosphorylation, upregulate cell surface expression of the activation marker cd25, and induce release of granzyme b. three recipients treated with the modified regimen with lfa3ig but with no anti - cd8mab successfully developed chimerism and achieved long - term survival (manuscript in preparation). Since our results suggested that tmem activation occurs after ktx, we speculated that a shorter interval between organ transplantation and dbmt might limit this response and increase the likelihood of inducing allograft tolerance . Therefore, we evaluated dbmt at one month after ktx in an attempt to identify the optimal timing of dbmt . As we anticipated, chimerism induction in recipients who received dbmt earlier after ktx was more successful . All seven recipients who received dbmt at one month developed excellent chimerism (data not shown). The fact that two of 13 recipients who received dbmt at four months failed to develop any detectable chimerism, suggested that tmem activation may indeed be lower earlier after ktx . However, to our surprise, no recipients of dbmt at one month achieved renal allograft tolerance (fig . The state of the inflammatory milieu during the peritransplant period has been shown to impact the molecular phenotype and function of alloreactive t cells . We therefore hypothesized that higher proinflammatory responses during the earlier post - transplant period adversely affected tolerance induction . Rt - pcr analyses of the peripheral blood mononuclear cells revealed that mrna levels of proinflammatory cytokines in the recipients who received dbmt at one month were significantly higher than those who received dbmt at four months . These results suggest that the presence of higher proinflammatory cytokines is detrimental to tolerance induction . Tolerance induction several months after organ transplantation (delayed tolerance) is feasible via the mixed chimerism approach with additional modifications to mitigate tmem responses that have been induced by the transplanted allograft . Timing of delayed dbmt also appeared to be critical for successful induction of allograft tolerance, which is affected by higher inflammatory responses during the early post - transplant period.
The dna in eukaryotic cells is wrapped around histones which can be post - translationally modified to regulate transcription . Large genomes, such as the mouse and human genomes, are replete with repetitive sequences including tandem repeats at centromeres and telomeres, and interspersed repeats (e.g. Lines, sines, alu repeats and ervs). In order to prevent aberrant transcription, these genomic repeats are packaged into constitutive heterochromatin, a condensed chromatin structure which restricts accessibility to transcriptional machinery . Aberrant transcription of these repeats can compromise genome integrity and maintaining the heterochromatic architecture at these sites is critical to genome stability (1). The passage of polymerases during dna replication or transcription poses a threat to chromatin and the timely restoration of nucleosomes is important for maintaining epigenetic memory (2). The replication of dna during s - phase is accompanied by the deposition of newly synthesised histones as chromatin is rapidly reassembled behind the replication fork (3). The canonical histones, h3.1/h3.2, are specifically expressed during this period and incorporated into chromatin in a replication - coupled manner . In contrast, the histone variant h3.3 is expressed throughout the cell cycle and deposition of this variant is replication - independent (46). In addition to the distinct expression patterns, h3.3 differs from the canonical h3.1/h3.2 counterparts at five amino acid residues . These substitutions in h3.3 mediate interactions with chaperone complexes which are unique to h3.3 (7,8) and facilitate the replication - independent deposition of this variant (4). Two major h3.3-specific chaperones have been identified; the hira complex which deposits h3.3 at genic regions (5,6) and the atrx / daxx complex which is responsible for h3.3 deposition at repetitive regions of the genome (911). The association of h3.3 with heterochromatin was first reported at the telomeres of mouse es cells (12). This was closely followed by the identification of a chaperone complex comprised of atrx and daxx (9,10). Atrx is a chromatin remodeller which is thought to localise to telomeres via interactions with g - quadruplex secondary structures (13,14) while daxx is an h3.3-specific chaperone (8). Both components of the atrx / daxx complex were found to be essential for the deposition of h3.3 at telomeres (9,10) and pericentric heterochromatin (pch; 11) though the functional significance of this pathway was unclear . More recent studies have now demonstrated that atrx / daxx mediated deposition of h3.3 is not limited to telomeric and pericentric repeats but occurs at heterochromatin distributed throughout the genome of mouse es cells (1518), including endogenous retroviral repeats (ervs; 1618), imprinted differentially methylated regions (dmrs) and selected intragenic methylated cpg islands (cgis; 15). Enrichment at these sites coincides with the h3k9me3 heterochromatin modification and disruption of atrx, daxx or h3.3 led to a loss of h3k9me3 at these regions (15,16,1820). This review will consolidate the major findings of these studies and outline the evidence to support a model where atrx / daxx deposits h3.3 to maintain h3k9me3 heterochromatin in the genome . The interaction between the atrx / daxx complex and histone h3.3 these highly repetitive regions of the genome are archetypal constitutive heterochromatin, and well documented as being enriched for h3k9me3, h4k20me3 and dna methylation . More recent chip - seq studies have demonstrated that atrx binding sites across the genome are generally associated with heterochromatic modifications (h3k9me3, h4k20me3, dna methylation; 15). Further supporting the idea that atrx localises to heterochromatin, a number of studies detected atrx (1518) and daxx (16,18) enrichment at erv repeats, particularly at iap repeats (intracisternal a particle; 16,18). Ervs are transposable elements which are known to be modified as silent heterochromatin, as aberrant expression results in genome instability (1). Consistent with this, atrx / daxx binding at ervs coincided with enrichment of h3k9me3 and the co - repressor complex kap1 (aka trim28) and setdb1 (aka eset; 16,17), a lysine methyltransferase which catalyses h3k9me3 . It is likely that the heterochromatin modifications at these regions direct atrx binding as the add domain of atrx has been demonstrated to specifically recognise h3k9me3 (2123). This interaction has been confirmed at the cellular level where atrx localisation to pch was demonstrated to be dependent on h3k9me3 catalysed by suv39h (23). Furthermore the monoallelic localisation of atrx to the methylated, heterochromatic allele of imprinted dmrs (15) demonstrates that chromatin modifications play an important role in directing atrx localisation . The atrx / daxx complex is known to be important for deposition of h3.3 at telomeres and pch . Chip - seq of endogenous h3.3 in wt, atrx ko (15,16) and daxx ko (16) cells demonstrated that this was also true for the methylated allele of imprinted genes (15) as well as certain tandem repeats and intragenic cgis (15). H3.3 was found to be generally enriched at atrx binding sites in wt cells and this enrichment was lost in atrx ko (15,16) and daxx ko cells (16). H3.3 was also found to be enriched at iap / erv retrotransposons (1517) although there is some disagreement about the requirement of atrx / daxx for h3.3 deposition at these sites . Two studies reported that atrx / daxx ko results in loss of h3.3 at iap / ervs (15,16) while one study reported increased h3.3 at iap / ervs in atrx ko cells (17). The primary difference between these studies is that chip was performed either against endogenous h3.3 (15,16) or an yfp - tagged h3.3 (9). We have eliminated bioinformatics analysis as a possible variable by using a single method to re - align all three data sets . (17), when we mapped the yfp - tagged h3.3 data set (9) to iapez repeats (figure 1), we detected a gain of yfp - h3.3 at this erv in atrx ko cells . However, the two h3.3 data sets generated using an antibody against endogenous h3.3 (15,16) did not recapitulate this result and h3.3 enrichment at iapez ervs was consistently lost in atrx ko cells (figure 1). H3.3 chip - seq reads from all three data sets were also mapped to the telomeres as a control . Atrx ko led to decreased h3.3 incorporation at telomeres in all three data sets although telomere enrichment was clearer for endogenous h3.3, compared to the yfp - tagged h3.3, in normal cells (figure 1). The relative enrichment profile of h3.3 at ervs compared to telomeres in the endogenous h3.3 chip data sets are in agreement with immunofluorescence analyses (10,12). In addition, upregulated iap expression was detected in both atrx ko (17) and h3.3 ko cells (16; see below), supporting a role for atrx / daxx - dependent deposition of h3.3 at ervs, in agreement with the endogenous h3.3 chip - seq data . Raw read files of yfp - h3.3 chip (9), endogenous h3.3 chip (15,16) and matched input sequencing from wt and atrx ko cells were downloaded from geo (35,36) and mapped to repeats with repeat enrichment estimator (37). Samples were normalised for total read counts by dividing mapped reads against total mappable reads . Results show normalised reads counts of h3.3 chip and matched input samples for each data set at (a) iapez repeats and (b) telomere repeats . Yfp - h3.3 was increased at iapez repeats in atrx ko relative to wt cells . All three data sets showed decreased h3.3/yfp - h3.3 at telomeres in atrx ko relative to wt cells . It is possible that the discrepancy between the endogenous h3.3 and yfp - h3.3 has arisen due to the introduction of the yfp tag . For example, h3.3 turnover has been demonstrated to be important for regulation (24) and the yfp tag may interfere with this process . However, it is clear that further experiments are required to reconcile the discrepancies between these data sets . Future analysis using a different epitope tag, a different antibody or h3.3 k9 mutant cells would be useful for clarifying the differences between these studies . Nonetheless, when taken together, these results suggest that atrx / daxx localises to selected heterochromatic regions in the genome and deposits h3.3 . This pathway has now been extended from telomeres and pch to also include methylated imprinted dmrs, ervs / iaps and selected short tandem repeats . In addition to the loss of h3.3, disruption of the atrx / daxx complex in mouse es cells also led to the loss of h3k9me3 at some genomic regions . Atrx ko led to a reduction in h3k9me3 at methylated imprinted dmrs, intragenic cgis (15) and iap repeats (16) as detected by chip - qpcr and chip - seq . Similarly, a reduction in h3k9me3 was also detected at iaps (16) and telomeres (18) in daxx ko cells . In addition, direct knockout of h3.3 also led to decreased h3k9me3 at iaps (16) and telomeres (19,20). Chip - rechip assays of h3.3 and h3k9me3 on a genome - wide basis (16) and specifically at telomeres (19) demonstrated that h3.3 at these heterochromatic sites were directly modified with k9me3 (h3.3 k9me3). Overall, these data support a model where atrx / daxx mediates deposition of h3.3 which can be modified with k9me3 to facilitate the maintenance of heterochromatin . The h3k9me3 modification is catalysed by suv39h1/2 at telomeres and pch (25,26) while setdb1 acts in a complex with kap1 to catalyse h3k9me3 at ervs (27,28) and methylated imprinted dmrs (28,29). The kap1/setdb1 complex was found to co - localise with atrx at ervs (17) and generally with atrx / daxx / h3.3/h3k9me3 across the genome (16). Consistent with previous studies, depletion of setdb1 led to reduced h3k9me3 at iaps / ervs (16,17) and also at telomeres (19), albeit to a lesser degree . A direct interaction between daxx and kap1 has been identified by co - immunoprecipitation (16) and suggests that daxx contributes to kap1 binding at some genomic sites . Although krab - znfs, such as zfp809 at ervs (30) and zfp57 at imprinted genes (29,31), are known to be the main determinant for kap1 localisation, it is possible that daxx may help to further stabilise these interactions . Overall, these studies suggest that the kap1/setdb1 complex is able to catalyse h3.3k9me3 at these sites . In addition, daxx (18) and h3.3 (19) were also found to interact with suv39h and depletion of suv39h led to a loss in h3.3 k9me3 at telomeres (19). Combined, these results demonstrate that both setdb1 and suv39h are able to catalyse the trimethylation of lysine 9 on h3.3 . Previous studies have shown that atrx ko leads to increased terra transcription at telomeres (9) and this was verified by sirna depletion of atrx and daxx (18). Knockout of histone h3.3 similarly led to increased terra transcription (19) confirming that the atrx / daxx deposition of h3.3 is important for suppressing telomere transcription . This has now been extended to other genomic loci; atrx ko cells failed to silence a mini - iap transgene (17) and resulted in aberrant allelic expression of imprinted genes (15) while h3.3 ko led to increased erv transcription in mouse es cells (16). It should be noted that multiple mechanisms may mediate transcriptional silencing at these genomic regions and the atrx / daxx / h3.3 complex is one of many heterochromatin factors which maintain silencing . The degree of transcriptional deregulation varies depending on which heterochromatin pathway is disrupted, however, aberrant transcription is a reliable indicator of alterations in the underlying chromatin structure . The importance of atrx / h3.3 in heterochromatin maintenance is further demonstrated by the decrease in other silent chromatin modifications including h4k20me3 at telomeres (19) and dna methylation at dmrs of imprinted genes (15). Taken together, these studies demonstrate that atrx / daxx deposition of h3.3 k9me3 is important for maintaining silent heterochromatin at a number of genomic sites . The interaction between the atrx / daxx complex and histone h3.3 was first described at telomeres and pch (911). These highly repetitive regions of the genome are archetypal constitutive heterochromatin, and well documented as being enriched for h3k9me3, h4k20me3 and dna methylation . More recent chip - seq studies have demonstrated that atrx binding sites across the genome are generally associated with heterochromatic modifications (h3k9me3, h4k20me3, dna methylation; 15). Further supporting the idea that atrx localises to heterochromatin, a number of studies detected atrx (1518) and daxx (16,18) enrichment at erv repeats, particularly at iap repeats (intracisternal a particle; 16,18). Ervs are transposable elements which are known to be modified as silent heterochromatin, as aberrant expression results in genome instability (1). Consistent with this, atrx / daxx binding at ervs coincided with enrichment of h3k9me3 and the co - repressor complex kap1 (aka trim28) and setdb1 (aka eset; 16,17), a lysine methyltransferase which catalyses h3k9me3 . It is likely that the heterochromatin modifications at these regions direct atrx binding as the add domain of atrx has been demonstrated to specifically recognise h3k9me3 (2123). This interaction has been confirmed at the cellular level where atrx localisation to pch was demonstrated to be dependent on h3k9me3 catalysed by suv39h (23). Furthermore the monoallelic localisation of atrx to the methylated, heterochromatic allele of imprinted dmrs (15) demonstrates that chromatin modifications play an important role in directing atrx localisation . The atrx / daxx complex is known to be important for deposition of h3.3 at telomeres and pch . Chip - seq of endogenous h3.3 in wt, atrx ko (15,16) and daxx ko (16) cells demonstrated that this was also true for the methylated allele of imprinted genes (15) as well as certain tandem repeats and intragenic cgis (15). H3.3 was found to be generally enriched at atrx binding sites in wt cells and this enrichment was lost in atrx ko (15,16) and daxx ko cells (16). H3.3 was also found to be enriched at iap / erv retrotransposons (1517) although there is some disagreement about the requirement of atrx / daxx for h3.3 deposition at these sites . Two studies reported that atrx / daxx ko results in loss of h3.3 at iap / ervs (15,16) while one study reported increased h3.3 at iap / ervs in atrx ko cells (17). The primary difference between these studies is that chip was performed either against endogenous h3.3 (15,16) or an yfp - tagged h3.3 (9). We have eliminated bioinformatics analysis as a possible variable by using a single method to re - align all three data sets . (17), when we mapped the yfp - tagged h3.3 data set (9) to iapez repeats (figure 1), we detected a gain of yfp - h3.3 at this erv in atrx ko cells . However, the two h3.3 data sets generated using an antibody against endogenous h3.3 (15,16) did not recapitulate this result and h3.3 enrichment at iapez ervs was consistently lost in atrx ko cells (figure 1). H3.3 chip - seq reads from all three data sets were also mapped to the telomeres as a control . Atrx ko led to decreased h3.3 incorporation at telomeres in all three data sets although telomere enrichment was clearer for endogenous h3.3, compared to the yfp - tagged h3.3, in normal cells (figure 1). The relative enrichment profile of h3.3 at ervs compared to telomeres in the endogenous h3.3 chip data sets are in agreement with immunofluorescence analyses (10,12). In addition, upregulated iap expression was detected in both atrx ko (17) and h3.3 ko cells (16; see below), supporting a role for atrx / daxx - dependent deposition of h3.3 at ervs, in agreement with the endogenous h3.3 chip - seq data . Raw read files of yfp - h3.3 chip (9), endogenous h3.3 chip (15,16) and matched input sequencing from wt and atrx ko cells were downloaded from geo (35,36) and mapped to repeats with repeat enrichment estimator (37). Samples were normalised for total read counts by dividing mapped reads against total mappable reads . Results show normalised reads counts of h3.3 chip and matched input samples for each data set at (a) iapez repeats and (b) telomere repeats . Yfp - h3.3 was increased at iapez repeats in atrx ko relative to wt cells . All three data sets showed decreased h3.3/yfp - h3.3 at telomeres in atrx ko relative to wt cells . It is possible that the discrepancy between the endogenous h3.3 and yfp - h3.3 has arisen due to the introduction of the yfp tag . For example, h3.3 turnover has been demonstrated to be important for regulation (24) and the yfp tag may interfere with this process . However, it is clear that further experiments are required to reconcile the discrepancies between these data sets . Future analysis using a different epitope tag, a different antibody or h3.3 k9 mutant cells would be useful for clarifying the differences between these studies . Nonetheless, when taken together, these results suggest that atrx / daxx localises to selected heterochromatic regions in the genome and deposits h3.3 . This pathway has now been extended from telomeres and pch to also include methylated imprinted dmrs, ervs / iaps and selected short tandem repeats . In addition to the loss of h3.3, disruption of the atrx / daxx complex in mouse es cells also led to the loss of h3k9me3 at some genomic regions . Atrx ko led to a reduction in h3k9me3 at methylated imprinted dmrs, intragenic cgis (15) and iap repeats (16) as detected by chip - qpcr and chip - seq . Similarly, a reduction in h3k9me3 was also detected at iaps (16) and telomeres (18) in daxx ko cells . In addition, direct knockout of h3.3 also led to decreased h3k9me3 at iaps (16) and telomeres (19,20). Chip - rechip assays of h3.3 and h3k9me3 on a genome - wide basis (16) and specifically at telomeres (19) demonstrated that h3.3 at these heterochromatic sites were directly modified with k9me3 (h3.3 k9me3). Overall, these data support a model where atrx / daxx mediates deposition of h3.3 which can be modified with k9me3 to facilitate the maintenance of heterochromatin . The h3k9me3 modification is catalysed by suv39h1/2 at telomeres and pch (25,26) while setdb1 acts in a complex with kap1 to catalyse h3k9me3 at ervs (27,28) and methylated imprinted dmrs (28,29). The kap1/setdb1 complex was found to co - localise with atrx at ervs (17) and generally with atrx / daxx / h3.3/h3k9me3 across the genome (16). Consistent with previous studies, depletion of setdb1 led to reduced h3k9me3 at iaps / ervs (16,17) and also at telomeres (19), albeit to a lesser degree . A direct interaction between daxx and kap1 has been identified by co - immunoprecipitation (16) and suggests that daxx contributes to kap1 binding at some genomic sites . Although krab - znfs, such as zfp809 at ervs (30) and zfp57 at imprinted genes (29,31), are known to be the main determinant for kap1 localisation, it is possible that daxx may help to further stabilise these interactions . Overall, these studies suggest that the kap1/setdb1 complex is able to catalyse h3.3k9me3 at these sites . In addition, daxx (18) and h3.3 (19) were also found to interact with suv39h and depletion of suv39h led to a loss in h3.3 k9me3 at telomeres (19). Combined, these results demonstrate that both setdb1 and suv39h are able to catalyse the trimethylation of lysine 9 on h3.3 . Previous studies have shown that atrx ko leads to increased terra transcription at telomeres (9) and this was verified by sirna depletion of atrx and daxx (18). Knockout of histone h3.3 similarly led to increased terra transcription (19) confirming that the atrx / daxx deposition of h3.3 is important for suppressing telomere transcription . This has now been extended to other genomic loci; atrx ko cells failed to silence a mini - iap transgene (17) and resulted in aberrant allelic expression of imprinted genes (15) while h3.3 ko led to increased erv transcription in mouse es cells (16). It should be noted that multiple mechanisms may mediate transcriptional silencing at these genomic regions and the atrx / daxx / h3.3 complex is one of many heterochromatin factors which maintain silencing . The degree of transcriptional deregulation varies depending on which heterochromatin pathway is disrupted, however, aberrant transcription is a reliable indicator of alterations in the underlying chromatin structure . The importance of atrx / h3.3 in heterochromatin maintenance is further demonstrated by the decrease in other silent chromatin modifications including h4k20me3 at telomeres (19) and dna methylation at dmrs of imprinted genes (15). Taken together, these studies demonstrate that atrx / daxx deposition of h3.3 k9me3 is important for maintaining silent heterochromatin at a number of genomic sites . A substantial proportion of mammalian genomes is comprised of repetitive dna which includes telomeres, centromeres and ervs . Aberrant transcription of these repeats is deleterious to the organism and maintaining heterochromatic silencing at these repeats is critical for protecting genomic integrity . Chromatin modifications, such as dna methylation, h3k9me3 and h4k20me3, are known to be important for maintaining silencing . In addition, a recent set of studies have now also identified the histone variant h3.3 as an important component of heterochromatin in mouse es cells . The complex which deposits h3.3 at heterochromatin is comprised of daxx, an h3.3 specific chaperone, and a chromatin remodeller, atrx . This chaperone complex both targets h3.3 to specific heterochromatic sites and interacts with histone modifiers to maintain the k9me3 heterochromatin mark . The add domain of atrx preferentially interacts with h3k9me3 which likely drives the localisation of atrx / daxx / h3.3 to heterochromatin modified regions including telomeres, ervs and the methylated imprinted dmrs . Daxx is then able to interact with either the kap1/setdb1 co - repressor complex or suv39h, both of which catalyse h3k9me3 . Atrx / daxx is therefore able to target heterochromatin for deposition of h3.3 and recruit methyltransferases to mediate direct modification of k9me3 on h3.3 . This represents a self - reinforcing pathway which is able to protect heterochromatin integrity throughout the cell cycle (figure 2). (a) heterochromatic regions such as telomeres, iap ltrs and methylated imprinted dmrs are distributed throughout the genome and enriched for h3k9me3 . (b) atrx recognises h3k9me3 and acts with daxx to deposit h3.3 to replace histones which are lost . Atrx / daxx / h3.3 are able to act continuously through the cell cycle to ensure constant maintenance of h3k9me3 heterochromatin . These studies have identified the atrx / daxx / h3.3 complex as an important contributor to heterochromatin silencing in mouse es cells however, a number of outstanding questions remain . One possible clue could lie in the replication - independent expression and incorporation of the h3.3 variant . This would suggest that atrx / daxx / h3.3 are particularly important outside of s - phase when the canonical histones are unavailable . Future analyses with highly quantitative time - lapse imaging experiments would provide useful information of the dynamics of h3.3, including turnover and deposition of newly synthesised h3.3, and recruitment of other chromatin repressors at these sites . Furthermore, while it is evident that depletion of atrx / daxx / h3.3 leads to loss of heterochromatin, the overall phenotypic consequences remain unclear . This is particularly pertinent to alt cancers where atrx mutations have been strongly linked to increased recombination at telomeres (32,33). Several studies have demonstrated that disruption of heterochromatin facilitates aberrant telomere recombination (34). Detailed analyses of how heterochromatin structure, including h4k20me3 and dna methylation, is disrupted in atrx / daxx / h3.3 deficient cells would provide insights into how this pathway might promote genome instability in cancer . Australia research council (arc) future fellowship award from the arc, australia [to l.h.w . ].
Lower urinary tract symptoms (luts) are common, especially in older men, and are most often caused by benign prostatic hyperplasia (bph) which results in benign prostatic enlargement (bpe) and bladder outlet obstruction (boo). Patients with severe luts unresponsive to pharmacological management, and those with bph complications are candidates for surgical therapy . Transurethral resection of the prostate (turp) has been considered the surgical gold standard in terms of efficacy and re treatment rate . However, recent scientific and technological advances have challenged the traditional surgical approach to bph . The morbidity and late complications of turp were the major impetus driving the development of new techniques such as bipolar turp and laser enucleation or vaporization . Open prostatectomy, recommended by the european association of urology for patients with prostate volumes> 80 ml, is associated with a prolonged hospital stay and serious complications such as severe blood loss and infection . Additionally, patients may require subsequent surgical revision . Despite the wide acceptance of open prostatectomy; blunt dissection from the capsule, particularly in the apical area, may be technically challenging for the less experienced surgeons and is frequently associated with complications . Laparoscopic, and more recently robotic techniques, have provided a minimally invasive alternative to open prostatectomy, with equal efficacy, faster recovery, and shorter hospital stay . There is less pain and improved cosmetic results, although operative time (ot) and estimated blood loss are dependent on surgical expertise . These endoscopic procedures allow improved visualization and reduced morbidity and are well established in the management of prostate carcinoma, providing a rationale for their use in the treatment of bph [35]. Articles for this review were identified through pubmed searches from january 2004 through december 2013 . Various algorithms were used including: benign prostatic hyperplasia, adenomectomy, simple prostatectomy, laparoscopic adenomectomy, and robotic assisted simple prostatectomy . The final reference list was approved by authors rs and tb based on originality and relevance to the scope of this review article . Prior to endoscopic procedures, patients underwent a detailed history assessment, a thorough physical examination, and an appropriate laboratory assessment . The severity of luts was evaluated by most authors using the international prostate symptom score (ipss) questionnaire and the maximal flow rate (qmax). For precise postoperative functional evaluation, some authors used the sexual health inventory for men (shim), typically 3, 6, and 12 months postoperatively [6, 7]. Prostate biopsy was performed as a standard procedure in some studies, but in other studies it was only performed in selected cases [8, 9]. Peri and postoperative assessment included ot, blood loss, transfusion requirements, catheterization time, and duration of hospital stay . Complications were evaluated based on their severity, usually according to clavien et al . . The longest follow up period was several years in patients after la, and up to 10 months in patients after robotic assisted adenomectomy [11, 12]. In 2002, mariano et al . Described the first la in a 71year old man with an ultrasonography estimated 173ml prostate and a 26.6 ng / ml prostate specific antigen (psa) level . The procedure was performed with five intraperitoneal trocars, with anterior prostate capsulotomy and placement of hemostatic sutures at the 5 and 7 o'clock positions after adenoma removal . Van velthoven et al . Described their technique of la, which included hemostatic control of the lateral venous vesicoprostatic pedicles, transverse anterior incision of the prostatic capsule, adenoma enucleation using the harmonic scalpel, and reconstruction of the posterior bladder neck and prostatic capsule . The authors reported very good functional outcomes; mean qmax was increased by 4.3 ml / s postoperatively . In 2005, sotelo et al . Stepwise, the technique included transverse cystotomy just proximal to the prostatovesical junction, subcapsular plane development, prostatic adenomectomy, prostatic fossa trigonization, and prostatic capsule suture repair . The mean ot was 156 min and blood loss was 516 ml (1002500 ml). A 7 ml / s increase in qmax was noted postoperatively . Since these initial reports, laparoscopic simple prostatectomy has been more widely adopted, offering surgeons an extra or intraperitoneal approach, following a transcapsular or transvesical route . In selected patients, finger assistance has been used for rapid enucleation of large adenomas [2, 16, 17, 8]. One technique involves an incision under the umbilicus with carbon dioxide insufflation into the extraperitoneal space via a veress needle to 12 mmhg . A second technique involves making a 2cm vertical midline incision above the pubic arch followed by blunt dissection of the preperitoneal and retzius space with an index finger and a 700ml self dilating balloon . In a third technique the retroperitoneal space is then bluntly dissected with an 8001200ml infusion of sterile saline solution into the balloon . The operation can also begin with the primary insertion of a 10mm infraumbilical port and laparoscope . Then, dissection of the preperitoneal space is completed with the aid of the laparoscope and insufflation . Usually, 4 trocars, 5 mm or 12 mm, are inserted in a fan shape, to introduce a needle for suturing, as in extraperitoneal radical prostatectomy . A single 10mm port is inserted infraumbilically as the camera port (figure 1). The dorsal vein complex is assessed and then carefully coagulated using bipolar forceps cranially, keeping an appropriate distance from the puboprostatic ligaments . In a fourth technique, two hemostatic sutures are applied to these vessels . Using the bladder catheter or a special metal guide inserted into the urethra as a reference point, the interface between the bladder neck and prostate base is identified . If necessary, two cross stitch hemostatic sutures are placed on the lateral surface of the prostate at the level of the bilateral vesicoprostatic vessels . The prostatic capsule is opened 34 cm transversally and 1 cm distal to the bladder neck . The capsular incision is carried to a depth that first reveals the off white tissue of the adenoma . Monopolar scissors and the suction irrigation cannula are used to develop the plane between the prostatic adenoma and the capsule (figure 2). The anterior plane is then developed, followed by lateral and posterior dissection . In some cases, where improved exposure is required, the incision is extended in the shape of an inverted t on the prostatic capsule . To enucleate the lateral lobes, a harmonic scalpel is used to develop the surgical avascular capsular plane in the distal projection towards the apex, the lateral projection to the posterior plane, and the cranial projection to the bladder neck, in a fashion similar to that used in open surgery . In a modified version of the procedure, two lateral stay sutures are used between the cut prostatic capsule and the cooper ligament, providing a clear visualization of the fossa and the cleavage plan . The adenoma is excised, and then one or two corresponding specimens are placed outside the capsule in the lateral prostatic fossa, e.g., in close proximity to the obturator fossa, to be removed later . Hemostasis is obtained with stitches for transcapsular arteries and bipolar or monopolar electrocoagulation for minor vessels . The prostatic fossa is inspected for any remaining adenoma nodules . To facilitate the re epithelialization of the prostatic fossa and to achieve more effective control of hemostasis, the prostatic fossa is trigonized using 24 stitches between the sacral lip of the bladder neck and posterior surgical capsule . An interrupted or running suture is used for prostatic capsule reconstruction, followed by the introduction of a bag via the lateral 10mm port for collection of the fragmented adenoma . When the adenoma is too large to be removed as a whole, morcellation is performed before extraction . A drain (redon type) is inserted via the port, the infraumbilical incision is enlarged to enable intact retrieval, and the bag with the specimen is removed . The prostatic capsule can also be opened through a longitudinal opening in the anterior aspect of the bladder and extended to the anterior aspect of the prostatic capsule using bipolar diathermy, scissors, or a harmonic scalpel . Stay sutures are placed between the edges of the open bladder on each side of the cooper ligament . Most published series describe the extraperitoneal technique for la, but a transperitoneal approach has also been described . With the patient in a steep trendelenburg position, the pneumoperitoneum is created, and five intraperitoneal trocars are placed in a w fashion . After the peritoneum is incised and the retzius space is dissected, the bladder and prostate are taken down . A midline incision covering the anterior aspect of the prostatic capsule and bladder neck exposes the adenoma . Retrigonization of the mucosa and hemostatic sutures of the bladder neck are performed with intracorporeal sutures . Some authors described another modification with an extraperitoneal transvesical approach [17, 23]. In this case, a transverse cystotomy incision is made proximal to the junction of the bladder and prostate . In this manner, next, a circular incision is made on the vesicle mucosa overlying the prostate lobes, and made deeper to reach the prostatic adenoma . The procedure ends with closure of the transverse cystotomy to ensure water tightness . In some patients, when the layer for surgical exposure of a large adenoma is unclear, or if the surgeon is inexperienced, the adenoma can be dissected using the finger assisted technique [2, 7, 16, 18, 22]. After opening the prostatic capsule and developing the plane of cleavage between the prostatic adenoma and capsule, gas flow is stopped and the index finger is introduced through a 23 cm suprapubic incision into this developed plane . This can be performed easily and is assisted by digital rectal examination (dre) [18, 19, 23]. The specimen is removed via the incision, and insufflation is restarted after closure of the suprapubic incision [2, 22]. Increased prostate weight, and hence prostate volume, have been correlated with increased ot . Excellent visualization and bloodless resection of the adenoma limits complications and decreases ot, especially with larger lesions . The use of vascular control, bladder neck and capsular incisions, and ultrasonic scissors as a sharp and blunt dissection instrument allow easier enucleation of larger adenomas . There have been no reports of conversion to open surgery due to unexpected difficulties corresponding to prostate size . In a recently published series, after excluding the first 10 cases of la, univariate analysis of the next 78 patients with a large prostate (> 90 ml) showed a correlation of prostate volume and ot with complications . Even with a large prostate (> 90 ml), la provides excellent operative and perioperative results and patient satisfaction (table 1). Series (with> 15 cases) on laparoscopic simple prostatectomy n number of patients; t transperitoneal approach; e extraperitoneal approach; catheter catheterization time after the procedure; i pss international prostate symptom score; qmax the maximal flow rate; nr not received a major benefit of la includes improved control of bleeding, possibly because of improved visualization and vascular compression from insufflated gas . Morbidity and pain are reduced compared to those in the open, procedure as incisions are smaller and there is no need for retraction . La has better esthetic results, reduced need for analgesics, fewer wound infections, shorter hospital stay, and an earlier return to normal activities compared with the open procedure (table 1). Intraoperative complications, mostly bleeding, are rare (<2.5%) with no effect on clinical outcome and a decreased need for transfusion [4, 22]. Intraoperative blood loss does not usually correlate with the amount of enucleated tissue (table 1). Decreased blood loss is achieved via gas compression of the venous system, aiding hemostasis, and allowing more precise dissection and coagulation of the adenomatous cleavage plane [11, 13]. Furthermore, better visualization improves hemostasis . The early complication rate of 14% is acceptable, particularly since most are less than clavien grade ii . Long term complications rarely occur (2.5% at the 12month follow up, <5% at the 30month follow up), and mainly include new obstructive urinary symptoms including urinary tract infections such as pyelonephritis, prostatitis, or epididymitis that are usually treated with conservative methods, and short presphincteric urethral stenosis usually treated with endoscopic urethrotomy . Most researchers observed a significant increase in the qmax in comparison to preoperative values (mean increased qmax 14.4 ml / s) and a marked decrease in ipss (mean decrease 17.2) postoperatively . Ipss, ipss in the quality of life domain, and the expanded prostate cancer index composite (epic) questionnaire score remained stable at 3, 6, and> 12 months postoperatively . Erectile function, evaluated with iief5 or other tools, did not change significantly as a result of la . Persistent retrograde ejaculation was a consequence of the surgery, but had no significant impact on sexual function . Psa levels were significantly different preoperatively and postoperatively, and later psa levels remained stable during follow up . Most investigators use the extraperitoneal technique based on the millin technique employed in open procedures [7, 11, 14, 15, 1719]. With this approach, the risk of bladder tamponade by clots is avoided along with the additional anesthesiology risks associated with the steep trendelenburg position that is necessary in the transperitoneal approach . The transperitoneal technique was used by some investigators [11, 13, 17], but is associated with a risk of ileus, peritonitis, and bowel injuries along with urine leakage and possible urine peritonitis from the bladder suture . No studies have compared extra and transperitoneal laparoscopic adenomectomy in the way laparoscopic or robotic radical prostatectomy has been compared [2426]. However, in la, these two approaches give comparable results (table 1). La has a relatively short learning curve in comparison with radical laparoscopic prostatectomy, and has been estimated at 510 surgeries . If conversion is required during a procedure using the preperitoneal access, no new access is needed because the surgery can be continued within the same space . As the number of surgeries performed and laparoscopic skills increase, there is a clear reduction in ot [11, 15, 21]. Studies comparing la and open surgery have shown that both procedures offer the same benefits in terms of functional results, but that la performed by experienced surgeons provided greater perioperative benefits such as reduced bleeding and transfusions, shorter irrigation and catheterization times, shorter hospital stays, lower analgesic requirements, shorter recovery times, and improved cosmetic results [4, 20, 21, 27] (table 2). Comparative series between open and laparoscopy adenomectomy catheterization time after the procedure the introduction of the da vinci robotic surgical system [intuitive surgical, inc ., sunnyvale, ca, usa] for urological procedures, including radical prostatectomy, has been a major step towards a minimally invasive approach . In 2008, sotelo et al . Used a newly developed technique for rasp in 7 patients with a mean prostate volume of 77.66 ml . The functional outcomes were very good with reduction in the ipss by 14.5 points and an increase in the qmax by 37.75 ml / min the authors concluded that robotic simple prostatectomy is a feasible and reproducible procedure for symptomatic bph . In the same year, yuh reported rasp using a technique similar to conventional millin surgery . Many clinical studies and case series using rasp have been published, but all have a small sample size and non comparative designs . Port placement and surgical access in rasp are similar to those used in robotic assisted radical prostatectomy . After exposure of the retropubic space, the endopelvic fascia is opened bilaterally to expose the puboprostatic ligaments . The dorsal venous complex is ligated and access to the adenoma is achieved through the transvesical [2831] or prevesical approach [9, 12]. In the prevesical approach, the plane between the adenoma and the prostatic capsule is identified and dissected, and the prostatic urethra is carefully transected to prevent external sphincter damage . Finally, the adenoma is removed and retrigonization is achieved by suturing the posterior edge of the bladder neck mucosa to the posterior edge of the urethra . In another technique, a horizontal cystotomy of the prostatic capsule is made . After removing the adenoma, some authors suggest a modification of retrigonization by folding the posterior prostatic capsule, suturing the anterior prostatic capsule to the anterior bladder wall, and performing a modified van velthoven continuous vesicourethral anastomosis . Patients treated with rasp show significant increases in qmax and reductions in ipss after surgery . Ot is usually slightly longer or comparable to that in la, although in some reports, ot was> 3 h. blood loss has also been comparable to that reported in la, and in most cases no blood transfusions were necessary [30, 32]. Hospital stays were short, and most patients were discharged 12 days after surgery (range 13.2 days) [6, 9, 30, 32]. Reported follow up periods have been short and it is difficult to precisely define the long term outcome and complications . The laparoendoscopic single site (less) procedure is also used to remove prostatic adenomas [33, 34]. The initial procedures used classical laparoscopic instruments with modifications, including the use of a port device for pre or transvesical access to the prostate [3538]. To avoid the basic limitations of less, collision of the robot's arms and small operative space, single site instruments designed for the da vinci surgical system were used to perform single port transvesical enucleation of the prostate (step) [36, 37]. In recent years, several clinical studies and case reports have been published on less [35, 3742]. Preliminary functional outcomes are encouraging, but the procedure is associated with a high risk of complications, and its role has yet to be determined . Prior to endoscopic procedures, patients underwent a detailed history assessment, a thorough physical examination, and an appropriate laboratory assessment . The severity of luts was evaluated by most authors using the international prostate symptom score (ipss) questionnaire and the maximal flow rate (qmax). For precise postoperative functional evaluation, some authors used the sexual health inventory for men (shim), typically 3, 6, and 12 months postoperatively [6, 7]. Prostate biopsy was performed as a standard procedure in some studies, but in other studies it was only performed in selected cases [8, 9]. Peri and postoperative assessment included ot, blood loss, transfusion requirements, catheterization time, and duration of hospital stay . Complications were evaluated based on their severity, usually according to clavien et al . . The longest follow up period was several years in patients after la, and up to 10 months in patients after robotic assisted adenomectomy [11, 12]. In 2002, mariano et al . Described the first la in a 71year old man with an ultrasonography estimated 173ml prostate and a 26.6 ng / ml prostate specific antigen (psa) level . The procedure was performed with five intraperitoneal trocars, with anterior prostate capsulotomy and placement of hemostatic sutures at the 5 and 7 o'clock positions after adenoma removal . Van velthoven et al . Described their technique of la, which included hemostatic control of the lateral venous vesicoprostatic pedicles, transverse anterior incision of the prostatic capsule, adenoma enucleation using the harmonic scalpel, and reconstruction of the posterior bladder neck and prostatic capsule . The authors reported very good functional outcomes; mean qmax was increased by 4.3 ml / s postoperatively . In 2005, sotelo et al . Stepwise, the technique included transverse cystotomy just proximal to the prostatovesical junction, subcapsular plane development, prostatic adenomectomy, prostatic fossa trigonization, and prostatic capsule suture repair . The mean ot was 156 min and blood loss was 516 ml (1002500 ml). A 7 ml / s increase in qmax was noted postoperatively . Since these initial reports, laparoscopic simple prostatectomy has been more widely adopted, offering surgeons an extra or intraperitoneal approach, following a transcapsular or transvesical route . In selected patients, finger assistance has been used for rapid enucleation of large adenomas [2, 16, 17, 8]. One technique involves an incision under the umbilicus with carbon dioxide insufflation into the extraperitoneal space via a veress needle to 12 mmhg . A second technique involves making a 2cm vertical midline incision above the pubic arch followed by blunt dissection of the preperitoneal and retzius space with an index finger and a 700ml self dilating balloon . In a third technique the retroperitoneal space is then bluntly dissected with an 8001200ml infusion of sterile saline solution into the balloon . The operation can also begin with the primary insertion of a 10mm infraumbilical port and laparoscope . Then, dissection of the preperitoneal space is completed with the aid of the laparoscope and insufflation . Usually, 4 trocars, 5 mm or 12 mm, are inserted in a fan shape, to introduce a needle for suturing, as in extraperitoneal radical prostatectomy . A single 10mm port is inserted infraumbilically as the camera port (figure 1). The dorsal vein complex is assessed and then carefully coagulated using bipolar forceps cranially, keeping an appropriate distance from the puboprostatic ligaments . In a fourth technique, a special metal guide inserted into the urethra as a reference point, the interface between the bladder neck and prostate base is identified . If necessary, two cross stitch hemostatic sutures are placed on the lateral surface of the prostate at the level of the bilateral vesicoprostatic vessels . The prostatic capsule is opened 34 cm transversally and 1 cm distal to the bladder neck . The capsular incision is carried to a depth that first reveals the off white tissue of the adenoma . Monopolar scissors and the suction irrigation cannula are used to develop the plane between the prostatic adenoma and the capsule (figure 2). The anterior plane is then developed, followed by lateral and posterior dissection . In some cases, where improved exposure is required, the incision is extended in the shape of an inverted t on the prostatic capsule . To enucleate the lateral lobes, a harmonic scalpel is used to develop the surgical avascular capsular plane in the distal projection towards the apex, the lateral projection to the posterior plane, and the cranial projection to the bladder neck, in a fashion similar to that used in open surgery . In a modified version of the procedure, two lateral stay sutures are used between the cut prostatic capsule and the cooper ligament, providing a clear visualization of the fossa and the cleavage plan . Any bladder stones are removed during the intervention through a capsular incision . The adenoma is excised, and then one or two corresponding specimens are placed outside the capsule in the lateral prostatic fossa, e.g., in close proximity to the obturator fossa, to be removed later . Hemostasis is obtained with stitches for transcapsular arteries and bipolar or monopolar electrocoagulation for minor vessels . The prostatic fossa is inspected for any remaining adenoma nodules . To facilitate the re epithelialization of the prostatic fossa and to achieve more effective control of hemostasis, the prostatic fossa is trigonized using 24 stitches between the sacral lip of the bladder neck and posterior surgical capsule . An interrupted or running suture is used for prostatic capsule reconstruction, followed by the introduction of a bag via the lateral 10mm port for collection of the fragmented adenoma . When the adenoma is too large to be removed as a whole, morcellation is performed before extraction . A drain (redon type) is inserted via the port, the infraumbilical incision is enlarged to enable intact retrieval, and the bag with the specimen is removed . The prostatic capsule can also be opened through a longitudinal opening in the anterior aspect of the bladder and extended to the anterior aspect of the prostatic capsule using bipolar diathermy, scissors, or a harmonic scalpel . Stay sutures are placed between the edges of the open bladder on each side of the cooper ligament . Most published series describe the extraperitoneal technique for la, but a transperitoneal approach has also been described . With the patient in a steep trendelenburg position, the pneumoperitoneum is created, and five intraperitoneal trocars are placed in a w fashion . After the peritoneum is incised and the retzius space is dissected, the bladder and prostate are taken down . A midline incision covering the anterior aspect of the prostatic capsule and bladder neck exposes the adenoma . Retrigonization of the mucosa and hemostatic sutures of the bladder neck are performed with intracorporeal sutures . Some authors described another modification with an extraperitoneal transvesical approach [17, 23]. In this case, a transverse cystotomy incision is made proximal to the junction of the bladder and prostate . In this manner, next, a circular incision is made on the vesicle mucosa overlying the prostate lobes, and made deeper to reach the prostatic adenoma . The procedure ends with closure of the transverse cystotomy to ensure water tightness . In some patients, when the layer for surgical exposure of a large adenoma is unclear, or if the surgeon is inexperienced, the adenoma can be dissected using the finger assisted technique [2, 7, 16, 18, 22]. After opening the prostatic capsule and developing the plane of cleavage between the prostatic adenoma and capsule, gas flow is stopped and the index finger is introduced through a 23 cm suprapubic incision into this developed plane . This can be performed easily and is assisted by digital rectal examination (dre) [18, 19, 23]. The specimen is removed via the incision, and insufflation is restarted after closure of the suprapubic incision [2, 22]. Increased prostate weight, and hence prostate volume, have been correlated with increased ot . Excellent visualization and bloodless resection of the adenoma limits complications and decreases ot, especially with larger lesions . The use of vascular control, bladder neck and capsular incisions, and ultrasonic scissors as a sharp and blunt dissection instrument allow easier enucleation of larger adenomas . There have been no reports of conversion to open surgery due to unexpected difficulties corresponding to prostate size . In a recently published series, after excluding the first 10 cases of la, univariate analysis of the next 78 patients with a large prostate (> 90 ml) showed a correlation of prostate volume and ot with complications . Even with a large prostate (> 90 ml), la provides excellent operative and perioperative results and patient satisfaction (table 1). Series (with> 15 cases) on laparoscopic simple prostatectomy n number of patients; t transperitoneal approach; e extraperitoneal approach; catheter catheterization time after the procedure; i pss international prostate symptom score; qmax the maximal flow rate; nr not received a major benefit of la includes improved control of bleeding, possibly because of improved visualization and vascular compression from insufflated gas . Morbidity and pain are reduced compared to those in the open, procedure as incisions are smaller and there is no need for retraction . La has better esthetic results, reduced need for analgesics, fewer wound infections, shorter hospital stay, and an earlier return to normal activities compared with the open procedure (table 1). Intraoperative complications, mostly bleeding, are rare (<2.5%) with no effect on clinical outcome and a decreased need for transfusion [4, 22]. Intraoperative blood loss does not usually correlate with the amount of enucleated tissue (table 1). Decreased blood loss is achieved via gas compression of the venous system, aiding hemostasis, and allowing more precise dissection and coagulation of the adenomatous cleavage plane [11, 13]. Furthermore, better visualization improves hemostasis . The early complication rate of 14% is acceptable, particularly since most are less than clavien grade ii . Long term complications rarely occur (2.5% at the 12month follow up, <5% at the 30month follow up), and mainly include new obstructive urinary symptoms including urinary tract infections such as pyelonephritis, prostatitis, or epididymitis that are usually treated with conservative methods, and short presphincteric urethral stenosis usually treated with endoscopic urethrotomy . Most researchers observed a significant increase in the qmax in comparison to preoperative values (mean increased qmax 14.4 ml / s) and a marked decrease in ipss (mean decrease 17.2) postoperatively . Ipss, ipss in the quality of life domain, and the expanded prostate cancer index composite (epic) questionnaire score remained stable at 3, 6, and> 12 months postoperatively . Erectile function, evaluated with iief5 or other tools, did not change significantly as a result of la . Persistent retrograde ejaculation was a consequence of the surgery, but had no significant impact on sexual function . Psa levels were significantly different preoperatively and postoperatively, and later psa levels remained stable during follow up . Most investigators use the extraperitoneal technique based on the millin technique employed in open procedures [7, 11, 14, 15, 1719]. With this approach, the risk of bladder tamponade by clots is avoided along with the additional anesthesiology risks associated with the steep trendelenburg position that is necessary in the transperitoneal approach . The transperitoneal technique was used by some investigators [11, 13, 17], but is associated with a risk of ileus, peritonitis, and bowel injuries along with urine leakage and possible urine peritonitis from the bladder suture . No studies have compared extra and transperitoneal laparoscopic adenomectomy in the way laparoscopic or robotic radical prostatectomy has been compared [2426]. However, in la, these two approaches give comparable results (table 1). La has a relatively short learning curve in comparison with radical laparoscopic prostatectomy, and has been estimated at 510 surgeries . If conversion is required during a procedure using the preperitoneal access, no new access is needed because the surgery can be continued within the same space . As the number of surgeries performed and laparoscopic skills increase, there is a clear reduction in ot [11, 15, 21]. Studies comparing la and open surgery have shown that both procedures offer the same benefits in terms of functional results, but that la performed by experienced surgeons provided greater perioperative benefits such as reduced bleeding and transfusions, shorter irrigation and catheterization times, shorter hospital stays, lower analgesic requirements, shorter recovery times, and improved cosmetic results [4, 20, 21, 27] (table 2). Comparative series between open and laparoscopy adenomectomy from published series the introduction of the da vinci robotic surgical system [intuitive surgical, inc ., sunnyvale, ca, usa] for urological procedures, including radical prostatectomy, has been a major step towards a minimally invasive approach . In 2008, sotelo et al . Used a newly developed technique for rasp in 7 patients with a mean prostate volume of 77.66 ml . The functional outcomes were very good with reduction in the ipss by 14.5 points and an increase in the qmax by 37.75 ml / min the authors concluded that robotic simple prostatectomy is a feasible and reproducible procedure for symptomatic bph . In the same year, yuh reported rasp using a technique similar to conventional millin surgery . Many clinical studies and case series using rasp have been published, but all have a small sample size and non comparative designs . Port placement and surgical access in rasp are similar to those used in robotic assisted radical prostatectomy . After exposure of the retropubic space, the endopelvic fascia is opened bilaterally to expose the puboprostatic ligaments . The dorsal venous complex is ligated and access to the adenoma is achieved through the transvesical [2831] or prevesical approach [9, 12]. In the prevesical approach, the plane between the adenoma and the prostatic capsule is identified and dissected, and the prostatic urethra is carefully transected to prevent external sphincter damage . Finally, the adenoma is removed and retrigonization is achieved by suturing the posterior edge of the bladder neck mucosa to the posterior edge of the urethra . In another technique, a horizontal cystotomy of the prostatic capsule is made . After removing the adenoma, some authors suggest a modification of retrigonization by folding the posterior prostatic capsule, suturing the anterior prostatic capsule to the anterior bladder wall, and performing a modified van velthoven continuous vesicourethral anastomosis . Patients treated with rasp show significant increases in qmax and reductions in ipss after surgery . Ot is usually slightly longer or comparable to that in la, although in some reports, ot was> 3 h. blood loss has also been comparable to that reported in la, and in most cases no blood transfusions were necessary [30, 32]. Hospital stays were short, and most patients were discharged 12 days after surgery (range 13.2 days) [6, 9, 30, 32]. Reported follow up periods have been short and it is difficult to precisely define the long term outcome and complications . The laparoendoscopic single site (less) procedure is also used to remove prostatic adenomas [33, 34]. The initial procedures used classical laparoscopic instruments with modifications, including the use of a port device for pre or transvesical access to the prostate [3538]. To avoid the basic limitations of less, collision of the robot's arms and small operative space, single site instruments designed for the da vinci surgical system were used to perform single port transvesical enucleation of the prostate (step) [36, 37]. In recent years, several clinical studies and case reports have been published on less [35, 3742]. Preliminary functional outcomes are encouraging, but the procedure is associated with a high risk of complications, and its role has yet to be determined . Despite its recent development, la has become a well established option for the surgical treatment of bph patients . La is standardized and reproducible and offers good functional results and a minimal complication rate along with other benefits of minimally invasive surgery . Rasp offers the benefits of robotic surgery, and a shorter hospital stay, faster recovery, and quicker return to work than la . Additionally, robotic surgery offers a number of benefits including stereoscopic vision and 6 degrees of freedom . Longer terms studies of the functional outcomes, complications, and cost analysis of rasp will further define this procedure's place in the urological surgeon's armamentarium.
Cellular allograft rejection is one of the commonest causes of graft dysfunction in the early as well as late posttransplant period . Chemokines induce migration of lymphocytes bearing specific chemokine receptors, which is central to allograft rejection, as has been emphasized in many experimental and human studies. [46] the chemokine receptor cc chemokine receptor 5 (ccr5) is expressed on the alloaggressive th1 cells, which are responsible for activating macrophages and mediating cytotoxicity via fas - fas ligand in the graft . This study aimed to determine the distribution of ccr5-positive cells in the graft and correlate it with the degree of cellular rejection . In this prospective study, 28 patients who had presented with graft dysfunction and demonstrated features of acute cellular rejection (acr) on biopsy [figure 1a and b] were included . C4d immunostaining was performed in all cases to exclude a concomitant antibody - mediated rejection . Cases of graft dysfunction attributable to calcineurin inhibitor toxicity or viral infection on biopsy were also excluded . (a) foci of tubulitis (h and e, 200); (b) foci of tubulitis (pas, 200); (c) lymphocytes in interstitium and foci of tubulitis (anti - ccr5, 200); (d) lymphocytes in interstitium and foci of tubulitis (anti - cd3, 200) sequential slides were subjected to immunohistochemistry for cd3 (rabbit polyclonal abcam ab5690 1:100) and ccr5 (rabbit polyclonal abd serotec ahp568 1:150) using conventional techniques . The slides were examined by two pathologists (akd and rg). In the same area of the section, both cd3- and ccr5-positive cells were counted separately in the tubules, interstitium, and glomeruli, and expressed as number of cells per high power field (hpf). Banff grading of acr depends on the quantity of leukocytes infiltrating the interstititum, tubules, and arterioles . Thus, we correlated ccr5-positive t lymphocytes with the total grade of rejection as well as the individual components such as tubulitis, intimal arteritis, and interstitial inflammation using statistical tools of correlation such as spearman correlation and pearsons correlation . Sequential slides were subjected to immunohistochemistry for cd3 (rabbit polyclonal abcam ab5690 1:100) and ccr5 (rabbit polyclonal abd serotec ahp568 1:150) using conventional techniques . The slides were examined by two pathologists (akd and rg). In the same area of the section, both cd3- and ccr5-positive cells were counted separately in the tubules, interstitium, and glomeruli, and expressed as number of cells per high power field (hpf). Banff grading of acr depends on the quantity of leukocytes infiltrating the interstititum, tubules, and arterioles . Thus, we correlated ccr5-positive t lymphocytes with the total grade of rejection as well as the individual components such as tubulitis, intimal arteritis, and interstitial inflammation using statistical tools of correlation such as spearman correlation and pearsons correlation . On histopathologic examination, the cases showed acr of variable banff grades, as detailed in table 2a and b. none of the protocol biopsies showed features to suggest borderline / acr . Clinical characteristics of the study population histological and immunohistochemical analysis of banff grade i cases histological and immunohistochemical analysis of banff grade ii - iii cases immunohistochemical analysis revealed that the cases had a significantly higher number of cd3-positive (35.16 vs. 2.82) and ccr5-positive cells (15.48 vs. 0.64) compared to the control group (p = 0.010), and the pattern of distribution between ccr5- and cd3-positive cells in sequential sections was closely comparable (p <0.01)[figure 1c and d]. In cases with rejection, the ccr5-positive lymphocytes were observed to be significantly more in the tubulointerstitium than in the foci of glomerulitis (15.48 vs. 0.32; p = 0.007) and also more in the foci of tubulitis compared to the interstitium, although this did not reach levels of significance (p = 0.351). Quantitation of cd3- and ccr5-positive cells infiltrating the arterial intima could not be performed due to very small numbers of infiltrating cells; however, few infiltrating ccr5-positive cells were noted [figure 2]. Correlation of chemokine receptor 5-positive cells in foci of tubulitis with interval after transplant a positive correlation was found between ccr5-positive cells and grading of rejection by banff 2007 (p <0.05), that is, higher grade of rejection had higher number of ccr5-positive cells . Percent of t cells expressing ccr5 in the foci of tubulitis positively correlated with the severity of tubulitis, and also with the severity of interstitial inflammation (p <0.05). No significant correlation with chronic features such as interstitial fibrosis and tubular atrophy was noted . No correlation was noted with the serum creatinine levels at the time of the biopsy, and the number of ccr5-positive cells did not predict response to antirejection therapy determined by follow - up serum creatinine levels (p = 0.134). Thus, although the number of ccr5-positive cells correlated with the grade of rejection, it did not correlate with the degree of graft dysfunction or response to therapy within rejection cases . The patient population was divided into two groups: one containing tacrolimus and the other containing cyclosporine . Patients on tacrolimus containing regimens were observed to have less total number of ccr5-positive cells, particularly in the foci of tubulitis (p <0.05). In relation to the time interval after transplantation, the mean count of t cells expressing ccr5 in the foci of tubulitis was significantly more in rejection occurring within 6 months of transplantation than in that occurring after a period of 6 months (p <0.05). In cases with rejection, the ccr5-positive lymphocytes were observed to be significantly more in the tubulointerstitium than in the foci of glomerulitis (15.48 vs. 0.32; p = 0.007) and also more in the foci of tubulitis compared to the interstitium, although this did not reach levels of significance (p = 0.351). Quantitation of cd3- and ccr5-positive cells infiltrating the arterial intima could not be performed due to very small numbers of infiltrating cells; however, few infiltrating ccr5-positive cells were noted [figure 2]. Correlation of chemokine receptor 5-positive cells in foci of tubulitis with interval after transplant a positive correlation was found between ccr5-positive cells and grading of rejection by banff 2007 (p <0.05), that is, higher grade of rejection had higher number of ccr5-positive cells . Percent of t cells expressing ccr5 in the foci of tubulitis positively correlated with the severity of tubulitis, and also with the severity of interstitial inflammation (p <0.05). No significant correlation with chronic features such as interstitial fibrosis and tubular atrophy was noted . No correlation was noted with the serum creatinine levels at the time of the biopsy, and the number of ccr5-positive cells did not predict response to antirejection therapy determined by follow - up serum creatinine levels (p = 0.134). Thus, although the number of ccr5-positive cells correlated with the grade of rejection, it did not correlate with the degree of graft dysfunction or response to therapy within rejection cases . The patient population was divided into two groups: one containing tacrolimus and the other containing cyclosporine . Patients on tacrolimus containing regimens were observed to have less total number of ccr5-positive cells, particularly in the foci of tubulitis (p <0.05). In relation to the time interval after transplantation, the mean count of t cells expressing ccr5 in the foci of tubulitis was significantly more in rejection occurring within 6 months of transplantation than in that occurring after a period of 6 months (p <0.05). As the renal allograft is recognized as foreign, an allospecific immune response is triggered which is largely mediated by chemokines . An increase in concentration of the cc chemokine regulated and normal t cell expressed and secreted (rantes) in the allograft has been demonstrated in cases of cellular rejection. [911] this chemokine is secreted by activated fibroblasts, and mesangial and tubular epithelial cells in the graft, and induces the migration of cd3-positive t cells expressing the ccr5 receptor . Studied the distribution of ccr5-positive cells by immunohistochemistry in cases of glomerulonephritis, interstitial nephritis, and transplant rejection . In the 18 transplant cases (9 acute and 9 chronic), ccr5-positive cells were seen in all interstitial areas, with mononuclear cell infiltrates constituting approximately 90% of the cd3-positive cells in cases of acute rejection and approximately 65% in cases of chronic rejection . Seven of the nine cases of acute rejection were of banff grade i. however, no correlations with histopathology were made . In clinical correlations, they found that the mean ccr5-positive cells per cortical hpf was significantly higher in cases with creatinine more than 1.2 mg / dl (p <0.01). In another study, segerer et al . Showed an increase in mrna expression of ccr5, ccr2b, and cxcr4 in allograft infiltrating leukocytes, compared to that in controls . Compared ccr5 and cxcr3 expression in 13 cases of acute rejection and an equal number of cases without rejection in biopsies . They found significantly higher numbers of ccr5-positive cells in acute rejection (160.9 33.1/30 hpfs) compared to no rejection cases (36.2 10.7, p <0.007), similar to that in this present study . Their cases varied from banff grade i a to ii b; however, no histopathologic correlations were made . We also found significantly increased number of ccr5-positive cells in patients on cyclosporine compared to those on tacrolimus containing regimen . Daniel seron et al . Reported increased cd3- and cd68-positive allograft infiltrating cells in patients on cyclosporine compared to those on tacrolimus, similar to our study . However, ours is the first study correlating cc chemokine receptor 5 in these two groups and shows significantly more ccr5-positive cells in patients on cyclosporine containing regime compared to those on tacrolimus . One important observation in cases 1 and 8 [table 2a] and cases 4, 11, and 14 [table 2b] was the presence of very few or no ccr5-positive cells . A possible explanation to this could be that ccr5, which is an early marker of rejection responsible for recruitment of the leukocytes, may not be observed in large numbers once rejection is for a longer time and well established . Almost all of these cases had intimal arteritis (except case 1), pointing toward increased severity of the cases . In this study, it was again confirmed that ccr5-positive cells are significantly increased in rejecting compared to stable grafts and are found predominantly in the foci of interstitial inflammation and tubulitis . In addition, histopathologic correlations showed significant increase in the infiltrating ccr5-positive cells with worsening banff grades . Early acr had significantly more ccr5-positive t cells in foci of tubulitis compared to late acr . With animal model experiments demonstrating that blockade of ccr5 receptors leads to retardation of rejection episodes, the findings of this study also indicate a future role of anti - ccr5 therapy in the treatment of rejection, particularly early episodes . In addition, as ccr5 represents the actively alloaggressive subset of t cells and increases with worsening of rejection, the authors propose that it be added to the ever - expanding list of activation markers for t cells, and future studies on cases of borderline rejection are warranted.
Mucormycosis is a rare but serious infection due to filamentous fungi of the division of mucorales, the class zygomycetes . They occur most often in immunocompromised patients with diabeties or haematological malignancies with prolonged neutropenia they represent the third cause of fungal infection in these patients after invasive candidiasis and aspergillosis . We report five cases of confirmed mucormycosis in patients hospitalized in the infectious diseases department of sousse, tunisia, between 2000 and 2013 . The main data are summarized in table 1 . The patiuents were four men and one woman, mean age 60 years (2577 years). Three patients had type 2 diabetes, one patient had acute leukemia (al) in treatment failure with prolonged neutropenia and one patient was immunocompetent . Mucormycosis was rhino - cerebral, rhino - orbital, auricular (left otitis media), pulmonary and cutaneous . Clinical signs included fever (all cases), peripheral facial palsy (pfp) and an orbital edema (two cases each), subcutaneous frontal abscess (fig . 1) with mental confusion, ear pain with purulent otorrhea and hearing loss left, productive cough with dyspnea and necrotizing fasciitis of the left leg . The diagnosis of mucormycosis was made after average 17 days of hospitalization (257 days). Computed tomography (ct) of the facial bones showed pansinusitis in two patients, associated with subcutaneous frontal abscess in a patient . Chest ct showed bilateral alveolar - interstitial infiltrate with alveolar consolidation in the right lower lobe (fig . 2). The magnetic resonance imaging (mri) showed bilateral predominantly frontal right lesion hyperintense on t2-flair in a patient (fig . The direct examination showed live mycological wide non septate hyphae with irregular diameter, indicative of mucorales, in three cases . Mucorales were isolated from the following samples: pus from the subcutaneous frontal abscess obtained by needle puncture, pus ear swab obtained on five different samples spaced several days, sinus biopsy, bronchial biopsy and intraoperative biopsy the soft tissue . It was identified as lichteimia corymbifera (formerly absidia corymbifera) in three cases and rhizopus arrhizus in two cases . All patients were treated with intravenous amphotericin b 0.7 to 1 mg / kg / day . Apart from one patient who died after four days of hospitalization, the mean duration of treatment was 32 days (1546). Hypokalemia was observed in one patient in the course of moderate acute renal failure at the end of treatment in two patients . In two cases, the evolution was marked by the early death in two patients (inter- hemispheric cerebral hematoma complicated with coma and respiratory distress in one case, and extensive pneumonia with respiratory distress in one case) and persistent sequelae in two other patients (pfp in two cases and hearing loss in one case). In one patient who had al, treatment with amphotericin b and surgical debridement resulted in a local improvement, but the patient died one month after his discharge due to his underlying disease . In our study, mucormycosis was observed more often in immunocompromised patients (four out of five cases) and the most common sites were rhino - cerebral and rhino - orbital . Indeed, in 90% of cases, mucormycosis occur in immunocompromised patients, mainly diabetic ketosis or hematological malignancy with neutropenia . The observed auricular localization in one of our patients, immunocompetent is exceptional . A tunisian retrospective study compiling 17 cases of mucormycosis between 1992 and 2007, diabetes and rhino - orbitofrontal cerebral localization was noted in all patients . In rhino - sinus mucormycosis, ct is the investigation of choice to study the invasion of bone and soft tissue abscesses, or hematoma, and extension to the central nervous system . Mri is more sensitive than ct for the investigation of possible cerebrovascular thrombosis . In pulmonary mucormycosis, chest radiograph or better chest ct typically show alveolar condensations sometimes excavated or nodular infiltrates frosted glass with or without halo sign . The reference method is the direct examination and culturing of the pathological product: puncture fluid (pus, serous fluid), tissue biopsy . Mucorales hyphae are short, little or no septate, thick - walled and often branched at right angles . The identification of the genus and species is of epidemiological interest but does not guide antifungal therapy . These characters are not specific where the recent use in specialized laboratories in molecular biology techniques such as pcr improved the etiological diagnostic . The most common mucormycosis genera are rhizopus (47%) and mucor (18%). Lichteimia was isolated in three of five patients . In the literature, this type is more frequently isolated from male patients . In addition to the mycological diagnosis, histological study of biopsy fragments are useful for the diagnosis of mucormycosis and allows the confirmation in case of mucorales filament presence in tissues and vessels where they are responsible for thrombosis with infarction and hemorrhage . In our study, the mucorales were isolated from pus obtained by percutaneous puncture or multiple swab samples in two cases, and on biopsy fragments in three cases . The rapid equilibration of ketoacidosis in diabetics, transfusion of hematopoietic growth factors in long - term neutropenia and hyperbaric oxygen therapy may be useful . Reference antifungal therapy is liposomal amphotericin b, 5 to 10 mg / kg / day . Other antifungal posaconazole or caspofungin can be used in combination with liposomal amphotericin b in case of treatment failure or as a substitute for serious side effects . Mortality is higher in case of diagnostic delay of more than five days and monocytopenia in patients with active malignant blood diseases . The genus or species of mucorales offending does not appear to influence the outcome . In a previous tunisian study, mortality was 65% . In our study, all patients were treated with amphotericin b deoxycholate as liposomal amphotericin b is not available in our country and two patients had surgical excision . The diagnosis of mucormycosis should be suspected in any diabetic patient with neutropenia or neutropenic presenting with rhino - orbitofrontal brain or lung unimproved by appropriate antibiotic therapy . Diagnosis is suspected on clinical and radiological features and confirmed by mycological and pathological examination . Morbidity and mortality are high due to the invasive nature of the frequent underlying malignancy, hence the importance of early and appropriate management.
Generation of 3-mb - pp13-mb - pp1 was synthesized using a previously described route with slightly modified conditions (18). P116, a zap-70-deficient jurkat - derived t cell line, was obtained from r. abraham (the burnham institute, la jolla, ca). 293 cells, a kidney epithelial cell line, were obtained from the american type culture collections ., 20 10 cells were transfected with 5 g of expression construct and 25 g of empty vector . Stable p116 clones expressing zap-70 constructs were selected with blasticidin (10 g / ml; invitrogen). Transient transfections of 293 cells were carried out in 24-well plates using lipofectamine plus reagent (invitrogen) according to the manufacturer's instructions . Plasmids a quikchange site - directed mutagenesis kit (stratagene) and standard pcr techniques were used to prepare zap-70 mutations m414a (zap-70) and m414a / c405v (zap-70) in the plasmid pbluescript (invitrogen). The mutated versions and wild - type human zap-70 were then subcloned into expression vector pef6.a (invitrogen) via ecori digest . For transfections, the following previously described plasmids were used: lck (19), flag - tagged lat (20), cd8- (21), hemagglutinin - tagged rat plc1 (22), and hemagglutinin - tagged murine tec kinase (22). The diacylglycerol kinase (dgk) construct was a gift from gary koretzky . An enhanced green fluorescent protein plasmid from invitrogen was used in cotransfection experiments . Antibodies ascites of c305, an anti - jurkat tcr -chain monoclonal antibody was used for tcr stimulations (23). For stimulation via cd28 the following antibodies were used for western blotting: plc1-py783, lat - py132 (biosource), zap-70-py319, thr / tyr for phospho - p44/42 mapk (cell signaling), lck (1f6 from j. b. bolen), anti - phosphotyrosine (4g10; upstate biotechnology), -tubulin (sigma), lat (abcam), slp-76 (santa cruz biotechnology), active p38, active jnk (promega), plc1 mixed monoclonal antibodies, and tec antibody (upstate biotechnology). The following antibodies have been described previously: 2f3.2 (anti - zap-70) (24) and 6b10.2 (anti - tcr-) (25). Flow cytometry assays for cd69 experiments, 6 h after transfection, cells were washed in rpmi and resuspended at 1 10 cell / ml . Cells were incubated with dmso (vehicle) or 3-mb - pp1 and stimulated with anti - tcr antibody (1:1000 c305) or phorbol 12-myristate 13-acetate (pma) (25 ng / ml). Cells were left overnight at 37 c with 5% co2 and then stained with allophycocyanin - conjugated cd69 (bd biosciences). Cells were then fixed with bd cytofix (bd biosciences), washed twice with facs buffer, and then permeabilized in caltag fix / perm medium b and stained for intracellular zap-70 (caltag). For intracellular flow cytometry analysis of perk, p116 stable lines were incubated with anti - tcr antibody (1:2000 c305) for a 30-min time course, with vehicle (dmso) or 3-mb - pp1, in a 96-well round bottom plate . Cells were fixed by adding bd cytofix, washed twice with facs buffer, and then permeabilized with 100% ice - cold methanol . Cells were then washed three times, stained with primary perk antibody, washed twice, and then stained with allophycocyanin - conjugated goat anti - rabbit secondary (jackson immunoresearch). Cell were stimulated with anti - tcr antibody (1:2000 c305) in the presence of dmso, 3-mb - pp1, or pp2 luciferase assays stable cell lines were transfected with 20 g of nfat / ap-1 luciferase and 20 g of vector only . Approximately 16 h after transfection, cells were stimulated with anti - tcr antibody (1:2000 c305) or pma (50 ng / ml) and ionomycin (1 m). Six hours later, the cells were harvested, lysed, and assayed for luciferase activity using a mithras lb 940 (berthold technologies). Superantigen stimulation and interleukin-2 (il-2) measurement superantigen - loaded antigen - presenting cells were prepared by incubating raji b cells with 100 ng / ml staphylococcus enterotoxin (toxin technology). 10 raji cells (staphylococcus enterotoxin) were then incubated with an equal number of zap-70 or zap-70 cells per well in a 96-well plate in a total volume of 0.2 ml of medium (rpmi with 5% fetal calf serum supplemented with penicillin, streptomycin, and glutamine) treated with the indicated concentrations of 3-mb - pp1 . After 18 h at 37 c with 5% co2, the il-2 concentration was determined by using human il-2 elisa ready - set - go! Cells were treated with pma (20 ng / ml) plus ionomycin (1 m) as a control . Stimulations and immunoblot analysis for 293 cell experiments, inhibitor or dmso control was added at the time of transfection, and cells were incubated for 24 h. cells were then harvested, spun down, and lysed in 2 concentrated sds sample buffer . Lysates were cleared by ultracentrifugation at 440,000 g for 30 min at 24 c . Supernatants were then collected, and dithiothreitol was added to a final concentration of 1% . P116 stable lines were stimulated (25 10/ml) in rpmi with anti - tcr antibody (1:2000 c305) for the indicated time . For most experiments, cells were lysed as noted in 2 concentrated sds sample buffer and subjected to ultracentrifugation as described above . For immunoprecipitation experiments, 10 10 cells were stimulated and lysed in ice - cold lysis buffer (10 mm tris, ph 7.6, 150 mm nacl, 1% nonidet p-40, and a mixture of protease and phosphatase inhibitors). Postnuclear supernatant was used for immunoprecipitation with antibody bound to protein a or protein g beads (amersham biosciences). Samples were analyzed by sds - page, and immunoblotting was performed using primary and horseradish peroxidase - conjugated secondary antibodies . Proteins were detected by chemiluminescence (western lightning) using a kodak image station (kodak). Generation of analog - sensitive zap-70 allele we generated an analog - sensitive zap-70 allele by mutating the gatekeeper methionine to alanine to create greater access in the atp pocket of the kinase domain . The resulting m414a mutant is referred to as analog - sensitive allele 1 (as1) (fig ., we introduced a secondary mutation, c405v, in conjunction with m414a, in an attempt to restore stability to the -sheet in the n - terminal kinase domain, which could potentially be compromised by the parental m414a mutation (26). 1b). As seen in fig . 1a, the mutation of the gatekeeper methionine to the smaller alanine residue generates more space in the catalytic domain, allowing room for binding of bulky pp1 analogs . In these studies, we chose to use the pp1 analog 3-mb - pp1 over other pp1 analogs, based upon its potent inhibition of zap-70 and the lack of effect on wild - type cells in a screen for t cell activation (data not shown). 3-mb - pp1 contains an extra methylene bridge to the phenyl substituent of pp1 and a 3-methyl substituent that has been shown to be important for reducing binding to wild - type kinases (fig . 1c) (27). To test the cellular activity of the zap-70 mutants and their sensitivity to 3-mb - pp1, we transiently transfected 293 cells with zap-70, or either one of the zap-70 constructs, along with lck to activate zap-70 and the transmembrane adaptor lat, which serves as a zap-70-specific substrate . As shown in fig 2a, zap-70 was insensitive to the addition of 3 or 6 m 3-mb - pp1 . In contrast, both zap-70 and zap-70 were inhibited by 3-mb - pp1 in a dose - dependent manner as measured by decreased lat phosphorylation . Importantly, the 293 transfection data also showed that the analog - sensitive zap-70 exhibited reduced catalytic activity when compared with the wild - type; however, both alleles were able to phosphorylate lat in the absence of inhibitor . We estimate the analog - sensitive zap-70 mutant has an average 2.1-fold reduction in cellular activity relative to wild - type, based upon quantification of the intensity of the total level of zap-70 relative to that of the phosphorylated lat in a series of immunoblots (data not shown). To further characterize the sensitivity of zap-70 to 3-mb - pp1, we transiently transfected the zap-70 constructs into the zap-70-deficient jurkat - derived t cell line, p116 . P116 fails to effectively initiate signaling events downstream of zap-70, including protein tyrosine phosphorylation, ca mobilization, ras / mapk activation, nfat - directed transcription, and expression of a variety of downstream genes such as cd69 (28, 29). We tested the transiently transfected cells for surface cd69 expression, mediated via the ras / mapk pathway, after overnight stimulation with an anti - tcr antibody (c305) in the presence or absence of 10 m 3-mb - pp1 . Both zap-70- and zap-70-expressing cells efficiently up - regulated cd69 following tcr stimulation in the presence of vehicle (dmso). However, the activation of cells expressing zap-70 was markedly impaired in the presence of 3-mb - pp1 (fig . No 3-mb - pp1-mediated effect was seen with either of the zap-70 alleles after pma stimulation, which bypasses proximal tcr signaling . Thus, the zap-70 is functional and is inhibited by 3-mb - pp1 in a t cell system . Moreover, although lck and many downstream kinases are required for cd69 induction, only the cells expressing the zap-70 alleles are sensitive to inhibition by 3-mb - pp1 . A, ribbon structure of a portion of the kinase domain of zap-70 (left) (protein data bank code 2ozo (35)) and zap-70 (m414a was manually introduced in pymol) (right). Zap-70 generation . A, ribbon structure of a portion of the kinase domain of zap-70 (left) (protein data bank code 2ozo (35)) and one of the principal challenges to developing inhibitor systems is avoiding off - target effects . Therefore, we further tested the specificity of 3-mb - pp1 inhibition by analyzing its effect on other relatively upstream kinase / substrate pairs implicated in tcr signaling in 293 transient transfection assays (fig . Lck activity was monitored by cotransfecting a cd8- chimera, which contains the cytoplasmic domain of and the extracellular domain of cd8, as a substrate . Both lck and tec kinase activity were only minimally affected even after incubation with high doses (9 - 10 m) of 3-mb - pp1 . Zap-70 is required to both initiate and maintain tcr - mediated increases in cytoplasmic free calcium ([ca]i)zap-70-deficient p116 cells fail to increase [ca]i after tcr stimulation . Therefore, we wanted to test the effect of 3-mb - pp1 on [ca]i . We first generated p116 clones that stably expressed zap-70, zap-70, or zap-70 to reconstitute tcr signaling . Both of the zap-70 clones expressed more zap-70 than the parental jurkat line (supplemental fig . 1). Therefore, we selected zap-70 and zap-70, which express comparable amounts of zap-70 to the wild - type clones . In addition, we utilized zap-70 because it expresses zap-70 equivalently to parental jurkat cells and, therefore, allowed us to rule out any potential artifact of zap-70 overexpression . To test the requirement of zap-70 for initiating [ca]i increases, cells were pretreated with a 6 m dose of 3-mb - pp1 or vehicle for 75 s and then stimulated with anti - tcr antibody . In both zap-70 and zap-70 stably transfected cells, 3-mb - pp1 treatment blocked tcr - mediated signaling, and there was only a negligible effect on zap-70 cells (data not shown; fig ., src family kinases such as lck in jurkat t cells are also required for inducing [ca]i increases because of their ability to mediate itam phosphorylation and zap-70 activation . Treatment with the src family kinase inhibitor pp2 had a comparable effect to that of inhibiting zap-70 in both wild - type and mutant stables, whereas only zap-70 was sensitive to blockade by 3-mb - pp1, suggesting a high degree of specificity . There was also no effect of 3-mb - pp1 treatment on ionomycin - dependent calcium release in zap-70 or zap-70 cells (data not shown). This block in ca signaling was interesting but was anticipated from studies of the parental p116 cells . However, we were more interested in determining the requirement of the zap-70 catalytic function for maintaining [ca]i elevation . The inhibitor system provides a powerful tool for answering such questions because 3-mb - pp1 can be added post - stimulation . To test this idea directly, we treated the stably transfected cells with 3-mb - pp1 after the maximal ca response was achieved (75 s after stimulation). Interestingly, under these conditions, 3-mb - pp1 treatment completely abrogated tcr - mediated [ca]i increase in analog - sensitive stables, returning [ca]i to base line within 20 s (fig . This suggests an ongoing requirement for zap-70 catalytic function beyond signal initiation by the tcr . The striking inhibition of calcium flux was also noted in a dose - response analysis to 3-mb - pp1 . As shown in fig . 4c, itis clear that the calcium flux of zap-70 cells is sensitive to even very low doses (including 0.5 and 1.0 m) of 3-mb - pp1 . A, 293 transient transfection of zap-70 constructs in the presence or absence of 3-mb - pp1 . Cells were lysed in 2 concentrated sds - page sample buffer and analyzed by immunoblotting with antibody against phosphotyrosine (top panel). Total zap-70, lck, and lat levels were determined by blotting with specific antibodies . Unless noted otherwise, all experiments shown in figs . 2, 3, 4, 5, 6, 7 and 8 are representative of at least three independent experiments . B, facs analysis of zap-70 and cd69 expression after stimulation of p116 cells transiently transfected with either zap-70 or zap-70 . Six h after transfection, cells were left unstimulated or stimulated with the anti - tcr antibody c305 or with pma (25 ng / ml) for 16 h in the presence of vehicle (dmso) or 3-mb - pp1 . Cells were stained for surface cd69 and then fixed, permeabilized, and stained for intracellular zap-70 . The numbers represent the percentage of zap-70cd69 (upper right) and zap-70cd69 (lower right). Although zap-70 staining was used in the experiment shown here, future experiments confirmed this result using cotransfected green fluorescent protein as a surrogate marker of zap-70 . Zap-70 cellular activity is selectively inhibited by 3-mb - pp1 . A, 293 transient transfection of zap-70 constructs in the presence or absence of 3-mb - pp1 . Cells were lysed in 2 concentrated sds - page sample buffer and analyzed by immunoblotting with antibody against phosphotyrosine (top panel). Total zap-70, lck, and lat levels were determined by blotting with specific antibodies . Unless noted otherwise, all experiments shown in figs . 2, 3, 4, 5, 6, 7 and 8 are representative of at least three independent experiments . B, facs analysis of zap-70 and cd69 expression after stimulation of p116 cells transiently transfected with either zap-70 or zap-70 . Six h after transfection, cells were left unstimulated or stimulated with the anti - tcr antibody c305 or with pma (25 ng / ml) for 16 h in the presence of vehicle (dmso) or 3-mb - pp1 . Cells were stained for surface cd69 and then fixed, permeabilized, and stained for intracellular zap-70 . The numbers represent the percentage of zap-70cd69 (upper right) and zap-70cd69 (lower right). Although zap-70 staining was used in the experiment shown here, future experiments confirmed this result using cotransfected green fluorescent protein as a surrogate marker of zap-70 . A, 293 transient transfection of lck and cd8-, serving as the kinase substrate, in the presence or absence of 3-mb - pp1 . Total lck, cd8-, tec, and plc1 were determined by blotting with specific antibodies . A, 293 transient transfection of lck and cd8-, serving as the kinase substrate, in the presence or absence of 3-mb - pp1 . Total lck, cd8-, tec, and plc1 were determined by blotting with specific antibodies . The requirement of zap-70 for [ca]i elevation was striking, but these measurements were made during short time intervals following the initiation of tcr signaling . Therefore, it was important to monitor nfat transcriptional events in order to determine the long - term effect of this failure to mobilize intracellular calcium . We transfected a nfat / ap-1 luciferase reporter into our stable lines and monitored the activity after 6 h of tcr stimulation . As expected from the calcium data, nfat activity was severely and equivalently diminished by 3-mb - pp1 treatment in both the zap-70 and zap-70 lines after anti - tcr antibody stimulation (fig . In addition, the response was dose - dependent . The highest dose at 6 m, which effectively blocked calcium responses, inhibited almost all nfat - driven luciferase activity (5% remaining activity compared with vehicle treated). Zap-70 inhibition suppresses superantigen - mediated il-2 induction the inhibition of calcium increases and nfat - transcription in 3-mb - pp1-treated cells demonstrated the utility of this analog approach and exposed the requirement of zap-70 to both initiate and maintain the calcium response after tcr antibody stimulation . However, we also wanted to test the system using a more physiological approach . Therefore, we decided to measure il-2 production following stimulation with staphylococcus enterotoxin e superantigen - loaded antigen - presenting cells . This stimulation method is more physiological than antibody treatment because it requires antigen - presenting cells and also because humans t cells respond to superantigens in pathologic situations such as toxic shock syndrome or food poisoning . As shown in fig . 5b thus, our findings show that this analog approach works in a physiologic context and that zap-70 is required to mediate a superantigen response to an enterotoxin . Interestingly, the zap-70 cells were less sensitive to 3-mb - pp1 than in the nfat - transcriptional assay shown in fig . 5a . Il-2 production is a more integrated response and therefore may be less sensitive to lower levels of zap-70 inhibition . Cells were loaded with indo-1 dye and stimulated with anti - tcr antibody and either pretreated (a) or treated post - maximal ca flux (b) with 6 m 3-mb - pp1 or vehicle (v; dmso). In a, pp2 was added at a final concentration of 20 m . The experiment in a is representative of multiple independent experiments, but pp2 was added as a control in two experiments . C, zap-70 cells were pretreated with a range of 3-mb - pp1 concentrations as indicated . Cells were loaded with indo-1 dye and stimulated with anti - tcr antibody and either pretreated (a) or treated post - maximal ca flux (b) with 6 m 3-mb - pp1 or vehicle (v; dmso). In a the experiment in a is representative of multiple independent experiments, but pp2 was added as a control in two experiments . C, zap-70 cells were pretreated with a range of 3-mb - pp1 concentrations as indicated . The cells were then left unstimulated, stimulated with anti - tcr antibody, or stimulated with 50 ng / ml pma plus 1 m ionomycin for 6 h at 37 c and then assayed for luciferase activity . The nfat response was first calculated as a percentage of the maximum as determined by pma plus ionomycin treatment . Then the activity was determined for each 3-mb - pp1 concentration relative to untreated (no 3-mb - pp1 or 0 m 3-mb - pp1). B, zap-70 and zap-70 cells were incubated with staphy - lococcus enterotoxin e - loaded antigen - presenting cells for 18 h in the presence of a range of 3-mb - pp1 concentrations before il-2 concentration was determined by enzyme - linked immunosorbent assay . The cells were then left unstimulated, stimulated with anti - tcr antibody, or stimulated with 50 ng / ml pma plus 1 m ionomycin for 6 h at 37 c and then assayed for luciferase activity . The nfat response was first calculated as a percentage of the maximum as determined by pma plus ionomycin treatment . Then the activity was determined for each 3-mb - pp1 concentration relative to untreated (no 3-mb - pp1 or 0 m 3-mb - pp1). B, zap-70 and zap-70 cells were incubated with staphy - lococcus enterotoxin e - loaded antigen - presenting cells for 18 h in the presence of a range of 3-mb - pp1 concentrations before il-2 concentration was determined by enzyme - linked immunosorbent assay . Data are representative of two experiments . Decreased phosphorylation of the lat - slp-76-plc1 signalosome upon zap-70 inhibition zap-70 has been thought to have at least two direct downstream targets, the adaptor proteins lat and slp-76 . Both lat and slp-76 are key signaling adaptors, and cells that lack expression of either protein fail to propagate many of the downstream tcr signals . In zap-70-deficient p116, neither lat nor slp-76 is efficiently phosphorylated; and, the same cells overexpressing a kinase - inactive form of zap-70 also fail to exhibit these phosphorylated substrates (1, 28). Lat and slp-76 interact indirectly and are responsible for forming a complex of signaling molecules downstream of the tcr . Slp-76 then recruits a number of important molecules including plc1, guanine nucleotide exchange factor vav, and the tec family kinase, interleukin-2-inducible t cell kinase (itk), the latter two of which appear to require slp-76 phosphorylation (31). In addition to its indirect interaction with slp-76 via gads, lat also interacts with numerous signaling molecules through its phosphorylated tyrosines, including the adaptor grb2 (growth factor receptor - bound protein 2) as well as plc1 . The formation of a lat- and slp-76-containing signalosome serves as a nucleation point for tcr signaling events . Phosphorylation of these adaptors, mediated by zap-70, plays a critical role in the formation and functional activity of this signaling complex . Because phosphorylation of lat and slp-76 by zap-70 is so critical for initiation of many of the downstream signaling events, we wanted to examine the phosphorylation status of lat and slp-76 after treatment with 3-mb - pp1 in the analog - sensitive clones . However, to first control for more proximal events that are not zap-70-dependent, we monitored lck - dependent tcr phosphorylation in zap-70 and zap-70 cells . 6a, tcr phosphorylation was not significantly altered after incubation with the inhibitor in either cell line, thereby reinforcing the idea that 3-mb - pp1 specifically targets zap-70-dependent events . It is important to note that the increase in total tcr immunoprecipitated from 3-mb - pp1-treated zap-70 cells was not reproducible . We then examined lat tyr phosphorylation, because plc1 has been shown to bind to this tyrosine when phosphorylated (32). Once activated, plc1 cleaves phosphatidylinositol 4,5-bisphosphate (pip2) into diacylglycerol and inositol 1,4,5-trisphosphate (ip3), where ip3 binds receptors on the endoplasmic reticulum that lead to release of ca stores . Mutation of tyr to phenylalanine has been shown to decrease plc1 phosphorylation and its binding to lat, in addition to diminishing the overall [ca]i increase (30, 32). 6b, induced phosphorylation of lat tyr is markedly inhibited after cotreatment with anti - tcr antibody and 5 - 10 m 3-mb - pp1 . In addition to lat phosphorylation, total slp-76 phosphorylation was reduced over a 2-min time course (fig . Vehicle - treated zap-70 mutant cell samples often exhibited a somewhat decreased phosphorylation relative to zap-70 cells, which we hypothesized was due to the decreased catalytic activity of the analog - sensitive mutant . Despite the decreased magnitude of phosphorylation in the untreated zap-70 cells, moreover, in very preliminary studies, we have been able to reconstitute t cell development of zap-70 null mice with the zap-70 clone expressed as a transgene, suggesting that the reduced catalytic activity of the mutant zap-70 is not substantially functionally impaired . P116 stable lines were incubated with vehicle or 5 m 3-mb - pp1 (unless indicated otherwise) and treated with either anti - tcr antibody or left unstimulated . A, after stimulation, 10 10 cells were lysed in 1% nonidet p-40 lysis buffer, and tcr was immunoprecipitated . Immunoprecipitated lysates were immunoblotted for total phosphotyrosine and then stripped and blotted for total tcr. B, cells were lysed in 2 concentrated sds - page sample buffer and analyzed by immunoblotting with phosphotyrosine - specific lat ptyr and total zap-70 antibodies . Approximately 0.4 10 cell equivalents were loaded onto a sds - polyacrylamide gel . C, after stimulation, slp-76 was immunoprecipitated as described in a. immunoprecipitated lysates were immunoblotted for total phosphotyrosine and slp-76 . Results are representative of three independent experiments, which were carried out using either zap-70 or zap-70 . D, lysates were prepared as described in b and immunoblotted with antibodies specific for plc1 ptyr, zap-70 ptyr, and tubulin . P116 stable lines were incubated with vehicle or 5 m 3-mb - pp1 (unless indicated otherwise) and treated with either anti - tcr antibody or left unstimulated . A, after stimulation, 10 10 cells were lysed in 1% nonidet p-40 lysis buffer, and tcr was immunoprecipitated . Immunoprecipitated lysates were immunoblotted for total phosphotyrosine and then stripped and blotted for total tcr. B, cells were lysed in 2 concentrated sds - page sample buffer and analyzed by immunoblotting with phosphotyrosine - specific lat ptyr and total zap-70 antibodies . Approximately 0.4 10 cell equivalents were loaded onto a sds - polyacrylamide gel . C, after stimulation, slp-76 was immunoprecipitated as described in a. immunoprecipitated lysates were immunoblotted for total phosphotyrosine and slp-76 . Results are representative of three independent experiments, which were carried out using either zap-70 or zap-70 . D, lysates were prepared as described in b and immunoblotted with antibodies specific for plc1 ptyr, zap-70 ptyr, and tubulin . In particular, phosphorylation of tyr has been shown to facilitate the interaction between tyr and the c - terminal sh2 domain of plc1, which leads to enzyme activation (33). Recently, it was demonstrated that slp-76-bound itk phosphorylates plc1 on this key residue (34). As shown in fig . 6d, phosphorylation of tyr on plc1 was markedly reduced in 3-mb - pp1-treated zap-70 cells . The specificity of the 3-mb - pp1 toward zap-70 was further emphasized in this experiment by the finding that zap-70 tyr phosphorylation, mediated by lck, was not affected by the addition of the inhibitor (fig . Recent crystallographic studies have shown that the tyr residue, which is phosphorylated by lck, is involved in the autoinhibition of zap-70 (35). Together, the loss of lat, slp-76, and plc1 phosphorylation can account for the marked inhibition we observed in calcium mobilization . We noticed only a modest decrease in total itk phosphorylation in 3-mb - pp1-treated zap-70 and zap-70 cells (data not shown). This is in agreement with the fact that src kinases regulate tec kinase phosphorylation (36); however, itk phosphorylation has recently been shown to be defective in slp-76-deficient cells (34). The remaining slp-76 phosphorylation in our 3-mb - pp1 cells may be sufficient to support itk phosphorylation . Thus, these studies indicate that phosphorylation of two of the most important downstream zap-70 substrates is impaired upon 3-mb - ppl treatment of zap-70-expressing cells but that other substrates such as the tcr chain, itk, and zap-70 itself, which are all phosphorylated by lck, are not substantially affected . Maximal and persistent ras / mapk phosphorylation requires zap-70 catalytic activity given the substantial 3-mb - pp1-mediated inhibition of calcium and plc1 phosphorylation in zap-70-expressing cells, we wanted to examine other plc1-mediated signaling events, particularly activation of the ras / mapk pathway via the generation of diacylglycerol . We had already demonstrated during the initial screening of 3-mb - pp1 that inhibitor treatment diminishes cd69 up - regulation, a downstream transcriptional target of ras activation, in cells transiently transfected with zap-70 (fig ., all zap-70 lines failed to maximally up - regulate cd69 after 16 h of stimulation with anti - tcr antibody . However, there was no effect on pma - mediated activation, which bypasses proximal tcr signaling (fig . The failure to express surface cd69 in 3-mb - pp1-treated analog - sensitive cells was expected to correlate with the lack of activation of the mapk, erk . However, we repeatedly noticed that we had substantial, albeit diminished and delayed, erk phosphorylation at 2 min in 3-mb - pp1-treated zap-70 cells . It became clear, both by western blotting and intracellular staining of perk using flow cytometry, that erk phosphorylation was delayed and not maintainable in the inhibitor - treated zap-70 cells (fig . Analysis of erk phosphorylation by the use of a phosphospecific erk antibody on whole cell lysates examined by sds - page showed clear induction of erk phosphorylation by 1 min in untreated zap-70 cells and zap-70 cells, but there was no detectable perk phosphorylation above basal levels in inhibitor - treated zap-70 cells until 1.5 - 2 min after anti - tcr antibody stimulation (fig . This pattern of erk phosphorylation held true for other mapk members, p38 and jnk, albeit basal levels of pp38 were also reduced . We were especially interested in p38 induction given the direct role that zap-70 is thought to play in activating p38 in t cells (37). Analysis of perk by intracellular flow cytometry showed similar results at the early time points, with only a small percentage of the cells containing phosphorylated erk 5 min post - stimulation (fig . This minimal phosphorylation was not sustained . By 30 min, erk phosphorylation in the analog - sensitive cells returned to base line, whereas untreated cells were still substantially positive for perk (fig . There was no difference in erk phosphorylation between zap-70 and zap-70 cells treated with vehicle . A, facs analysis of cd69 expression after stimulating stable lines for 16 h with or without anti - tcr antibody or pma (25 ng / ml) in the presence or absence of 10 m 3-mb - pp1 . B, p116 stable lines were incubated for a 2-min time course with either vehicle or 5 m 3-mb - pp1 and treated with anti - tcr antibody or left unstimulated . Cells were lysed in 2 concentrated sds sample buffer and blotted for perk, pjnk, pp38, and tubulin . C, p116 stable lines were incubated with anti - tcr antibody over a 30-min time course with vehicle (filled histogram) or 5 m 3-mb - pp1 (open histogram). At the appropriate time points, d, same as in c except cells were treated with either vehicle (filled), 5 m 3-mb - pp1 (solid line), or 10 m 3-mb - pp1 (dotted line). E, zap-70 was transiently transfected with 20 g of green fluorescent protein vector and either empty vector (left) or 20 g of dgk (right) and stimulated and stained as described in c. transfected cells were identified by gating on gfp cells . The histograms are labeled as follows: unstimulated (dotted line); 5 min c305 + dmso (filled); 5 min c305 + 5 m 3-mb - pp1 (solid line). Zap-70 catalytic activity is required for complete and persistent ras / mapk phosphorylation . A, facs analysis of cd69 expression after stimulating stable lines for 16 h with or without anti - tcr antibody or pma (25 ng / ml) in the presence or absence of 10 m 3-mb - pp1 . B, p116 stable lines were incubated for a 2-min time course with either vehicle or 5 m 3-mb - pp1 and treated with anti - tcr antibody or left unstimulated . Cells were lysed in 2 concentrated sds sample buffer and blotted for perk, pjnk, pp38, and tubulin . C, p116 stable lines were incubated with anti - tcr antibody over a 30-min time course with vehicle (filled histogram) or 5 m 3-mb - pp1 (open histogram). At the appropriate time points, d, same as in c except cells were treated with either vehicle (filled), 5 m 3-mb - pp1 (solid line), or 10 m 3-mb - pp1 (dotted line). E, zap-70 was transiently transfected with 20 g of green fluorescent protein vector and either empty vector (left) or 20 g of dgk (right) and stimulated and stained as described in c. transfected cells were identified by gating on gfp cells . The histograms are labeled as follows: unstimulated (dotted line); 5 min c305 + dmso (filled); 5 min c305 + 5 m 3-mb - pp1 (solid line). Although we were able effectively to eliminate increases in [ca]i with 3-mb - pp1, treatment with the same inhibitor still generated a substantial, yet abbreviated, pattern of erk phosphorylation . This discrepancy between the ca data and the erk phosphorylation was interesting because it either suggested that the level of plc1 activity post - treatment was sufficient for some erk phosphorylation but not ca mobilization or that the residual erk phosphorylation was plc1-independent . Such a plc1-independent pathway could be mediated by grb2-sos recruitment to the phosphorylated tcr -chain, as had been suggested previously (38), although grb2-sos is also known to be recruited to phosphorylated lat (39, 40). Interestingly, the existence of a zap-70-independent pathway leading to erk phosphorylation has been reported . In these studies, p116 cells repeatedly exhibited delayed and transient erk phosphorylation; however, this required the use of high levels of cd3 cross - linking (40, 41). We used two approaches to test whether residual plc1 activity was the mediator of the erk activation . First, we increased the concentration of 3-mb - pp1 to 10 m and found that this eliminated the remaining erk phosphorylation (fig . Second, we transiently overexpressed diacylglycerol kinase (dgk), which converts diacylglycerol into phosphatidic acid by phosphorylation of the free hydroxyl group . We hypothesized that if the remaining erk phosphorylation were plc1-dependent, then overexpression of dgk would eliminate the remaining erk in the 3-mb - pp1-treated zap-70 cells . Overexpression of dgk did, in fact, eliminate the remaining erk phosphorylation at 5 min post - stimulation in the presence of 3-mb - pp1, thus providing additional evidence that the erk phosphorylation appears to be plc1-dependent (fig . Therefore, although [ca]i increases were severely inhibited by 3-mb - pp1 treatment, similar doses of the inhibitor still allowed for enough plc1 activity to initiate the mapk cascade to some extent . However, this level of residual erk phosphorylation was not sufficient to maintain long - term phosphorylation or promote maximal transcriptional changes . These data are consistent with the notion that the mechanisms to amplify or produce positive feedback on erk activation are more sensitive to low levels of plc1 activity than the mechanisms that regulate the calcium pathway . Superagonist cd28 antibody is zap-70-dependent one of the benefits of utilizing a small molecule inhibitor system is to be able to test the role of the given protein in a variety of pathways where its function is controversial or not easily determined . For instance, zap-70 is well known for its critical role in proximal tcr signaling, but its function in cd28 superagonist signaling has been unclear . Conventional anti - cd28 antibodies require tcr coengagement to induce proliferation, but there is a class of anti - cd28 antibodies, termed stimulating or superagonists, that can stimulate t cell il-2 production and proliferation in the absence of tcr antibodies . One human cd28 superagonist was recently utilized in a clinical trial because it had been shown to induce development of regulatory t cells, but when administered to the healthy volunteers in a phase i clinical trial, the superagonist induced an unanticipated cytokine storm followed by multiorgan failure in volunteer subjects participating in this trial (42). Thus, understanding the biochemical mechanisms by which cd28 superagonist antibodies function might help to prevent such unforeseen, disastrous complications with immunologically active agents in the future . Although the use of stimulating anti - cd28 antibody has been shown to activate some common events downstream of the tcr, including slp-76 and vav phosphorylation, all tcr - mediated pathways are not induced (43). Moreover, initially, it was assumed that zap-70 did not function in superagonist stimulation because zap-70 did not appear to be phosphorylated after stimulation in primary rat cells (44). However, although a more recent study also did not observe zap-70 phosphorylation, the same group found that overexpression of a dominant negative zap-70 construct resulted in inhibition of the anti - cd28-induced il-2 production, thus suggesting an important yet unappreciated role for zap-70 in this signaling process (43). Therefore, given the controversial role for zap-70 signaling in this signaling cascade and the importance of understanding how superagonists function, we decided to utilize our inhibitor system to dissect the role of this kinase in superagonist stimulation . Previous researchers had not observed zap-70 phosphorylation following cd28 superagonist stimulation, but the stimulating cd28 an28.1/5d10 antibody induced detectable zap-70 tyr phosphorylation after a 1-min stimulation of jurkat t cells, albeit at a substantially lower level than anti - tcr - stimulated cells (fig . Interestingly, as has been reported previously, the same cells induced substantial plc1 tyr phosphorylation, with delayed kinetics when compared with tcr stimulation . Therefore, the presence of zap-70 phosphorylation in these cells suggests that zap-70 could play a role in the signaling cascade . To explore this idea further, we first used p116 to see if the cells could be activated by anc28.1 stimulation . 8a demonstrates that superagonist stimulation induces perk, so we decided to monitor cd69 as a readout for anc28.1-mediated ras activation, which has been reported previously to be induced in superagonist - treated cells (44). Although zap-70 cells clearly up - regulated cd69 after overnight stimulation with soluble anc28.1 at 1 g / ml, p116 failed to induce surface cd69 expression (fig . A, jurkat cells were stimulated for 1 or 5 min with either anti - tcr antibody or a final concentration of 1, 5, or 10 g / ml anc28.1/5d10 antibody at 37 c . Cells were lysed in 2 concentrated sds sample buffer and analyzed by immunoblotting for plc1 ptyr, zap-70 ptyr, perk, and tubulin . B, facs analysis of cd69 expression after stimulating zap-70 or p116 for 16 h with anc28.1 antibody (1 g / ml) or pma (25 ng / ml). The histograms are labeled as follows: unstimulated (dotted line); anc28.1 (solid line); pma (filled). C, cd69 analysis of zap-70 and zap-70 after anc28.1 antibody (1 g / ml) stimulation with or without 5 m 3-mb - pp1 . The histograms are labeled as follows: unstimulated (dotted line); anc28.1 + vehicle (filled); anc28.1 + 3-mb - pp1 (solid line). 7c, except cells were stimulated with anc28.1 antibody (1 g / ml) for 5 min with or without 5 m 3-mb - pp1 . A, jurkat cells were stimulated for 1 or 5 min with either anti - tcr antibody or a final concentration of 1, 5, or 10 g / ml anc28.1/5d10 antibody at 37 c . Cells were lysed in 2 concentrated sds sample buffer and analyzed by immunoblotting for plc1 ptyr, zap-70 ptyr, perk, and tubulin . B, facs analysis of cd69 expression after stimulating zap-70 or p116 for 16 h with anc28.1 antibody (1 g / ml) or pma (25 ng / ml). The histograms are labeled as follows: unstimulated (dotted line); anc28.1 (solid line); pma (filled). C, cd69 analysis of zap-70 and zap-70 after anc28.1 antibody (1 g / ml) stimulation with or without 5 m 3-mb - pp1 . The histograms are labeled as follows: unstimulated (dotted line); anc28.1 + vehicle (filled); anc28.1 + 3-mb - pp1 (solid line). 7c, except cells were stimulated with anc28.1 antibody (1 g / ml) for 5 min with or without 5 m 3-mb - pp1 . The induction of zap-70 phosphorylation in jurkat and the failure of p116 to be activated by anc28.1 clearly suggest that stimulating cd28 antibody treatment requires zap-70 . However, previous research by dennehy et al . (43) suggests that only tonic signaling via zap-70 might be required to prime the system . This hypothesis cannot be tested in p116 cells, which do not express zap-70 and thus would not exhibit zap-70-dependent tonic signals . The best way to test this hypothesis is with a small molecule inhibitor; therefore, we treated our zap-70 cells with 3-mb - pp1 concurrently with 1 g / ml anc28.1 antibody . This treatment inhibited cd69 up - regulation and perk phosphorylation in two analog - sensitive lines but had no effect on zap-70 cells (fig . Thus, the use of 3-mb - pp1 and the zap-70-expressing cells demonstrates the requirement of zap-70 in cd28 superagonist - mediated signaling as well as the utility of having an inhibitor system to study the role of zap-70 . We successfully generated two zap-70 alleles that retain kinase activity but are sensitive to inhibition by the pp1 analog 3-mb - pp1 . In both 293 and p116 cells, zap-70 reconstitutes zap-70 functions but is inhibitable by the addition of 3-mb - pp1 in a dose - dependent fashion . Failure to initiate or maintain calcium mobilization was associated with and a likely consequence of the inhibition of phosphorylation of the key zap-70 adaptor targets, lat and slp-76 . Without the phosphorylation of those important adaptor molecules, plc1 cannot be recruited to the membrane, phosphorylated, or activated properly to generate the ip3 required for [ca]i increase . The physiological relevance of this approach was, in part, verified by the ability of 3-mb - pp1 to inhibit il-2 production in superantigen - stimulated zap-70 cells . Interestingly, although il-2 production was inhibited, it was relatively insensitive to lower concentrations of 3-mb - pp1 as compared with other readouts such as calcium flux . This differential sensitivity in the various assays might be due to the difference in stimulations but also may be the result of the former being a more integrated response . Overall, we found that the half - maximal inhibitory concentration (ic50) varied among different readouts used throughout this study, although it was consistently within the range of 1 to 10 m . Such differences in the ic50 between different functional readouts have been reported for other kinases (45). Although phosphorylation of tyr on plc1 was not readily inducible nor were elevations of [ca]i detectable following 3-mb - pp1 treatment of zap-70 cells, we consistently observed evidence of erk phosphorylation in a percentage of these cells . The level of erk phosphorylation was delayed, reduced in magnitude, and not sustained when compared with zap-70 or untreated zap-70 cells . Nonetheless, this diminished response was observed reproducibly . It had been reported previously that zap-70-deficient cells, p116, are capable of activating erk, thus providing support for a zap-70-independent mechanism (40, 41). However, such zap-70-independent activation required high levels of tcr stimulation . It was also plausible to consider a possible plc1-independent mechanism, as grb2-sos have been reported to interact with the phosphorylated tcr chain (38) and also with phosphorylated lat (40). Thus, there could be separate zap-70- and plc1-independent mechanisms leading to the activation of the ras / mapk pathway . However, when we increased the dose of 3-mb - pp1 or, more importantly, transiently transfected dgk to convert the remaining diacylglycerol into phosphatidic acid, perk was no longer induced . It remains unclear why 3-mb - pp1 completely inhibited calcium mobilization but not erk phosphorylation . The experiments were performed under different conditions, with the cells being suspended in a larger volume in the ca assay than in the perk experiment . This larger volume increases the ratio of drug to cell even though similar concentrations of 3-mb - pp1 were used in the two different experiments . In addition, given the delay in erk phosphorylation in 3-mb - pp1-treated zap-70 cells, we hypothesized that a positive feedback loop over erk is strong enough to support transient erk phosphorylation in these cells . This positive feedback could involve the functional amplification loop of the two guanine nucleotide exchange factors, rasgrp and sos, that we have described previously (46). Thus, our studies herein support the notion that grb2-sos plays a less important role than rasgrp in tcr signaling leading to ras activation . Zap-70 not only has kinase function but also has been shown to serve as an adaptor for many proteins, including lck, vav, c - cbl, and crk, via its phosphorylated tyrosine residues . One of the outstanding questions has been: what downstream tcr signaling functions is zap-70 able to fulfill as an adaptor in the absence of its catalytic function? Following the addition of 3-mb - pp1 to zap-70 stable cell lines, we did not observe that any substantial tcr signaling functions were maintained . However, we did not determine whether the association of zap-70 with the other proteins was still inducible . Our laboratory had previously reported that the deletion of the region between the second sh2 domain and kinase domain of zap-70, termed interdomain b, which contains the key tyrosines that are known to interact with other proteins when phosphorylated, does not eliminate kinase activity (47). In addition, point mutations of tyrosines 315 and 319 to alanine or glutamic acid do not ablate kinase activity (1). The recently solved crystal structure of zap-70 supports a role for the interdomain b tyrosines in maintaining zap-70 in an inactive and closed conformation when unphosphorylated (35). Upon phosphorylation of zap-70 by lck thus, although biochemical evidence has shown that the zap-70 tyrosines promote binding with other important signaling molecules, these events are clearly not sufficient for substantial downstream signaling in the absence of zap-70 catalytic activity . Overall, these findings allow for the possibility that the role of zap-70 as an adapter might be significant only in the context of a catalytically active protein kinase . One of the benefits of using a small molecule inhibitor is to be able to study an enzymatic role in a process where having a genetically deficient system is undesirable . Although previous researchers did not observe zap-70 phosphorylation upon superagonist stimulation, we were able to detect induction of zap-70 phosphorylation, albeit weaker than after tcr antibody stimulation . Recently, a role for zap-70 has been implicated in superagonist stimulation, but it was hypothesized that only tonic signaling is required (43). Our data using 3-mb - pp1 and analog - sensitive zap-70 alleles suggest that zap-70 is actively required in this signaling cascade . Although the level of zap-70 phosphorylation is lower following stimulation with the cd28 superagonist, the induced signaling events appear to be zap-70-dependent, suggesting the presence of an amplification loop downstream of zap-70 . (43, 44) did not observe it in either rat primary t cells or jurkat cells . The stimulating cd28 antibody used in our current experiment was also different than the one used in those previous studies . Overall, these data support the general utility of this zap-70 inhibitor system and the general benefit of using multiple biological methods to dissect protein function ., broad expression of many of these enzymes makes them unappealing targets because inhibition of the kinase, when outside of the disease context, can lead to undesirable side effects . Zap-70 is an attractive target because it is expressed predominantly in t cells . In situations where overactive t cells are a substantial component of disease, such as in autoimmune diseases or transplantation, targeting zap-70 could provide a means to target the t cells while avoiding adverse effects on other cells . The system that we have generated for analyzing the effect of zap-70 inhibition will allow us to identify the utility of a zap-70 inhibitor in preclinical models in mice that we have recently reconstituted with the analogsensitive allele of zap-70 . Importantly, in preliminary studies, we have confirmed that peripheral t cells isolated ex vivo from the analog - sensitive transgenic animals are uniquely susceptible to inhibition by 3-mb - pp1, thus validating an in vivo system for our future studies . These studies together with crystal structures of the intact autoinhibited zap-70 protein and the activated isolated kinase domain may help in the development of a clinical zap-70 inhibitor (35, 48).
He was known to suffer from pxf and had undergone standard uneventful phacoemulsification with in - the - bag implantation of monofocal iol in both eyes . In the right eye, a single piece poly (methyl methacrylate) (pmma) iol (+ 19 d) and in the left eye, a single piece acrylic foldable iol (+ 19 d) were implanted . The surgeries were performed in the right and left eye at 9 and 8 years, respectively, before to this examination . All the previous dilated annual eye examinations had been normal, with no clinical evidence of iol subluxation . On clinical examination, his visual acuity was 20/60 in both the eyes . Slit lamp examination revealed anterior partial in - the - bag iol dislocation [figs . 1 and 2], capsular contraction and deformation of iol haptics . Intraocular pressure by goldmann applanation tonometer (gat) was 19 mmhg in both eyes . Pupils were dilated with 2.5% phenylephrine eye drops . Indirect ophthalmoscopy revealed normal ocular fundi . Constriction of pupils and iol repositioning was tried by instillation of 2.0% pilocarpine eye drops in the supine position, but the procedure was unsuccessful . The next day, iol along with the capsular bag were explanted surgically, [fig . 3] and secondary scleral suture fixation of single piece pmma iol (auro india) was done in the same sitting for the right eye . After 12 months of surgery, best corrected visual acuity was 20/30 in both eyes . The manifest refraction was 1.00 dc 180 and + 1.00 ds/1.50 dc 180 in the right and left eye, respectively . The eyes were quiet and iop was 19 mmhg (gat) in both eyes . Anteriorly dislocated in - the - bag acrylic foldable intraocular lens with (a) capsular contraction and (b) compressed haptic in - the - bag modified c loop poly (methyl methacrylate) intraocular lens with (a) ring of soemmering and (b) compressed haptic explanted intraocular lens with the capsular bag iol dislocation may occur in the immediate postoperative period and is mainly due to poor iol fixation . Other predisposing factors are prior vitrectomy surgery, trauma, high myopia, retinitis pigmentosa and connective tissue disorders such as marfan syndrome, homocystinuria, hyperlysinemia, scleroderma, weill the late dislocation of iol may occur anytime from 1 month to 8 years following surgery . Bilateral spontaneous in - the - bag iol dislocations were reported in the anterior or posterior chamber in two cases of gyrate atrophy, one case of retinitis pigmentosa, and in one patient with intermediate uveitis . A literature search revealed only one case report of bilateral spontaneous anterior partial in - the - bag iol dislocation in a patient with pxf . Various corrective surgical management approaches to dislocated iol including iol exchange or iol repositioning with scleral or iris fixation have been described . Apart from the more commonly used suture fixation, a sutureless, glued iol technique has also been described in a case of bilateral, anterior, in - the - bag subluxation of iol in a patient with retinitis pigmentosa . As all the mentioned surgical procedures are reported to be equally efficacious, the ultimate choice of surgery depends on the surgeons preference and experience . Bilateral anterior partial in - the - bag iol dislocation occurred spontaneously 9 years after cataract surgery . Iol haptics were found to be compressed either due to loss of biomechanical memory or due to contraction of the capsule . Except pseudophakia, during all previous eye examinations, no other signs indicative of future risk of iol dislocation were observed . The present method of management of iol exchange, limited anterior vitrectomy, and scleral suture fixation of iol was found to be a safe and effective surgical option in such a case . It is also observed that iol design and biomaterial had no role in the prevention of spontaneous dislocation of iol . We believe pxf, myopia, and capsular contraction together were responsible for spontaneous dislocation of the iols, and it is only the second such case report in literature . Such dislocations can be managed definitively by iol exchange and scleral suture fixation and limited anterior vitrectomy.
Pseudoaneurysm of the mitral - aortic fibrosa (pmaf) or intervalvular fibrosa is an uncommon condition, consisting of a pulsatile cavity in the mitral - aortic junction communicating with the left ventricular outflow tract (lvot). When intervalvular fibrosa pseudoaneurysm communicates to both the left ventricle (lv) and left atrium (la), eccentric jet flow from the anterior to posterior mitral annulus may occur during systole and mimic mitral regurgitation due to perforation in the anterior leaflet . A 24-year - old male was referred to our emergency room with a history of acute dyspnea of 2 days duration associated with orthopnea . At admission, he had nyha class iv dyspnea . He had no history of prolonged fever, cough, or chest pain . On physical examination, examination of the lungs and heart revealed extensive crackles, grade 4/6 pan systolic murmur at the apex and gallops . No skin lesions were noted and the extremities did not show any peripheral edema . There was no clinical evidence of acute rheumatic activity or infective endocarditis . The chest x - ray showed mild cardiomegaly with a cardiothoracic ratio of 0.55 with evidence of pulmonary edema . The erythrocyte sedimentation rate (esr) was 35 mm at 1 h. the c - reactive protein (crp) level was 2.2 mg / dl and antistreptolysin o (aslo) titer was normal . Two - dimensional transthoracic echocardiogram (tte) demonstrated a false aneurysm - like structure in the maif at the lvot [figure 1, video 1]. It also demonstrated the neck of pseudoaneurysm communicating with the lvot and also a defect in the aneurysmal wall communicating with the la . Color doppler examination showed a communication between the echo - free space and lvot and a turbulent flow through the defect in the aneurysmal wall into the la [figure 2, video 2] suggestive of supra - annular mitral regurgitation . The mitral, aortic valve, and tricuspid valves were normal and there were no vegetations over mitral or aortic valves and no annular abscess . The diagnosis of a pseudoaneurysm of the maif was confirmed using transesophageal echocardiography (tee). It demonstrated a large pseudoaneurysm of maif measuring 3.0 4.2 cm communicating with the lvot through a narrow neck [figure 3, video 3] and also rupture in its wall [figure 4, video 4] resulting in communication between the lvot and la causing a large shunt . Transthoracic echocardiography in a parasternal long - axis view showing an aneurismal sac posterior to aorta (arrow) transthoracic echocardiography in an apical four - chamber view showing an aneurismal sac in la with rupture (arrow) into la with color doppler showing a turbulent jet resulting in supra annular mr and valvular mr transesophageal echocardiography in a four - chamber view showing aneurismal sac with neck (arrow) communicating with the lvot transesophageal echocardiography in four chamber view showing aneurismal sac in maif with rupture (arrow) into la with color doppler showing a turbulent jet resulting in supra - annular mr maif is the junction between the left half of the non - coronary cusp and the adjacent third of the left coronary cusp of the aortic valve and the anterior mitral leaflet . Pseudoaneurysm of the maf is a rare but potentially life - threatening complication of aortic valve endocarditis, aortic valve surgery, or chest trauma . Aortic valve endocarditis is usually the main reason that predisposes the maif to perforate and form a pseudoaneurysm . Both direct extension of the infection from the aortic wall and the aortic jet striking the subaortic structures and anterior mitral leaflet may damage and infect the maif . The relatively avascular tissue of this region contributes to the extension of infection and subsequent distortion . Rupture of an maif pseudoaneurysm may give rise either to a pericardial tamponade, an eccentric jet of mitral regurgitation, or a direct communication between the lvot and the la . In some instances, the pseudoaneurysm remains intact and progressive enlargement may compress the coronary arteries causing symptomatic coronary obstruction. [68] these false aneurysms are prone to rupture, embolize, or even cause further destruction of the aortic or mitral valve apparatus . When the pseudoaneurysm communicates with the ascending aorta or lv or la; it may present as heart failure with a clinical picture similar to that of aortic or mitral regurgitation, respectively . Karalis and colleagues described 24 (44%) complications involving 55 consecutive cases of aortic endocarditis, including 8 abscesses and aneurysms in interfibrosa, 7 interfibrosa perforations into the adjacent la, and 9 anterior mitral aneurysms and perforations . However, in the present case, there was no evidence of the above - mentioned risk factors . Tee is known to be superior to tte in the visualization of valvular vegetations, abscesses, and other complications in patients with infective endocarditis . Tte may show the aneurysm located behind the aortic root by the occurrence of systolic expansion and diastolic collapse seen by echocardiography . This should be differentiated from a typical aortic ring abscess which does not show this phenomenon . Both tte and tee can detect a pseudoaneurysm of the maif with sensitivity rates of 43% and 90%, respectively . 3d echocardiography may provide excellent three - dimensional anatomical information and also help in guiding the appropriate surgical therapy . The recommended treatment for pmaf is surgery even in asymptomatic patients in order to prevent the development of complications . Resection and repair of the defect can be done with or without aortic valve replacement or homograft replacement of the aortic root . Surgery for this condition is technically complex since patients often have a background of prior surgery; hence the associated morbidity and mortality are not negligible . If the anatomy of the maif aneurysm is suitable even percutaneous device closure of the aneurysm can be an option . To the best of our knowledge, it is rare for maif aneurysm to develop in the absence of aortic valve disease, infective endocarditis, prosthetic aortic valve, or trauma . Maif aneurysm can rupture into la and can result in large lv to left atrial shunt . Both tte and tee are quite sensitive in identifying the relationship as well as complication resulting from rupture into surrounding structures, and also help in guiding the appropriate surgical therapy.
Mental, emotional, and autonomic functions of the brain arise from interactions between the nearly 100 billion neurons that comprise this organ in humans . On average, synapses are asymmetric, complex, and highly dynamic [1, 2]. The plasticity of synapses and dendritic spines, the morphological structures that are the loci of most excitatory synapses in the central nervous system (cns), are widely believed to underlie learning and memory and are frequently altered in neurodevelopmental and neurodegenerative diseases [3, 4]. Thus, understanding the development, dynamics, and elimination of synapses is crucial for human health . A dizzying array of signals coordinates these processes, and thus receptors are an integral component of the synaptic regulatory machinery [14]. Receptors also represent the most accessible point at which to manipulate these processes pharmacologically . G - protein coupled receptors (gpcrs) comprise a superfamily of approximately 800 members in humans, including many important drug targets . They exhibit a characteristic seven - transmembrane (7tm) core structure by which gpcrs interact with and activate a variety of heterotrimeric g - proteins, which in turn activate or repress intracellular signaling cascades . Adhesion - gpcrs are a gpcr subfamily with 33 members in humans that are characterized by an extended n - terminal extracellular segment connected to the core gpcr structure by a distinctive gpcr autoproteolysis - inducing (gain) domain, which is present in all adhesion - gpcrs except gpr123 [8, 9]. The n - terminal segments of most adhesion - gpcrs contain multiple domains capable of binding to other cells or the extracellular matrix [810]. These include at least 16 different types of domain, with multiple types frequently occurring within the same protein; domains include cadherin - like repeats, thrombospondin - like repeats, rhamnose - binding lectin domains, and calnexin domains . Adhesion - gpcrs can be divided into 9 subfamilies based on phylogenetic analysis of the gpcr moiety; members of the different subfamilies generally also have related complements of n - terminal adhesive domains [9, 10]. Gain domains mediate autoproteolytic cleavage of adhesion - gpcrs during translation in the er at a site within the gain domain called the gpcr proteolysis site (gps) [11, 12]. After cleavage, the n- and c - terminal fragments (ntfs, ctfs) of most adhesion - gpcrs remain noncovalently associated [9, 10]. However, this scenario is complicated . Some adhesion - gpcrs do not undergo autoproteolysis, and some that do may even swap ntfs with other adhesion - gpcrs resulting in hybrid adhesion - gpcrs [9, 13, 14]. It has been widely believed that the ntfs may repress the signaling mediated by ctfs, and that ligand binding relieves this inhibition, possibly by causing dissociation of the ntf from the ctf [8, 15]. Recently, a peptide agonist sequence named stachel was identified on the c - terminal side of the gps of adhesion - gpcrs . This sequence, which is specific for a given adhesion - gpcr, can activate g - protein dependent signaling through the adhesion - gpcr when it is unmasked by removal of the ntf or conformational changes in the protein (either of which is presumably ligand - induced). Identification of the gain domain and stachel sequence are both recent findings, illustrating a rapid advance in the knowledge of adhesion - gpcr biology after years lagging behind other gpcrs . Adhesion - gpcrs function in various tissues throughout organisms [8, 9], but an important driving force of recent rapid advances in adhesion - gpcr biology has been the discovery that adhesion - gpcrs regulate the development and function of many aspects of the nervous system . These include migration of neuronal precursors, axon guidance, myelination of axons, vascularization of the brain, and synapse formation and function [8, 9]. In this brief review, we highlight the roles of the brain - specific angiogenesis inhibitor (bai) subfamily of adhesion - gpcrs at neuronal synapses . Adhesion - gpcr nomenclature arose over a long period of time and in a nonsystemic manner . Thus, the members of the bai subfamily, bai13, would now be named adgrb13 . This new nomenclature is not yet in standard use, and we will use the traditional names for adhesion - gpcrs, noting the new designations of adhesion - gpcrs we discuss . For general information on adhesion - gpcr function bai1, bai2, and bai3 (adgrb13) comprise a subfamily of adhesion - gpcrs that are highly expressed in the brain [9, 17]. Bais are large proteins, approximately 200 kda in size, with each possessing a long n - terminal region containing multiple adhesive thrombospondin type 1 repeats (tsrs), a hormone - binding domain, and the autoproteolysis - inducing gain domain (figure 1). Bais also contain an extended intracellular region c - terminal to the conserved 7tm gpcr domain that terminates in a pdz - binding motif, qtev (gln - thr - glu - val). Bai1 contains an additional tsr (five in total), an integrin - binding rgd (arg - gly - asp) motif, and a c - terminal proline - rich region not present in the other two bai family members (figure 1). Bais are widely expressed in postnatal and adult brain, with bai1 and bai2 mrna levels peaking at postnatal day 10 (p10), while the level of bai3 mrna is highest 1 day after birth . Bai1 protein is present in neurons, glia, and macrophages, with particularly high expression in cortical and hippocampal pyramidal neurons [2226]. Less is known about the cellular distribution of bai2 and bai3 proteins, although bai3 is abundant in cerebellar purkinje cells [2729]. In neurons, bai1 and bai3 are both enriched in the postsynaptic density (psd), suggesting a role for these proteins in synapse development and/or function [25, 30, 31]. Like most adhesion - gpcrs, bais possess a gain domain, but their ability to undergo autoproteolytic cleavage appears to be cell - type specific and not required for proper surface trafficking . For instance, while bai1 is cleaved at the gps site in mouse brain and human malignant glioma cells [11, 3234], uncleaved full - length bai1 is also clearly present in hippocampal and cortical neurons . Cleavage of the bai1 gain domain generates a secreted 120 kda fragment called vasculostatin-120 (vstat120), which is capable of inhibiting angiogenesis and tumor formation [32, 33]. Bai1 is also cleaved at a second site n - terminal to the gain domain by matrix metalloproteinase 14 (mmp-14). This cleavage event generates a 40 kda fragment called vasculostatin-40 (vstat40), which also has antiangiogenic activity . The antiangiogenic effects of vstat120 and vstat40 are primarily mediated by the tsrs, which bind to the scavenger receptor cd36 and induce proapoptotic signaling . While proteolytic cleavage of bai proteins is thought to both modulate the function of the full - length receptors and release their ntfs, which can exert their own physiological effects, more work needs to be done to understand how cleavage is regulated and what precise consequences it has on bai function . Research in the last decade has revealed a number of important roles for bai family members in diverse cellular processes [17, 36]. As indicated above, bai proteins can function as potent inhibitors of angiogenesis and tumor progression . Bai1 expressed in macrophages has also been shown to bind to phosphatidylserine (ps) and lipopolysaccharide (lps) and mediate the engulfment of apoptotic cells and gram - negative bacteria, respectively [24, 37]. Bai1 promotes engulfment in response to ps or lps binding by activating the associated elmo / dock180 signaling module, which in turn activates the small gtpase rac1 and induces rac1-dependent actin cytoskeletal remodeling required for internalization of apoptotic cells or bacteria [24, 37]. The ability to bai1 to bind to ps is also important for myoblast fusion, and loss of bai1 results in a reduction in myofiber size and impaired muscle regeneration in mice . The tsrs on the n - terminus of bai family members are essential for their capacity to regulate these diverse cellular processes, and therefore proteolysis of the bai extracellular domain may dramatically alter bai function . Despite the recent advances in our understanding of bai function, until recently, little was known about the roles of bai adhesion - gpcrs in neurons . Over the last few years, bais have emerged as important regulators of synaptogenesis and synaptic plasticity . Below, we consider the synaptic functions of each of the bai family members in turn . Bai1 is enriched in, though not exclusively localized to, the psd in dendritic spines in hippocampal neurons; this has been shown by biochemical fractionation and immunocytochemistry in rat hippocampal neurons and mouse brains [25, 31]. This enrichment indicated that bai1 might play a role in synaptic formation or function, and this problem was attacked in two different ways . In both cases, our approach was to acutely knock down bai1 both in vitro using cultured rat hippocampal neurons and in vivo using in utero electroporation of shrnas directed against bai1 . In both systems, we found that bai1 plays a key role in dendritic spine formation . Knockdown of bai1 in cultured primary hippocampal neurons resulted in a loss of spine and synapse density with a shift of remaining spines to an immature elongated morphology . In vivo knockdown also resulted in a dramatic loss of spine density and a shift toward less mature spines in the somatosensory and the cingulate cortices . Bai1's prospinogenic and prosynaptogenic activities are mediated through its interactions with the cell polarity complex tiam1/par3 through its c - terminal pdz - binding motif (figure 2). Tiam1 is an activator of the small gtpase rac1, which directs the actin cytoskeletal remodeling that drives spine and synapse development . Tiam1 couples rac1-dependent spine and synapse formation to extracellular signals, including glutamate (via nmda receptors), ephrin - b (via ephb receptors), and bdnf (via trkb receptors). Bai1 anchors the tiam1/par3 complex to dendritic spines where localized rac1 activation promotes the formation of dendritic spines and subsequent excitatory synaptogenesis . Of note, although other rac1 activators such as elmo / dock180 bind to bai1, rac1 activation leading to spinogenesis requires only tiam1, as bai1 mutants lacking the tiam1/par3-interacting motif cannot rescue the knockdown phenotype, whereas mutants that do not interact with elmo / dock180 can . Consistent with these results, knockout mouse studies recently revealed a requirement for bai1 in spatial learning and synaptic plasticity . Bai1-null mice have severe deficits in both hippocampus - dependent spatial learning and memory along with enhanced long - term potentiation (ltp) and impaired long - term depression (ltd). An interesting result arising from this study was the discovery that bai1 contributes to proper synapse formation through its ability to stabilize the expression of the postsynaptic scaffold protein psd95 . It was determined that bai1 binds to and inhibits the e3 ubiquitin ligase mdm2, thereby preventing the psd95 degradation that was responsible for the spatial learning and plasticity phenotypes observed in bai1-null mice (figure 2). Although both of these studies agreed that bai1 plays a role in synapse function, there were important differences in the results . Our results using shrnas against bai1 led to stark and obvious loss of spines, while the results with the bai1-null mice showed no difference in spine density . There are obvious differences in the techniques used that could have given rise to these differences, and we will return to this issue below . Bai1's c - terminal pdz - binding motif also interacts with a variety of other synaptic molecules . Proteomic analysis reveals that the c - terminal segment of bai1 can bind to pdz - domain - containing proteins such as sap97 (dlg1), densin-180, magi-1/bap1, magi-2, and magi-3 . However, the exact functions of the majority of these interactions are not well understood . One potentially interesting bai1-binding protein is the insulin receptor substrate 53 (irsp53), which binds to a proline - rich region in bai1's intracellular c - terminal segment and is also enriched in the psd [43, 44]. Since irsp53 is itself a downstream effector of rac1 and cdc42 and a regulator of dendrite spine morphogenesis, future studies that explore the effects of irsp53-bai1 interactions could elucidate key mechanisms of spinogenesis and synaptogenesis . Irsp53's potential role in autism spectrum disorder (asd) makes this an even more interesting interaction to investigate . Like bai1 roles for bai2 in neurogenesis and synaptogenesis have been suggested but not well established experimentally . Bai2-deficient mice were found to display increased resistance to social defeat stress and reduced immobility in the tail suspension test, two behavioral assays that assess depressive behavior in rodents . Bai2-deficient mice were also shown to exhibit increased neurogenesis in the dentate gyrus of the hippocampus, where bai2 is highly expressed [47, 48]. These two observations are likely related since enhanced adult neurogenesis has been shown to positively correlate with resistance to depression . It is also consistent with reports that bai2 suppresses the expression of vascular endothelial growth factor (vegf), as vegf stimulates adult neurogenesis in the dentate gyrus . Loss of bai2 could therefore increase vegf levels, resulting in enhanced neurogenesis and increased resistance to stress . This idea will need to be further investigated . Furthermore, since stress and depression are known to induce synapse loss, while antidepressants promote synaptogenesis, in future studies it will be interesting to investigate the possible roles of bai2 at synapses . Biochemical fractionation studies have revealed that like bai1, bai3 localizes to excitatory synapses in the brain [30, 53]. Furthermore, overexpression studies examining the localization of bai3 in transfected hippocampal neurons have shown that it is highly enriched in spines where it colocalizes with the postsynaptic marker psd95 . Indeed, recently bai3 was shown to regulate excitatory synapse connectivity and formation in the mouse cerebellum [28, 29] (figure 2). Knockdown of bai3 using lentivirus - delivered shrna in p7 pups induced clear deficits in connectivity between cerebellar climbing fibers and their target purkinje cells and between parallel fibers and purkinje cells by p21 . Dendritic spine density and vglut1-positive synaptic contacts were both decreased in purkinje cells with reduced bai3 levels . Similarly, mice lacking bai3 specifically in purkinje cells show a significant decrease in the number of vglut2-positive puncta in the cerebellum . Bai3's role at climbing fiber synapses is mediated through its interactions with a class of secreted complement proteins known as the c1q - like complement (c1ql) family . C1ql proteins are broadly expressed in the brain with different spatial and temporal expression patterns shown by family members c1ql14 . In particular, c1ql1 is highly expressed during the first 2 postnatal weeks in various neuronal populations, particularly in the hippocampus, cerebral cortex, and cerebellum . Transient c1ql1 secretion in the cerebellum promotes purkinje cell spinogenesis, and the effect of modulating c1ql1 expression on purkinje cell spinogenesis depends on the expression levels of bai3 . Critically, the c1ql1-bai3 interaction promotes developmental synapse refinement and triggers elimination of surplus climbing fiber synapses, helping to select and maintain a single winning climbing fiber . Bai3 expression in purkinje cells is required for this process, and the climbing fiber is the source of c1ql . Moreover, continued expression of bai3 is necessary for maintenance of climbing fiber synapses, and adult mice lacking c1ql, which possess excess climbing fiber synapses per purkinje cell, eliminate these extra synapses when c1ql is introduced into the animals . C1ql1 interacts with bai3 through the n - terminal cub domain, which is unique to bai3 . Bai3 also interacts with another c1ql family member, c1ql3, through its tsrs . Incubating cultured hippocampal neurons with c1ql3 was shown to decrease excitatory synaptic density, and this effect was reversed by adding the isolated tsrs of bai3 to the culture . This result suggests a role for bai3/c1ql3 in hippocampal synapse development akin to the bai3/c1ql1-mediated pruning function in the cerebellum described above . It is not known if bai3 also plays an earlier role in promoting synapse formation in the hippocampus . Further, since the tsrs in bai3 are present in all bais, it is possible that c1ql3 also interacts with bai1 and bai2, but this remains to be investigated . Bai3's role in synapse elimination during cerebellar development could shed some light on the differences observed in the shrna - transfected versus bai1-null mice described above . If proper spine formation requires a competition to sort out the winning synapse, expression profiles of relevant proteins in participating neurons might contribute to the resolution of this competition . In the neurons in which bai1 was removed via shrna, only the transfected cells had a deficit in bai1, and they represented a small fraction (<5%) of the total population . If they were in competition with bai1-expressing neurons for the establishment of synapses, and bai1 promotes winning the competition, then the bai1 knockdown neurons would be at a decided disadvantage relative to the vast majority of neurons expressing normal levels of bai1 . This state of affairs would hold for both the cultured neurons and the in vivo preparations . On the other hand, the neurons examined in the bai1 null mice existed on a background of bai1 null neurons . Therefore, the unmarked neurons would not have an advantage in preserving synapses and this may explain why no loss of dendritic spines was observed . Such argument by analogy can only go so far, and compensation by other bai family members could also be a factor, but this hypothesis warrants further investigation . In addition to the roles that bais play in synaptogenesis and synaptic function, there is evidence that additional adhesion - gpcrs function in these roles . Latrophilins are an adhesion - gpcr subfamily comprised of 3 members latrophilins 13 (lphn13 or adgrl13) and eltd1 (adgrl4) in humans and represent one of only two subfamilies conserved in invertebrates . Latrophilins were identified as receptors for the black widow spider toxin -latrotoxin, which causes a massive ca - mediated exocytosis of neurotransmitter - containing vesicles from the presynaptic side of the synapses . Lphn1 and lphn3 are largely restricted to the brain, while lphn2 is expressed in many tissues . In addition to their gain domains, lphns contain a hormone receptor motif, an olfactomedin - like domain, and a rhamnose - binding lectin domain in their ntfs . Lphn1 is thought to mediate its effects on synapse formation via interactions with teneurin-2/lasso [57, 58], neurexin-1/2, and fibronectin leucine - rich transmembrane proteins (flrts). Presynaptic lphn1 binds to teneurin-2 via its lectin domain with nanomolar affinity in a manner regulated by alternate splicing of lphn1 [57, 58]. The lphn1/teneurin interaction leads to presynaptic ca increases, and disruption of the interaction using the teneurin - binding segment of the lphn1 ntf decreases both excitatory and inhibitory synapse density in rat hippocampal neurons . Lphn1's interaction with neurexins also has nanomolar affinity and is regulated by alternate splicing of neurexins but is largely mediated by lphn1's olfactomedin domain . This interaction is especially intriguing because neurexins and their canonical binding partners, neuroligins, form trans - synaptic complexes and are strongly implicated in asd . It is not yet known what function the lphn1/neurexin interaction serves at synapses, but given the known roles of both proteins, it is likely to be of high interest . Lphn3 has received increased attention of late due to a strong emerging correlation with attention deficit / hyperactivity disorder (adhd) in humans [61, 62]. Lphn3 binds to flrt-3 via its olfactomedin domain and to teneurin-1 via its olfactomedin and lectin domains [63, 64]. Presynaptic lphn3 interacts with postsynaptic flrt-3 to promote synapse formation in hippocampal neurons and in cortical synapses from layers 2/3 to layer 5 [63, 64]. Interestingly, flrt-3 and teneurins vary in their distributions throughout the layered structure of the cortex, suggesting that lphn3 could serve different functions in different regions of the brain by interacting with distinct ligands . In short, lphns are implicated in both presynaptic function and in directing synapse formation by forming complexes with transmembrane ligands in neuronal membranes . The celsr adhesion - gpcr subfamily is characterized by the presence of atypical cadherin repeats, calcium - binding egf - like domains, laminin g domains, and a hormone receptor motif in their ntfs in addition to the gain domain [9, 10]. Like latrophilins, this subfamily is conserved in invertebrates, with flamingo in drosophila melanogaster, fmi-1/2 in caenorhabditis elegans, and celsr13 (adgrc13) in humans [9, 10]. Adhesion - gpcrs of the flamingo / celsr subfamily function in many aspects of nervous system development, including neural tube closure, axon guidance, and the formation of dendritic arbors [9, 6568]. These effects are mediated through the now classical interaction of these proteins with the cellular planar cell polarity (pcp) machinery, as well as camp- and ca - dependent mechanisms [9, 65, 66, 68]. Synaptic defects are observed when expression of celsr - subfamily adhesion - gpcrs is altered or repressed, but it is difficult to determine whether these are direct effects on synaptic formation and/or maintenance, or whether they arise secondarily from malformation of axons and dendrites . Loss of flamingo leads to formation of ectopic neuromuscular junctions, or synapses between axons and muscle, in drosophila . It also leads to malformed en passant synapses in this system, though these synapses are functional . Further, aging animals lacking flamingo exhibit a decrease in neuromuscular junctions, though this appears to be an effect of axonal degeneration . Numerous questions remain to be answered in order to determine the specific roles of celsr subfamily adhesion - gpcrs in synaptic formation and function . Finally, very large gpcr 1 (vlgr1 or adgrv1) has been implicated in the formation of cochlear synapses, though its specific role remains unclear . Identification of adhesion - gpcrs involved in synaptic formation and function as well as elucidation of the mechanisms and signals that underlie these roles are important challenges for both adhesion - gpcr and synaptic biology . Given the important roles that bai adhesion - gpcrs play in promoting synapse development and plasticity and inhibiting angiogenesis and tumor formation, it is not surprising that they have been implicated in a number of human diseases . For instance, single nucleotide polymorphisms (snps) and copy number variations in the human bai3 gene have been associated with schizophrenia [7173], bipolar disorder, and drug addiction, brain disorders characterized by synapse abnormalities . Furthermore, bai3 expression is affected by lithium treatment, which is often used to treat patients with bipolar disorder and schizophrenia [74, 76]. The human bai1 gene is also located in a hot spot for de novo germline mutations in patients with autism, and bai1 expression is upregulated in mouse models of rett and mecp2 duplication syndromes . Conversely, bai1 expression is downregulated in glioblastoma and is inversely correlated with neovascularization in colorectal and lung cancers . The growing evidence that bais play critical roles in human disease suggests that they may make good therapeutic targets in the future . Gpcrs are generally considered to be the most successful therapeutic targets for a broad spectrum of diseases . Indeed, greater than 50% of the current therapeutic agents on the market target these proteins [79, 80]. Greater insight into the regulation and function of bais could therefore facilitate the development of novel therapies for the treatment of brain disorders and cancer . After years of relative obscurity, there have been rapid recent advances in understanding the biology of bais and other adhesion - gpcrs . These molecules are intriguing because they tend to have multiple ligand binding domains that suggest that they are signal integrators, recognize large, complex substrates, and/or detect coincidences . The complexities added by ntf swapping, signaling by both gpcr - dependent and -independent modes, splice variants, and potential formation of higher level complexes are only beginning to be understood in a functional context . These complexities lend themselves to neuronal and synaptic function, given the role that these cells and structures play in storing and processing information . Bais in particular are demonstrating key roles in synaptic function, though they play other roles in and out of the brain as well . A full appreciation of bai function will require the identification of all bai ligands, complete elucidation of bai expression patterns and localization, identification of all binding partners and modes of signaling, and dynamic measurements of these properties . These are exciting challenges that hold great promise for increasing our understanding of synaptic function, as well as treating synaptic dysfunction.
Cancer is a huge burden of our societies with an overall worldwide incidence of 182,3 cases per 100.000 inhabitants and an overall mortality of 102,4/100.000 according to the international agency for research on cancer of the world health organization (estimated age - standardized incidence and mortality rates (asr) for both sexes). Highest incidence rates are reported for breast, colorectal and cervical cancer in women and lung and prostate cancer in men . Current treatment options comprise of surgery, chemotherapy or radiation plus more recently introduced targeted therapies . Targeted therapies aim to specifically address malignantly transformed cells while sparing healthy tissues . Thus, receptors, which are important during embryonic development and readopted by cancer cells, belong to the most promising targets . One of the most prominent molecules of that kind is the human epidermal growth factor receptor-2 (her-2). Her-2 is a receptor tyrosine kinase, mediating signals for cell proliferation, cell mobility and survival . In the absence of a known ligand her-2 is very important during embryonic development, e.g. It plays a role in ductal morphogenesis of the mammary gland, but it is almost not expressed on adult tissue, except the heart . On the contrary, her-2 is overexpressed in breast, ovarian, gastric, colorectal, pancreatic, and endometrial cancers . Another closely related receptor tyrosine - kinase is the epidermal growth factor receptor (egfr). Its overexpression is associated with head and neck squamous cell carcinoma (hnscc), non - small - cell lung cancer (nsclc), colorectal cancer (crc), breast and pancreatic cancer, but also with certain types of brain cancer . In contrast to her-2, egfr senses the epidermal growth factor (egf) and other important growth signals, such as transforming growth factor- (tgf-) or amphiregulin [11 - 14]. Egfr is physiologically required for promoting cell proliferation and dna repair, but can also lead to tumor growth, progression, and evasion of apoptosis via the activation of plc--pkc, ras - raf - mek, pi-3k - akt - mtor and jak2-stat3 pathways . Overall, egfr and her-2 together with her-3 and her-4 belong to the erbb - family, which derives its name from the homology to the erythroblastic leukemia viral oncogene protein (v - erb - b,). Currently two forms of targeted therapies against egfr and her-2 are in clinical use: i) blocking the intracellular receptor tyrosine kinase with small molecules and ii) attacking the extracellular domains of the receptor with monoclonal antibodies . Small molecules targeting egfr comprise erlotinib (tarceva, roche) and gefitinib (iressa, astrazeneca) plus the dual kinase inhibitors lapatinib (tykerb, glaxosmithkline) and afatinib (gilotrif, bhringer ingelheim), the latter inhibiting her-2 as well (, see table 1). Especially the reversible inhibitors gefitinib, being fda - approved in may 2003 and erlotinib, with fda - approval in november 2004, are successfully applied in non - small - cell lung cancer . Although gefitinib was recalled from that indication in the us, it is still widely used in japan, where patients display a higher rate of egfr - mutations in nsclc, and also received marketing authorization in the european union in 2009 . Moreover, erlotinib is approved for the treatment of advanced pancreatic cancer and several next generation irreversible egfr - tyrosine kinase inhibitors, like canertinib, are under investigation for their efficacy in breast, colorectal, lung, pancreatic, renal, head and neck, gynecologic and prostate cancer . The most prominent tyrosine kinase inhibitor (tki) for her-2 is lapatinib (tykerb, glaxosmithkline), the above mentioned reversible dual inhibitor of her-2 and egfr, which was fda - approved in march 2007 for the treatment of advanced breast cancer . Also in this case, irreversible inhibitors, like neratinib or again canertinib are widely investigated . In contrast to small molecules that intracellularly interfere signaling via blocking the kinase activity, monoclonal antibodies directed against egfr and her-2 aim to extracellularly inhibit ligand binding or dimerization of these receptors, respectively . For targeting egfr, two monoclonal antibodies are currently in clinical use, cetuximab (erbitux, merck kgaa), which was fda - approved in february 2004 and panitumumab (vectibix, amgen), which received fda - approval in september 2006 (, table 1). In particular cetuximab, a human - murine chimeric igg1 antibody has become an indispensable cornerstone in the treatment of advanced - stage metastatic crc and advanced hnscc . Cetuximab finds its epitope within the ligand - binding site of egfr (extracellular domain iii) and can thus block binding of growth signals . Panitumumab works mechanistically similar; it can also prevent egf - binding via sterical hindrance, but on a different epitope on domain iii, though partially overlapping . Panitumumab is successfully applied in the treatment of metastatic colorectal cancer . For all above mentioned therapeutics, wild type (wt) kirsten rat sarcoma viral oncogene homolog (kras)-status of the patient is of uttermost importance, as it could be demonstrated, that acquired kras mutations lead to resistance against egfr targeting . As kras is a downstream effector - protein in the egfr - signaling pathway, mutations that lead to constitutive activation of kras counteract growth signal inhibition of all egfr targeting drugs . Therefore kras - status is meanwhile routinely determined for every human patient before any egfr - specific treatment is initiated . Also in case of her-2 targeting, two monoclonal antibody therapies are fda - approved: trastuzumab (herceptin, roche) and pertuzumab (perjeta, genentech). Especially trastuzumab, being fda approved in september 1998, proved to be highly successful for the treatment of metastatic breast cancer and has later received importance also for treatment of metastatic gastric cancer and tumors of the gastroesophageal junction (gej,). Trastuzumab has been so successful in breast cancer therapy, that very recently, in february 2013, also a drug - conjugated trastuzumab derivate, trastuzumabemtansine (t - dm1, kadcyla, roche) was approved by the fda for treatment of advanced breast cancer, fuelling the emerging field of antibody - drug conjugates . The other her-2 targeting antibody, pertuzumab received fda - approval in june 2012 and is also applied for treatment of metastatic breast cancer, as specified in table 1 . Trastuzumab and pertuzumab target different epitopes on her-2: trastuzumab binds to subdomain iv on the extracellular domain (ecd) of her-2, whereas pertuzumab targets subdomain ii . As her-2 has no endogenous ligand, the mechanisms of action of trastuzumab and pertuzumab differ from those of the mentioned egfr - targeting immunoglobulins . Trastuzumab sterically prevents the formation of her-2 homodimers or highly active heterodimers with other erbb - family members, mainly her-3 and egfr (. Moreover, upon trastuzumab binding, her-2 gets endocytosed and shedding of the receptor is inhibited, which otherwise would lead to an actively - signaling p95-remnant on the cancer cell surface . Pertuzumab, on the other hand, binds more distant from the cell membrane, is more efficient in preventing heterodimer formation and in contrast to trastuzumab, which inhibits ligand - independent dimerization, pertuzumab especially inhibits ligand - induced her-2 heterodimers . This different mode of action prompted researchers to investigate a combination therapy of both antibodies in preclinical models, resulting in more complete her-2 blockade, and efficacy in cases where cancer had progressed after trastuzumab monotherapy . Also in clinical settings, this combination therapy proved to be highly effective and peaked in june 2012 in the first fda approval of a dual her-2 targeting regimen for metastatic breast cancer . The mechanism of action of monoclonal antibodies, however, cannot be confined to their growth signal inhibitory capacity only; as all mentioned monoclonal antibodies feature fully functional human constant regions, thus they are also able to attract immune effector cells to the site of the tumor and trigger immune cell mediated cancer cell death . Among the attracted immune cells are monocytes and macrophages dominant, which are known for their tumoricidic potential in mediating antibody - dependent cell - mediated cytotoxicity (adcc) via insertion of granzyme b and caspase enzymes . Moreover, our group could demonstrate that monocytes are also able to mediate high levels of antibody - dependent cell - mediated phagocytosis (adcp) upon trastuzumab treatment . Other attracted cell types expressing fc - receptors for antibody binding, are nk - cells and neutrophilic granulocytes, which have been shown to bear high tumoricidic potential with regard to adcc . Moreover, professional antigen - presenting cells, that also express fcreceptors such as dendritic or langerhans cells can be attracted and can induce activation of tumor - reactive t - cells upon facilitated antigen uptake and presentation . This mechanism has been demonstrated to lead to tumor regression in a xenograft model of cetuximab treatment in concert with reconstituted immune cells . Upon a closer look, all mentioned egfr or her-2 targeting antibodies belong to the igg class of immunoglobulins, with cetuximab, trastuzumab and pertuzumab being igg1 antibodies and panitumumab, being an igg2 . In fact, all currently fda - approved monoclonal antibodies for therapy of cancer are gamma - immunoglobulins . This is indeed astonishing as the human immunoglobulin repertoire consists of 5 different classes: iga, igd, ige, igg and igm . Based on their different constant domains, all of them bear distinct physiological properties with respect to distribution, tissue penetration and function, such as complement - activation or adcc and adcp mediation: igm, the primary antibody response against pathogens is highly active in opsonization, which leads to pathogen clearance by phagocytic cells . Iga protects body surfaces, such as the respiratory, gastrointestinal or genitourinary tract and is abundantly found in secrets like tear fluid or saliva, where it exerts neutralizing functions . The function of iga in mother s milk is of special interest as it protects the infant against pathogens in the mother s environment, which are also of high risk to the child . Igg is the most abundant antibody isotype in the bloodstream, as it constitutes 70% of all serum immunoglobulins . Igg antibodies, which can be further subdivided in igg1 - 4 in humans, exert systemic immune - protection by binding to bacteria, viruses and fungi with high affinity . In general, igg1 and igg3 antibodies are mainly produced in response to protein antigens, whereas igg2 and igg4 antibodies react against pathogens with polysaccharide capsules, like streptococcus pneumoniae . The biological role of igd, however, remains still enigmatic; discovered late (in 1965) in the serum of a myeloma patient, it was long neglected, because it is only cleaved in minor amounts . Membrane - bound igd on the surface of b - cells though, was found to regulate b - cell activation . Despite thorough investigation in recent years, the physiological and pathophysiological role of igd is still unclear; however, an interesting aspect is, that igd was found to bind to certain bacterial proteins with relatively high affinity, but not via its antigen binding - site, as rather through sugar residues on its constant region . Moreover, it has recently been shown, that circulating igd can activate antimicrobial, proinflammatory and b cell - stimulating effects in basophilic granulocytes . Ige, finally, is the most recently discovered immunoglobulin subclass, as it was only first described in 1966 by teruko and kimishige ishizaka as a novel immunoglobulin in the serum of an atopic individual . At the same time, bennich and johannsson could purify a paraprotein from serum of a myeloma patient, which they termed ignd . They could soon link this novel ignd to asthma and it turned out to be the same protein as the group from japan had found . On a meeting in lausanne in february 1968, finally, under the guidance of the who immunoglobuline reference laboratory, it was decided to designate this novel immunoglobuline ige . Ige plays an important role in defense against parasites and is a key molecule in the pathophysiology of allergic diseases such as atopic dermatitis, asthma, food allergy or anaphylaxis . Upon crosslinking of ige - antibodies, bound to fcri - receptors on the surface of mast cells or basophils, ige is mostly known for its detrimental role in allergy, but several studies have for long pointed towards a natural tumor surveillance function of this antibody isotype . Interestingly, large epidemiologic studies could reveal an inverse association between the history of atopic diseases and cancer . In 2005, turner et al . Published a study enrolling 1.1 million us - american adults with self - reported, physician - diagnosed asthma or hay fever, who had no cancer at baseline and were followed up for 18 years . In this population, a relative - risk reduction for all cancer mortality could be observed [relative risk (rr) = 0.88; 95% confidence interval (ci) 0.83 - 0.93]. However, in a separate analysis of never - smokers, this effect still persisted, but was not significant anymore . In a following literature analysis of studies from the medline database from 1966 to august 2005, the same group described strong inverse associations for pancreatic cancer and glioma, whereas lung cancer was positively associated with asthma . However, methodical issues to these historical studies with regard to exposure assessment, confounding and bias were addressed by the authors . The most recent study investigating a possible association between ige and cancer was published in 2010: van hemelrijck et al . Reviewed 27 studies from pubmed and embase and surveyed a swedish cohort of 24.820 people, who underwent ige measurements . Here, the authors could show a weak inverse association in their cohort, and a pattern by cancer type in the meta - analysis of the historical studies . Another interesting observation was made when the anti - ige antibody omalizumab (xolair, novartis) underwent clinical trials and pooled phase i to iii data was evaluated . Omalizumab, which removes ige from the circulation, is currently approved for therapy of severe persistent asthma . In those mentioned phase i to iii trials with allergics undergoing omalizumab treatment, a slightly higher number of malignant neoplasms was observed in the anti - ige - treated group (20 of 4127=0.5% compared to 5 of 2236=0.2% in the control group). Malignancies that occurred in the treatment group comprised of breast, non - melanoma skin, prostate, melanoma and parotid cancer . Subsequently, busse et al . Analyzed 67 phase i to iv trials of omalizumab and could not confirm any possible association between omalizumab treatment and cancer in this extended study . Summarizing all epidemiologic observations one can only state, that a possible association between ige and cancer remains still unclear due to the lack of big prospective studies . They could isolate ige antibodies which were not only specific for a 50 kda pancreatic cancer antigen but were indeed able to mediate adcc of pancreatic cancer cells in vitro, pointing towards a benefitial role of ige - antibodies in defense against cancer . Pioneer studies with igg and ige antibodies of the same epitope specificity tested head - to - head revealed a higher potential of the ige in terms of cytotoxicity . The very first studies were performed with mov18igg and ige, antibodies that target the folate receptor (fr)-. Fr- (also known as folate - binding protein, lk26 trophoblastic antigen or gp38) is a glycosylphosphatidylinositol (gpi)-anchored membrane protein that binds folic acid and is regarded as a tumor - associated - antigen (taa) in gynecologic malignancies, due to its overexpression in more than 90% of epithelial ovarian cancers and in a subpopulation of uterine carcinomas . As folate is a necessary micronutrient of replicating cells, overexpression of fr- facilitates enhanced growth of cancer cells . For targeting of this receptor, gould et al could demonstrate in an ovarian cancer model, that mov18ige was able to mediate adcc of fr- expressing tumor cells in vitro and in vivo . In a mouse model using xenografted human fr- overexpressing cells, mice that received mov18ige treatment developed in the presence of human peripheral blood mononuclear cells (pbmcs) significantly smaller tumors than those treated with mov18igg . In a follow - up study, it could further be demonstrated, that also cytotoxic killing by monocytes can be efficiently triggered with ige . In a subsequent nude mouse study, where again fr- overexpressing tumors were grafted and pbmcs were reconstituted, the ige - treated group had shown monocytic infiltration of the tumor xenografts, which was still persistent after 3 weeks and led to significantly longer survival . Moreover, upon a closer look in an in vitro flow cytometric model, specific adcc of tumor cells, executed by monocytes upon mov18ige stimulation could be displayed . Finally phagocytosis of fr- positive tumor cells by monocytes armed with fr- specific ige could be displayed by fluorescence microscopy . Subsequently, we could demonstrate similar results for the her-2 system in close collaboration with prof . Karagiannis: upon generation of a recombinant trastuzumab - like ige, constituted of the same variable regions as original trastuzumab (being an igg1), it was shown in a flow cytometric assay, that the ige antibody is highly effective in mediating adcc of monocytes against her-2 overexpressing cells . Interestingly, in this model, the ige antibody mediated high levels of adcc but only background adcp, whereas the picture was completely opposite for the igg, which mediated killing of tumor cells almost exclusively via adcp . This could be a first hint towards distinct mechanisms of tumor cell killing mediated by different immunoglobulin classes; however, this still has to be confirmed in more extensive experiments and has to be investigated also for other cancer types . Apart from a possibly higher potential for mediating adcc, ige - based immunotherapies of cancer could have other beneficial effects: first, ige antibodies have a uniquely high affinity to their receptors on immune cells (ka~ 1010/m for fcri and ka~ 108 - 109/m for the cd23 trimer complex), which significantly exceeds the affinities of igg1 - 4 to their high - affinity receptor fcri . Thus, due to its rapid binding to fc-receptors on cells, ige is quickly removed from the circulation, which is advantageous in terms of side - effects because of the short duration of the compound in the bloodstream . Moreover, potential ige - immunotherapies would be effectively distributed to tumor tissues, as ige antibodies bound to fc-receptors on e.g. Mast cells can use those cells as shuttle systems to penetrate malignancies and as mast cells are tissue - resident immune cells, this transport would be highly efficient . Consistently, we would like to quote the review problems of delivery of monoclonal antibodies by reilly et al ., who wrote that the clinical success of monoclonal antibody - based cancer diagnosis and therapy depends, however, on solving a number of pharmacokinetic delivery problems . These include: (i) slow elimination of monoclonal antibodies from the blood and poor vascular permeability; (ii) low and heterogeneous tumour uptake, and state that those two substantial challenges of anti - tumor immunotherapy could be simply addressed by using ige . Other possible advantages include the high sensitivity of ige - effector cells to activation by antigens and the speed and amplitude of the response, which can most impressively be seen during allergic and anaphylactic reactions, typically beginning within minutes upon allergen exposure . That is at the same time also the biggest concern of using ige - based immunotherapies against cancer: recombinant ige, applied intravenously, always bears the risk of anaphylactic reactions; therefore, careful selection of the target epitope is of uttermost importance in this regard . During an anaphylactic reaction, preformed ige, that is bound to fc-receptors on the surface of mast cells or basophilic granulocytes is cross - linked by allergens, which induces release of stored granules, containing vasoactive amines (e.g. : histamine) or lipid mediators (e.g. Prostaglandin d2, platelet - activating factor, or leukotrienes). This rapid release can lead within minutes to fatal symptoms like asphyxiation from laryngeal swelling, circulatory collapse from hypotensive shock, cardiac arrest, or respiratory failure because of bronchoconstriction . In order to prevent such effects, the target structure for designing passive immunotherapies with ige - antibodies should not be expected to be cross - linking, which means, that the epitope should be monovalent andit should not circulate in the blood, or if, it should only circulate in a monomeric form . It should not circulate in the blood, or if, it should only circulate in a monomeric form . These requirements are fulfilled for the mentioned anti - fr- antibody mov18ige, but also for the anti - her-2 antibody trastuzumab - like ige . For both antibodies it could be demonstrated, that monomeric target molecules do not trigger mediator release of mast cells, which were preloaded with their specific ige - antibodies, respectively . Furthermore, rudman et al . Could demonstrate, that although serum levels of fr- were increased in ovarian cancer patients (up to 40 ng / ml) compared to healthy controls (mean=1,73; sd=3,45), basophilic granulocytes loaded with mov18ige were not significantly activated upon incubation with fr- even at a concentration of 300 ng / ml . On the other hand, both antibodies were shown to mediate mast cell degranulation upon incubation with tumor cells, displaying high numbers of target molecules in a repetitive manner on their surface . Here, cross - linking of fc-receptors is highly efficient, and therefore local anaphylaxis at the tumor site could be expected, which would again be beneficial, as it results in initiation of a strong immune response . As mast cells also store tumorinhibiting agents in their granules, e.g. Tumor necrosis factor (tnf)-, this degranulation could also result in direct tumor cell killing . Moreover, also other cells involved in anaphylactic reactions, such as eosinophils, have been shown to execute tumoricidic functions, e.g. Via secretion of granzyme a or eosinophilic peroxidase . Another big challenge of current immunotherapies with igg antibodies is that not all human fc-receptors are immune - activating, but one among them, fcriib is -inhibiting . Therefore, the tumoricidic effects of igg - based immunotherapies also depend on the net ratio of binding to activating and inhibiting receptors . As it has recently been shown for igg4, a subclass that shows relatively high binding affinity to fcriib, this antibody is not able to trigger immune cell - mediated tumor cell killing in vitro, despite being taa - specific . Moreover it was demonstrated, that igg4 antibodies significantly impaired the killing potential of igg1 antibodies of the same specificity in vitro and in vivo . Strategies to overcome this limitation include modification of the posttranslational glycosylation of the igg - constant regions heavy chains, as these sugar residues have been identified to be of high relevance for distinct binding affinities to different fc - receptors . For ige on the other hand, there are no inhibitory receptors, so again this isotype could contribute to overcome a current challenge of immunotherapies of cancer . However the fc-receptor - biology differs considerably between humans and mice, as the high affinity ige receptor fcri is only expressed on mouse basophils and mast cells, whereas it has been described in humans on mast cells, basophils, eosinophils, monocytes, langerhans and dendritic cells . This is a huge limitation of current mouse models in displaying all mentioned in vivo benefits and risks of ige - based immunotherapy of cancer and the great benefit of using a comparative oncology approach . Although human and veterinary medicine share the same goals and aims, namely to treat patients and promote health, currently both of them are distinct sciences with distinct studies taught on separate universities . Research is presently going on in one or the other, but there is little crosstalk between the two specialties . The concept of comparative medicine, however, aims to study naturally occurring diseases across species to improve both human and veterinary medicine . There is in fact no explicit reason for a strict separation of studies for humans or other mammals, as many pathophysiological processes have been shown to be similar and highly comparable, or as the german pathologist rudolf virchow stated: between animal and human medicine the object is different but the experience obtained constitutes the basis of all medicine . This view, which is also in line with the wider concepts of one medicine, or one health, bringing together aspects of health of humans, animals and also their environment is not really novel . Browsing through the history of medicine, this approach appears at many points, starting with hippocrates and plato in ancient greece . Even further back in time, physicians in ancient india were trained to treat humans and animals, especially cattle, elephants and horses and in ancient china, medical treatment for horses was highly elaborated as well . Also in europe and north america human and veterinary medicine were closely interconnected for a long period of time, peaking in the 18th, 19th and early 20th century . Many important findings were made by comparative observation and experimentation during that time, for instance edward jenner (1749 - 1823) noted that milkers who had been in contact with cowpox - infected cows did not develop smallpox (variola minor) but only the milder cowpox . He also observed that this protection could be mediated by inoculation of a small amount of pus from cowpox blisters, a method he called vaccination because of the latin word for cow, vacca . Similarly, louis pasteur (1822 - 1895) worked on cholera in humans and chicken, robert koch (1843 - 1910) researched on human and bovine tuberculosis, and the mentioned rudolf virchow (1821 - 1902) worked on trichinella infections in humans and pigs . Besides virchow, the most important proponent of comparative medicine was sir william osler, a canadian physician (1849 - 1919), who studied, worked and taught in london, berlin, vienna, toronto and montreal, and was one of the four founding professors of johns hopkins hospital in baltimore . Osler lectured at mcgill university for medical students as well as for students from the veterinary medical college with emphasis on comparative topics . His research in this field concentrated on infectious diseases in dogs, pigs and cattle and his deep interest is documented in many editorials of the journal of comparative medicine and surgery . Recent developments are no longer dependent on distinguished individuals, but comprise the establishment of special departments for comparative medicine within universities, such as at stanford, yale or at the university of california, davis . Whereas comparative medicine focused mainly on infectious diseases in previous centuries, modern approaches tend to tackle another big burden of our societies, cancer, via the principle of comparative oncology . The comparative oncology approach aims to speed up drug development simultaneously for human and animal cancer patients via clinical trials in pet patients, primarily cats and dogs . Although murine models have been proven to be highly effective with regards for understanding basic principles of malignant transformation, cancer signal transduction pathways or drug resistance formation, these models often poorly mimic human cancer for drug testing . For many compounds, the translation of a safe and efficacious agent in mice into an actual drug fails, due to the poor presentation of key features of human cancer in murine tumor models, such as genomic instability, long latency periods or the lack of intra - tumor heterogeneity . Moreover, the concept of toxicity studies, which are conducted in healthy animals, followed immediately by phase i and phase ii trials in humans, often leaves many important questions unanswered, before treating a relatively high number of human patients . On the other hand, also more and more extensive studies in animals cannot be the solution, as it would increase the ethical dilemma that potential benefits for humans stand against the costs sustained by animals . Thus, legislators and regulatory bodies state in their directives on drug development that the 3rs should be applied on animal experiments, which are: replacement, reduction, and refinement [143,144]. In line with these concepts, clinical studies in animal patients, which suffer from spontaneously developed tumors, would allow the investigation of drug effects on malignancies that developed naturally within intact immune systems, in the context of their original tumor microenvironment in pet animals that share similar environmental factors as their owners, for instance pollution . Such trials in veterinary patients could replace many preclinical experiments, refine our models and ultimately reduce animal experiments . The information obtained from these studies would be highly relevant and valuable, while the treated veterinary patients would be provided with cutting edge research simultaneously . Studies in this field of comparative oncology with treated dog patients, led to the advancement of surgical techniques, like limb sparing for sarcoma patients, elucidating hyperthermia or evaluating novel delivery strategies, like inhalation of cytokines or chemotherapies . (cotc), a multicenter initiative of twenty comparative oncology centers throughout the usa . Founded and centrally managed by the national cancer institute of the national institutes of health (nih), 11 clinical trials have been conducted so far, ranging from all fields of cancer therapy attempts like evaluation of rgd targeted delivery of phage expressing tnf - alpha to tumor bearing dogs (cotc001, closed trial) via preclinical comparison of three indenoisoquinolines candidates in tumor bearing dogs (cotc007b, open trial) to evaluation of the mtor inhibitor rapamycin in dogs with metastatic osteosarcoma one of the most successful example of a recent clinical comparative oncology trial was published in 2009, in which the tyrosine kinase inhibitor toceranib phosphate (palladia, su11654, pfizer), targeting kit, vascular endothelial growth factor receptor 2 (vegfr2) and platelet derived growth factor receptor - beta (pdgfr) was tested in dog cancer patients with recurrent mast cell tumors . This large clinical phase iii trial could demonstrate significant effects of toceranib phosphate with regard to overall response rates, median duration of objective responses and time to tumor progression, which has finally led to the approval of toceranib phosphate for mast cell tumors in dogs and to the approval of sunitinib (sutent, su11248, pfizer), a similar compound, for therapy of human renal cell cancer and gastrointestinal stromal tumors (gist,). Also for evaluation of ige - based immunotherapies of cancer, trials in dog cancer patients would be highly valuable, due to the fact that dogs, in contrast to mice, share important principles of the ige - biology with humans, underlined by the clinical observation that dogs also suffer from ige - mediated diseases, such as atopic dermatitis or food allergies . Thus, the introduction of passive immunotherapy in veterinary clinical oncology would be highly valuable to elucidate the full potential of ige - based immunotherapy of cancer . We contributed to this strategy by our recent molecular characterization of canine egfr and the generation of a recombinant canine ige of the exact cetuximab specificity [155, 156].
In 2013, approximately 9% of americans aged 12 years and older had used one or more illicit drugs in the past month, and 23% reported binge drinking in the past month . Much of this behavior may fall outside of diagnostic guidelines for dependent or disordered use, instead qualifying as risky or harmful use, which may go undetected and/or be asymptomatic . Screening, brief intervention, and referral to treatment (sbirt) is a method of integrating behavioral health and primary care to identify patients substance use and provide appropriately matched services . The sbirt typically begins with a preliminary screening (prescreening), and patients who prescreen positive are asked to complete a full screening . Patients whose full screenings suggest risky, harmful, or dependent levels of use are provided with services based on the outcomes of the screening tool(s) and the care provider s clinical judgment . These services include brief interventions, which increasingly utilize motivational interviewing as a mechanism to promote change, brief treatment, and referral to treatment . The substance abuse and mental health services administration has funded sbirt implementation in both inpatient and outpatient settings . Multiple studies have supported the effectiveness of sbirt for alcohol in outpatient primary care settings, although recent studies have disputed support for the widespread dissemination of sbirt to target illicit drug use, contradicting prior research . The joint commission published standards for national hospital inpatient quality in 2012 that included screening and brief intervention (sbi) for alcohol as an optional core measure . This standard matches literature supporting the efficacy and importance of sbirt for alcohol in inpatient care . Compared to these findings, comparatively little research has addressed sbirt s efficacy for drug use in inpatient hospital settings . There is still a need to examine the prevalence of alcohol and other drug use in inpatient and outpatient medical settings . Research currently suggests that individuals who receive inpatient care for substance use disorders typically experience mental health - related comorbidities and are more likely than other individuals to require recurrent acute care . Studies utilizing full screening tools generally have identified a higher prevalence and/or severity of alcohol consumption in inpatient care than in outpatient clinics . However, by nature of when they were conducted, such studies did not have access to the currently recommend sbirt prescreening questions . As previously described, sbirt now incorporates validated prescreening using single questions for alcohol and drugs to assess eligibility for longer screening tools . Little research has assessed differences in prescreening outcomes between inpatient and outpatient settings using those questions, especially within the same geographic region, which may influence substance use behavior . Another factor that may influence prescreening outcomes is the fact that sbirt programs often use the same prescreening tools but different administration methods (self - administered or interview based). Some individuals view substance use as socially unacceptable or immoral and may be less likely to answer interview - based questions accurately, though the alternative, self - administered forms, may produce less sensitive and specific data . However, linking substance use prevention and treatment to primary medical care treatment a principal component of sbirt may serve to reduce stigma and mitigate some of those effects . Finally, comorbidity between alcohol use, other drug use, and depression is well - established and should be included in models assessing prescreening outcomes . There is therefore also a need to assess the impact that prescreening administration methodology might have on prescreening outcomes . Given those gaps in knowledge, this article describes an analysis of secondary data from a safety - net health services organization that integrated sbirt for both alcohol and drug use into 10 outpatient primary care clinics and a centrally located inpatient general hospital . Of those 11 total sites, 4 utilized interview - based prescreening and 7 utilized self - administered prescreening . This study tested whether administration method (self - administered vs interview) and setting (inpatient versus outpatient) predicted prescreening outcomes in a large sample of primary care patients . Then, among patients who prescreened positive, it tested whether full screening scores differed by administration method and setting . Reporting these results represents an important step in evolving and directing future research on sbirt in primary care . Data for this study were collected between september 8th, 2014, and february 18th, 2015, from a safety - net health services organization in indianapolis, indiana . Data from outpatient settings were collected from 10 different primary care centers from the same county, while data collected from the inpatient setting were from patients admitted to the adult medicine wards at a centrally located hospital . To protect patient confidentiality, the inclusion criterion for this study was all adults (age 18 +) who attended any of the study sites during the study period and who also were eligible for their annual sbirt prescreen, meaning they had not been prescreened within a year or were a new patient within the system . In total, data for 14 447 unique patients were collected and organized in preparing this report, distributed among 10 outpatient clinics (n = 13 315) and 1 inpatient hospital (n = 1130). The overall sample was 63% female (n = 9076) and 37% male (n = 5363) and was 18% hispanic or latino (n = 2540), 41% african american (n = 5960), 31% white (n = 4442), and 2.8% individuals of other races (n = 416). = 3,629) and/or ethnicity (9.5%, n = 1358). As a standard of care, all patients attending the clinics and hospital wards during the study period completed the single - question prescreens for alcohol and illicit drugs / prescription drugs for nonmedical reasons and the patient health questionnaire-2 (phq-2) screening tool for depression . The questions were either completed by self - administration on a paper form in the waiting room (6 outpatient clinics, n = 9459 and the hospital site, n = 1130) or interview with a medical assistant in a triage exam room (4 outpatient clinics, n = 4986). Since this process was part of an implementation project, administration type was not randomly assigned to sites; rather, as part of the overall organizational planning process, clinic managers at each site met with a technical assistance team and selected the method that they felt best met the needs of their clinic s staff and patients . In clinics using self - administered prescreening forms, a spanish translation was available, and in clinics using the oral interview format, a spanish - speaking medical assistant was available . Patients who prescreened positive for alcohol or drugs met with a social worker or mental health counselor to complete full screening instruments matched to the positive prescreening result(s). The alcohol use disorders identification test (audit) was used for alcohol and the drug abuse screening test (dast) was used for drugs . Additional information regarding the structure of data collection for this sbirt program previously has been published . In order to determine whether prescreening outcomes for alcohol and drugs were independent from setting and administration, we created 2 binary logistic regression models to compute adjusted odds ratios with prescreening results set as the outcome variable and setting, administration method, gender, depression (phq-2), and use of other substances set as predictor variables . Race / ethnicity were not included as predictor variables because the substantial amount of missing data was nonrandom . Then, in order to determine whether the mean screening scores among patients who completed the audit and/or dast were different between those attending outpatient and inpatient settings and those receiving different prescreening methods, the researchers used analysis of variance, adjusting post hoc comparison based on whether the assumption of equal variances was met (tukey hsd) or not (games - howell). Overall percentages of positive prescreens by setting and administration type are available in table 1 . In the model for alcohol (see table 2), self - administered prescreens were 2.4 times more likely to be positive than interview prescreens, and prescreens completed by inpatients were 1.4 times more likely to be positive than those by outpatients . Further, males were 2.2 times more likely to prescreen positive than females, patients prescreening positive for depression were 1.5 times more likely to prescreen positive for alcohol, and patients prescreening positive for drugs were 5.1 times more likely to prescreen positive for alcohol . In the model for drugs (see table 2), prescreens completed by inpatients were 2.6 times more likely to be positive as those by outpatients . Males were 2.0 times more likely to prescreen positive than females, patients prescreening positive for depression were 2.2 times more likely to prescreen positive for drugs, and patients prescreening positive for alcohol were 5.0 times more likely to prescreen positive for drugs . Self - administered and interview are both subsets of the outpatient category . Adjusted odds ratios of positive prescreening by setting and administration type . 1 = outpatient; 2 = inpatient . 1 = female; 2 = male . 1 = negative; 2 = positive . Finally, within the subsample of individuals who prescreened positive for alcohol and who completed the audit screening instrument (n = 1433), the mean audit score for patients in the outpatient setting with a self - administered prescreening was 7.3, the mean audit score for patients in the outpatient setting with an orally administered prescreening was 8.2, and the mean audit score for patients in the inpatient setting was 11.3 . The overall comparison of means was significant (f = 27.5, p <.001), with both outpatient screening scores being significantly lower than the inpatient score (see table 3). However, within the subsample of individuals who prescreened positive for other drugs and who completed the dast screening instrument (n = 782), the mean dast score for patients in the outpatient setting with a self - administered prescreening was 2.6, the mean dast score for patients in the outpatient setting with an orally administered prescreening was 2.6, and the mean dast score for patients in the inpatient setting was 2.2 . The overall comparison of means was nonsignificant (f = 2.8, p = .062; see table 3). Abbreviations: sa, self - administered; anova, analysis of variance; audit, alcohol use disorders identification test; dast, drug abuse screening test . This study found evidence suggesting that setting and administration method may be associated with prescreening outcomes in some cases . First, the higher number of positive prescreens among inpatients identified in this study was conceptually consistent with prior research . This study adds to existing knowledge by verifying such findings using the currently suggested sbirt prescreening questions while controlling for important confounding factors (gender, prescreening administration type, depression, and other drug use). However, it cannot be concluded from these data alone that patients are more willing to admit to alcohol use in one medical setting versus another . In addition, the finding that patients using a self - administered form prescreened positive for alcohol more often than those completing an interview is consistent with prior research . As before, this is one of the first studies to examine whether this would hold true when using currently recommended sbirt prescreening forms . Interpretation of this finding is limited in scope, though, as this retrospective study was unable to randomize clinics to prescreening administration types; although some variables were controlled for and although all of the clinics in the study were located within the same county, it is possible that the prevalence of alcohol use was higher in some patient populations than in others . This concern was somewhat attenuated, but not eliminated, by aggregating groups of clinics (4 and 6) together for analyses . Interestingly, patients using a self - administered form did not prescreen positive for other drugs more or less often than those completing an interview, suggesting a possible inconsistency in how patients prefer to respond to the alcohol and drug prescreening questions . Any work that further investigates this topic should attend closely to this discrepancy to see if it is replicated, and, if so, why . A randomized experiment testing the impact of medical setting and prescreening administration method on responses to the sbirt prescreening questions may be warranted based on these findings and might usefully inform organizations attempting to situate behavioral health resources within a health care organization . In addition, mean scores on the audit and dast were not affected by prescreening method among outpatients . However, significant pairwise differences were observed between outpatient and inpatient settings across mean screening audit scores . The mean scores in the outpatient were 7.3 (self - administered) and 8.2 (interview), where a score of 8 is considered to be the cutoff at which a patient likely will benefit from a brief intervention and/or begin to experience harms related to alcohol . On the other hand, the mean score in the inpatient setting was 11.1, well into the range of scores indicating the need for a brief intervention (8 - 15), affirming prior findings that alcohol use severity may be higher in inpatient settings . For the dast, the mean scores in the outpatient setting were both 2.6 and 2.6, where a score of 1 to 2 indicates a low degree of problems related to drug abuse, and a score of 3 is the cutoff for a moderate degree of problems . The mean score in the inpatient setting was 2.2, but no significant differences were observed . Interestingly, these findings suggest that, in outpatient settings, method of prescreening may not impact sbirt s ability to detect clinically significant problems using a full screening tool . However, this finding cannot be generalized to inpatient settings, where only self - administration was utilized . First, because this study was conducted within a single health care organization, it has limited generalizability to other health care organizations; however, we suggest some generalizability to other urban safety net hospitals and health systems . Second, literacy levels vary, and it is difficult to determine the effects that literacy levels may have had on the results of the self - administered screening tools, and in what direction, if any, that may have biased the results . Finally, this study was unable to include race and ethnicity as control variables in the regression models, so it is unclear how cultural differences may have impacted elicitation of patient responses, although the sample included in this study was both racially and ethnically diverse . This study examined differences in sbirt prescreening and screening results by prescreening administration method and medical setting using currently recommended sbirt processes . Prior work in these areas was strong but did not have access to more recent advances related to prescreening . This work concurred with many previous findings regarding substance use prevalence and severity while suggesting several new directions for research.
Hepatitis b virus (hbv) is a public health problem and a major cause of mortality and morbidity (1). Almost 30% of the world population is infected with hbv, and more than 600,000 people die each year from acute disease or chronic sequelae secondary to hbv infection . Hepatitis b surface antigen (hbsag) is the earliest marker of acute infection and is helpful for the diagnosis of hbv infection . The carrier rate varies from 0.1 to 20% for this marker in different communities (2, 3). The world health organization (who) recommends that all blood donations should be screened for evidence of infections, such as human immunodeficiency virus (hiv), hepatitis b and c, and syphilis . Information provided by 164 countries to the who global database on blood safety showed that worldwide, more than 92 million blood samples are donated annually . Of these, an estimated 1.6 million units are excluded due to the presence of infectious markers, including hbsag (4, 5). In the multiple countries where pre - transfusion screening of blood donations for hbsag is conducted systematically, and the high - risk groups are rejected from blood donation, the prevalence of hbv in blood donors is less than that in the general population; this results in underestimation of the extent of this issue . However, the risk of transmission still exists in several developing countries (3, 6). The eastern mediterranean regional office (emro) is a one of the six regional offices of the who around the world, consisting of 22 member states with a total population of 605 million individuals (7). It is estimated that about 4.3 million people are infected with hbv in this region (8). Unsafe blood transfusion and poor public health awareness are the major risk factors for hbv infection in this part of the world (1). The who has defined prevalences of <2%, 2 8%, and> 8% as low, intermediate, and high prevalences of hbv, respectively (2). In the emro and some middle eastern countries, low intermediate and high prevalence of hbv have been reported across all age groups (1, 9). Several studies conducted in the emro and middle eastern (e and m) countries have investigated the prevalence of hbsag positivity in blood donors and have reported different values . A study conducted on approximately 15 million iranian blood donors over 10 years showed that about 1% of blood donors were hbsag positive, and the prevalence declined over the study period (10). (2014) (11) over 6 years showed that only 0.5% of blood donors were hbsag positive in iran . A study performed on more than six million turkish blood donors reported that more than 4% of this population was hbsag positive, while the study by tigen et al . (2015) (12) showed an hbsag prevalence of 1.5% in turkish blood donors (13). The prevalence increases in the blood donors of djibouti (14) and pakistan (15), with rates up to 10% and 6%, respectively . There have been several single - center studies on the prevalence of hbsag positivity in blood donors from e and m countries, but they have reported different estimates . In addition, there are no comprehensive and reliable data with a large sample size on the prevalence of hbsag positivity in these regions . Therefore, the present study aimed to determine the prevalence of hbsag positivity in blood donors of e and m countries using a systematic review and meta - analysis of published cross - sectional studies during 2000 2015 . An electronic literature search through two medline and embase databases (pubmed and ovid), scopus, and google scholar was carried out for articles published from january 1, 2000, to august 31, 2015, using different combinations of the following keywords in titles and/or abstracts: prevalence, epidemiology, these keywords were combined with the names of the following e and m countries: afghanistan, bahrain, cyprus, djibouti, egypt, iran, iraq, jordan, kuwait, lebanon, libya, morocco, oman, pakistan, palestine, qatar, saudi arabia, somalia, sudan, syria, tunisia, turkey, united arab emirates, and yemen . Iranian databases, such as iranmedex, magiran, medlib, and the scientific information database, were also searched using relevant english and persian keywords . If the full text of articles was not available, emails were sent to authors . If the authors did not respond after 1 month, informative abstracts were used for data extraction . Published studies in english, persian, french, and arabic were considered if they met the following criteria: 1) the studies were cross - sectional and 2) had clearly declared information on the number of hbsag - positive cases among healthy blood donors from e and m countries . The exclusion criteria were as follows: 1) studies on blood donors who had positive hbsag, 2) studies reporting confusing data or probable errors, and 3) studies with fewer than 1,000 samples . Studies without any information on the country were retained for reviewing . Author name or journal title had no effect on the choice to exclude or include the articles . Data extraction was conducted separately by one researcher (m.b . ), and critical evaluation for quality assessment of the articles was performed by another researcher who was not involved in the literature search (s.m.a . ). A meeting was scheduled before the critical evaluation, and researchers were justified about the questions . After the critical evaluation, the selected articles were checked by both the authors . The selected and included citations were reviewed, and data were extracted to excel spreadsheets, which included the first author s name, year of publication, name of country, sample size, male percentage, and hbsag prevalence and its standard error (se). If there were other parameters reported besides se, such as standard deviation, confidence interval, and/or p value, a suitable modification was performed to calculate se . Se was estimated using the following formula: se = (p (1 p)/n) (p = prevalence, n = sample size). The se of hbsag prevalence in each study was estimated based on the binomial distribution formula . Cochran s q - test of heterogeneity was performed to detect homogeneity among the studies . Higgins and thompson s i2 was also applied due to the inherent limitations of cochran s q in detecting true heterogeneity . Depending on whether homogeneity or a large value of i2 was found, a fixed or random effects model, respectively, was used to aggregate data from studies and produce the pooled estimates, with the metan command . Results were provided as forest plots with description of the findings in the plots, and the point estimations and their 95% confidence intervals (cis) were computed accordingly . An electronic literature search through two medline and embase databases (pubmed and ovid), scopus, and google scholar was carried out for articles published from january 1, 2000, to august 31, 2015, using different combinations of the following keywords in titles and/or abstracts: prevalence, epidemiology, these keywords were combined with the names of the following e and m countries: afghanistan, bahrain, cyprus, djibouti, egypt, iran, iraq, jordan, kuwait, lebanon, libya, morocco, oman, pakistan, palestine, qatar, saudi arabia, somalia, sudan, syria, tunisia, turkey, united arab emirates, and yemen . Iranian databases, such as iranmedex, magiran, medlib, and the scientific information database, were also searched using relevant english and persian keywords . If the full text of articles was not available, emails were sent to authors . If the authors did not respond after 1 month, informative abstracts were used for data extraction . Published studies in english, persian, french, and arabic were considered if they met the following criteria: 1) the studies were cross - sectional and 2) had clearly declared information on the number of hbsag - positive cases among healthy blood donors from e and m countries . The exclusion criteria were as follows: 1) studies on blood donors who had positive hbsag, 2) studies reporting confusing data or probable errors, and 3) studies with fewer than 1,000 samples . Studies without any information on the country were retained for reviewing . Author name or journal title had no effect on the choice to exclude or include the articles . Data extraction was conducted separately by one researcher (m.b . ), and critical evaluation for quality assessment of the articles was performed by another researcher who was not involved in the literature search (s.m.a . ). A meeting was scheduled before the critical evaluation, and researchers were justified about the questions . The selected and included citations were reviewed, and data were extracted to excel spreadsheets, which included the first author s name, year of publication, name of country, sample size, male percentage, and hbsag prevalence and its standard error (se). If there were other parameters reported besides se, such as standard deviation, confidence interval, and/or p value, a suitable modification was performed to calculate se . Se was estimated using the following formula: se = (p (1 p)/n) (p = prevalence, n = sample size). The se of hbsag prevalence in each study was estimated based on the binomial distribution formula . Cochran s q - test of heterogeneity was performed to detect homogeneity among the studies . Higgins and thompson s i2 was also applied due to the inherent limitations of cochran s q in detecting true heterogeneity . Depending on whether homogeneity or a large value of i2 was found, a fixed or random effects model, respectively, was used to aggregate data from studies and produce the pooled estimates, with the metan command . Results were provided as forest plots with description of the findings in the plots, and the point estimations and their 95% confidence intervals (cis) were computed accordingly . Based on the data gathered from titles and abstracts, out of 4,125 citations, 86 of them were potentially related to hbsag prevalence in blood donors from e and m countries . All of the papers were carefully examined to avoid including duplicate papers; two studies with duplicate data from the same group were excluded (16, 17). We found three publications that were not available online, and despite contacting their authors or publishers, we could not obtain their abstracts (22 - 24). In addition, there were nine studies with sample sizes of less than 1,000 people from egypt, iran, pakistan, somalia, sudan, and yemen that we excluded from the meta - analysis to avoid the effect of lower sample size (27 - 35). However, because we had no information in our meta - analysis on the hbsag prevalence in somalia or sudan, we reviewed three studies that reported the hbsag prevalence in these two countries (33 - 35) (figure 1). Therefore, the remaining 66 studies, involving 28,947,262 blood donors and assessing hbsag prevalence in e and m countries, were considered for the meta - analysis . Out of these, 18 studies related to iran (10, 11, 36 - 51), 14 to pakistan (15, 52 - 64), 10 to turkey (12, 13, 65 - 72), 7 to egypt (73 - 79), 7 to saudi arabia (80 - 86), 2 to iraq (87, 88), and 2 to yemen (89, 90). There was one study each from other countries, which included cyprus (91), djibouti (14), jordan (92), kuwait (93), lebanon (94), and morocco (95). There were no data available for the following countries: afghanistan, bahrain, libya, oman, palestine, qatar, saudi arabia, syria, tunisia, and the united arab emirates . Pooled prevalence of hbsag in blood donors of all e and m countries was 2.03% (95% ci: 1.79 2.26). When these countries were divided into groups, the prevalence in the emro countries was 1.99% (95% ci: 1.84 2.14), and that in middle eastern countries was 1.62% (95% ci: 1.36 1.88; tables 1 - 3). Abbreviations: e, emro countries; e and m, all countries from the two regions; m, middle eastern countries; nr, not reported . Abbreviations: e, emro countries; e and m, all countries from the two regions; m, middle eastern countries; nr, not reported . Abbreviations: e, emro countries; e and m, all countries from the two regions; m, middle eastern countries; nr, not reported . Pooled estimate by random effects meta - analyses . The pooled hbsag prevalences in blood donors considered in more than one study were 1.58% (95% ci: 1.39 1.78) in egypt (figure 2), 0.58% (95% ci: 0.4 0.76) in iran (figure 3), 0.67% (95% ci: 0.61 0.73) in iraq, 2.84% (95% ci: 2.62 3.06) in pakistan (figure 4), 3.02% (95% ci: 2.2 3.84) in saudi arabia (figure 5), 1.68% (95% ci: 0.42 2.94) in turkey (figure 6), and 5.05% (95% ci: 4.64 5.44) in yemen . The prevalences in countries considered in only one published article were 3% (95% ci: 2.54 3.45) in cyprus, 10.4% (95% ci: 10.2 10.59) in djibouti, 1.72% (95% ci: 1.53 1.9) in jordan, 1.92% (95% ci: 1.68 2.15) in kuwait, 0.92% (95% ci: 0.77 1.07) in lebanon, and 0.95% (95% ci: 0.9 0.99) in morocco . One study from somalia showed that among 115 healthy blood donors, 22 (19.1%) were hbsag positive (35). Two other studies from sudan reported that among 400 and 260 male blood donors, 25 (6.25%) and 26 (10%) donors were positive for hbsag, respectively (33, 34). Pooled prevalence of hbsag in blood donors of all e and m countries was 2.03% (95% ci: 1.79 2.26). When these countries were divided into groups, the prevalence in the emro countries was 1.99% (95% ci: 1.84 2.14), and that in middle eastern countries was 1.62% (95% ci: 1.36 1.88; tables 1 - 3). Abbreviations: e, emro countries; e and m, all countries from the two regions; m, middle eastern countries; nr, not reported . Abbreviations: e, emro countries; e and m, all countries from the two regions; m, middle eastern countries; nr, not reported . Abbreviations: e, emro countries; e and m, all countries from the two regions; m, middle eastern countries; nr, not reported . Pooled estimate by random effects meta - analyses . The pooled hbsag prevalences in blood donors considered in more than one study were 1.58% (95% ci: 1.39 1.78) in egypt (figure 2), 0.58% (95% ci: 0.4 0.76) in iran (figure 3), 0.67% (95% ci: 0.61 0.73) in iraq, 2.84% (95% ci: 2.62 3.06) in pakistan (figure 4), 3.02% (95% ci: 2.2 3.84) in saudi arabia (figure 5), 1.68% (95% ci: 0.42 2.94) in turkey (figure 6), and 5.05% (95% ci: 4.64 5.44) in yemen . The prevalences in countries considered in only one published article were 3% (95% ci: 2.54 3.45) in cyprus, 10.4% (95% ci: 10.2 10.59) in djibouti, 1.72% (95% ci: 1.53 1.9) in jordan, 1.92% (95% ci: 1.68 2.15) in kuwait, 0.92% (95% ci: 0.77 1.07) in lebanon, and 0.95% (95% ci: 0.9 0.99) in morocco . One study from somalia showed that among 115 healthy blood donors, 22 (19.1%) were hbsag positive (35). Two other studies from sudan reported that among 400 and 260 male blood donors, 25 (6.25%) and 26 (10%) donors were positive for hbsag, respectively (33, 34). The present study showed that the pooled prevalence of hbsag in blood donors of e and m countries was 2.03% (95% ci: 1.79 2.26), which is considered an intermediate prevalence as defined by the who . The prevalence in iran was 0.58%, which was lower than that in other e and m countries . Based on the world bank list of middle - income countries, among the 13 e and m countries from which data were used in our meta - analysis, 10 countries are members of middle - income countries (96). The who has estimated the prevalence of hbv in blood donors of middle - income countries to be in the range of 0.19 to 2.33%, which is comparable with our results (6). The results of two individual studies on 7,277 and 3,292 blood donors in the democratic republic of the congo showed that 9.2% (97) and 3.7% (98) of blood donors were found to be positive for hbsag, respectively . In addition, the prevalences of hbsag in blood donors were 18.6% in nigeria (99), 12% in ghana (100), 10.1% in equatorial guinea (101), 4.7% in ethiopia (102), and 14.96% in burkina faso (103), which shows that the prevalence of hbsag in most of the african countries was higher than that observed in our meta - analysis of e and m countries . The prevalence of hbsag among blood donors of the who south - east asian regions showed a different pattern . The values were 1.09% (104), 1.27% (105), and 1.7% (106) in india; 1.4% in bangladesh (107); 0.53% in nepal (108); and 2.6% in thailand (109), which indicate that the range of available data in these regions is somewhat comparable with the prevalence in e and m countries . A systematic review in 24 european countries (110) showed that the hbsag prevalence in first - time blood donors was 0.74% . In addition, another study conducted on more than 1 million blood donors in the united states (111) showed that the prevalence was only 0.04%, indicating that the infection rates in europe and us were lower than that in our region . Overall, it seems that the discussed differences in hbsag prevalence in blood donors reflect a disparity in prevalence among the population who are suitable to donate blood, the types of donors (such as voluntary unpaid blood donors from lower risk groups), and the quality of blood processing (6). According to our results, the pooled estimate of hbsag infection was higher in other e and m countries than iran, which exhibited a prevalence of 0.58% in its blood donors, while the prevalence increased for yemen and djibouti, by up to 5.05% and 10.4%, respectively . This lower prevalence in iran can be attributed to the effectiveness of the screening system, the selection of eligible donors, and the high vaccination coverage for hbv in the first years of life (11, 112). In addition, low public awareness, particularly for the general population (113), unsafe services for blood donation (114), and a higher rate of hbsag infection in the general population (115) may contribute to the higher prevalence of hbsag in other e and m countries . The strength of the current study was that the search strategy was performed with high sensitivity with all related keywords to include most of the available published studies . In addition, the large sample size of this study was another strength; it included 28,947,262 blood donors from e and m countries . The first limitation of this study was that the quantity and quality of data varied by country . To decrease the effect of studies with low sample sizes on the estimated prevalence, we excluded studies with fewer than 1,000 samples . Another limitation of this study was that some databases, such as web of science, were not searched due to sanction limitations in iran . In addition, university research projects and students theses were not included in our search . Based on the who classification of hbv prevalence, the prevalence of hbsag in blood donors of e and m countries represented an intermediate level (2.03%), while the individual prevalence rates in each of the emro (1.99%) and middle eastern (1.62%) countries was low . The available data showed a wide disparity in the prevalence of hbsag among the e and m countries . The lowest prevalence of hbsag was seen for iranian blood donors, while the highest rate was observed in djiboutian blood donors.
The most common debilitating disorders affecting society at large are pain and depression, which are the most prevalent among neurological and psychiatric disorders . Depression and pain co - exist in almost 80% of patients (1) and are associated with impaired health - related quality of life, often contributing to high mortality (2,3). It has been observed that patients suffering from inflammatory and neuropathic pain are almost 5 times more prone to develop depression or anxiety disorder as compared to the general population (46). However, the majority of patients who suffer from comorbid depression and pain are not responsive to pharmacological treatments that address the pain or depression, making this comorbidity disorder a heavy burden on patients and society (7). These clinical observations on the association of pain and depression have been confirmed in several animal models of depression and chronic pain based on genetics, stress, lesion, and pharmacological manipulation that show altered nociceptive response (8,9). Considering the significance of the complex interaction between pain and depression and its societal impact, a better understanding of the molecular basis for this association is needed for developing more effective therapeutics . In ancient times, this depression - pain comorbidity was treated through the use of extracts of the cannabis sativa plant, commonly known now as marijuana . Use of marijuana for addressing pain due to various reasons has become a hot topic in terms of possible addiction, drug abuse as well as regulatory issues . Although historically, the use of marijuana dates back to over 2000 bc, the biological action of the main psychoactive ingredient of marijuana, -tetrahydrocannabinol (-thc) has only recently been identified . The biological receptor of -thc on the cell surface has recently been identified and described (10,11). Characterization of this receptor led to understanding of the mode of action of -thc that underlies its wide spectrum of pharmacological effects, which encompass euphoria, calmness, appetite stimulation, sensory alterations and analgesia (10,11). Identification of the first endogenous cannabinoid - like substance, anandamide, in pig brain reiterated the significance of the so - called cannabinoid receptor and its endogenous ligands in the control of a wide variety of biological activities (12). The name' anandamide', derived from sanskrit (' ananda' meaning bliss) is given to n - arachidonoylethanolamine, for its cannabinomimetic effects . Another endogenous cannabinomimetic compound known as 2-arachidonoylglycerol (2-ag) was identified (13,14). Of note, considering that these compounds are cannabinomimetic and endogenous, acting on the cannabinoid receptors, they are known as endocannabinoids . Besides anandamide and 2-ag, there are other endogenously produced molecules that also likely influence the function of cb receptors . These molecules include oleamide (15), o - arachidonoyl ethanolamine, also termed virodhamine (16), 2-ag ether or noladin ether (17), and n - arachidonoyldopamine (18). However, their physiological role is not clear and thus whether they are true endocannabinoids has yet to be ascertained . In addition to -thc, almost 80 other phytocannabinoids are found in the cannabis extracts, with a structure similar to that of thc . Of these, thc is the most studied and was shown to activate cannabinoid receptor type 1 (cb1) and cb2 and affect many pathophysiological processes, including anti - nociception (19). However, because of its cb1-mediated unwanted cns effects, the clinical utility of thc is limited (19). Subsequent studies revealed that another phytocannabinoid, cannabidiol, with very low affinity to bind to cb1 and cb2 receptors, exerts positive pharmacological effects, such as anti - anxiety, anti - epileptic, anti - bacterial, anti - inflammatory, anticancer and also anti - diabetic properties without any psychoactivity (20). Nabiximols, a cannabis extract containing thc and cannabidiol at a 1:1 ratio, has been approved for the treatment of neuropathic pain, spasticity associated with multiple sclerosis and intractable cancer pain (21). In addition to the natural cannabinoids, synthetic cannabinoids, such as dronabinol, and its analogue nabilone, have been developed to address various types of pain . For instance, dronabinol and nabilone are currently used for chemotherapy - associated emesis in canada and usa and nabilone is indicated for anorexia associated with aids - related weight loss (22). In addition, findings of a clinical trial showed the efficacy of nabilone in diabetic neuropathy (23). Another synthetic drug, an antagonist / inverse agonist of cb1 receptor, rimonabant, initially approved for obesity and smoking cessation, was found to have depressive effects and was subsequently withdrawn . Endocannabinoids are lipophilic molecules synthesized' on demand' from membrane phospholipids, and released immediately, without storage in vesicles . Anandamide is produced in a two - step process involving n - arachidonoylation of the membrane phospholipid, phosphatidylethanolamine, to form n - arachidonoyl phosphatidylethanolamine (nape) by a calcium - dependent n - acyltransferase, followed by hydrolysis by a nape - selective phospholipase d (nape - pld) to form n - arachidonoylethanolamine (anandamide) (24,25). Anandamide levels are regulated by its breakdown through the action of fatty acid amide hydrolase (faah) (26). 2-ag is synthesized in a two - step process, in which diacylglycerol (dag) is first produced by the plc from inositol phospholipids, followed by the hydrolysis of dag to 2-ag by plasma membrane - associated sn1-dag lipase (dagl) (14). Once formed, 2-ag levels are regulated by monoacylglycerol lipase (magl), which accounts for ~85% of the hydrolysis and by / hydrolase domain containing 6 (abhd6) and abhd12, which also hydrolyze 2-ag to arachidonic acid and glycerol (27). In addition to hydrolysis, 2-ag is acted on by cyclooxygenase-2 (28) and lipoxygenase (29), to form prostaglandin glyceryl esters and other related bioactive compounds (fig . Two subtypes of cannabinoid receptors, cb1 and cb2, have been cloned and characterized (11,30). Cb1 receptors are most abundant in the central nervous system (cns), whereas cb2 receptors are present mostly in peripheral tissues with immune functions, and most densely in the spleen (31). In the cns, cb1 receptors are distributed densely in motor and limbic regions, in areas involved in pain transmission and modulation (e.g., periaqueductal grey, rostral ventromedial medulla, and spinal cord dorsal horn), as well as in the periphery (32). In the synapses, the cb1 and cb2 receptors are g - protein coupled receptors of gi / go subtype, and mediate the inhibition of neurotransmitter release these cb receptors inhibit adenylate cyclase . Only cb1 receptor activation, but not that of cb2 receptors, causes blockage of voltage - dependent n- and p / q - type calcium channels through the activation of potassium channels and mitogen - activated protein kinase . Although cb2 receptors are mostly localized in immune cells and peripheral tissues, their presence has been observed in some subsets of neurons in brain and thus these receptors likely participate in the modulation of neurotransmission (33). Endocannabinoids also bind to other receptors including transient receptor potential vanilloid 1, peroxisome proliferator - activated receptors, gpr55, and gpr119 (3436) and this non - cb1/2 receptor activity of endocannabinoids accounts for the differential effects of certain cannabinoid agonists and pharmacological modulators of endocannabinoid tone . Following activation of their receptors, endocannabinoids are removed from the synaptic junction / extracellular space by a process of cellular uptake and then their hydrolysis . It has been suggested that the uptake of anandamide is probably mediated via a specific' endocannabinoid membrane transporter', which is yet to be identified (37,38). Anandamide is hydrolyzed in post - synaptic neurons by faah, thus terminating the anandamide action at the time of its synthesis, whereas 2-ag is hydrolyzed in pre - synaptic neurons by magl, following cb1 receptor activation . Metabolism of anandamide by lipoxygenase and cycloxygenase enzymes yields oxygenated products with activity on non - cannabinoid targets (39). Endocannabinoids are lipophilic molecules synthesized' on demand' from membrane phospholipids, and released immediately, without storage in vesicles . Anandamide is produced in a two - step process involving n - arachidonoylation of the membrane phospholipid, phosphatidylethanolamine, to form n - arachidonoyl phosphatidylethanolamine (nape) by a calcium - dependent n - acyltransferase, followed by hydrolysis by a nape - selective phospholipase d (nape - pld) to form n - arachidonoylethanolamine (anandamide) (24,25). Anandamide levels are regulated by its breakdown through the action of fatty acid amide hydrolase (faah) (26). 2-ag is synthesized in a two - step process, in which diacylglycerol (dag) is first produced by the plc from inositol phospholipids, followed by the hydrolysis of dag to 2-ag by plasma membrane - associated sn1-dag lipase (dagl) (14). Once formed, 2-ag levels are regulated by monoacylglycerol lipase (magl), which accounts for ~85% of the hydrolysis and by / hydrolase domain containing 6 (abhd6) and abhd12, which also hydrolyze 2-ag to arachidonic acid and glycerol (27). In addition to hydrolysis, 2-ag is acted on by cyclooxygenase-2 (28) and lipoxygenase (29), to form prostaglandin glyceryl esters and other related bioactive compounds (fig . Two subtypes of cannabinoid receptors, cb1 and cb2, have been cloned and characterized (11,30). Cb1 receptors are most abundant in the central nervous system (cns), whereas cb2 receptors are present mostly in peripheral tissues with immune functions, and most densely in the spleen (31). In the cns, cb1 receptors are distributed densely in motor and limbic regions, in areas involved in pain transmission and modulation (e.g., periaqueductal grey, rostral ventromedial medulla, and spinal cord dorsal horn), as well as in the periphery (32). In the synapses, the cb1 and cb2 receptors are g - protein coupled receptors of gi / go subtype, and mediate the inhibition of neurotransmitter release . These cb receptors inhibit adenylate cyclase . Only cb1 receptor activation, but not that of cb2 receptors, causes blockage of voltage - dependent n- and p / q - type calcium channels through the activation of potassium channels and mitogen - activated protein kinase . Although cb2 receptors are mostly localized in immune cells and peripheral tissues, their presence has been observed in some subsets of neurons in brain and thus these receptors likely participate in the modulation of neurotransmission (33). Endocannabinoids also bind to other receptors including transient receptor potential vanilloid 1, peroxisome proliferator - activated receptors, gpr55, and gpr119 (3436) and this non - cb1/2 receptor activity of endocannabinoids accounts for the differential effects of certain cannabinoid agonists and pharmacological modulators of endocannabinoid tone . Following activation of their receptors, endocannabinoids are removed from the synaptic junction / extracellular space by a process of cellular uptake and then their hydrolysis . It has been suggested that the uptake of anandamide is probably mediated via a specific' endocannabinoid membrane transporter', which is yet to be identified (37,38). Anandamide is hydrolyzed in post - synaptic neurons by faah, thus terminating the anandamide action at the time of its synthesis, whereas 2-ag is hydrolyzed in pre - synaptic neurons by magl, following cb1 receptor activation . Metabolism of anandamide by lipoxygenase and cycloxygenase enzymes yields oxygenated products with activity on non - cannabinoid targets (39). Pain is an integrative experience that involves physiological, emotional and cognitive aspects and this experience varies among individuals . Laboratory animals, on which most of basic pain research is conducted, cannot report pain and in animals, pain is generally monitored by differentiating between the subjective experience and nociception, the measurable neuronal events underlying the pain (?). Nociceptive pathways are triggered by the transduction of noxious stimuli, such as heat and mechanical injury, into neuronal action potentials by sensory afferent neurons, such as mechanoreceptors in the peripheral nervous system . These action potentials travel through the axon of the primary afferent neuron, and the cell body, to a synapse in the superficial dorsal horn of the spinal cord (41). Inputs, from several cells types within the spinal cord, are integrated and passed onto ascending pathways to the brainstem, and subsequently to the thalamus . The thalamus then transmits the signal to higher brain regions involved in the sensory (e.g., the somatosensory cortex) and emotional / affective (e.g., the amygdala and cingulate cortex) aspects of pain . Due to the cross - talk between supra - spinal nociceptive regions, incoming nociceptive signals can be either enhanced or dampened by descending modulatory pathways projecting from the brain to the spinal cord (40,41). The endocannabinoid system is distributed throughout the spinal and supraspinal regions, and thus is able to effectively regulate neurophysiological activities, including affective and nociceptive processing (42). Clinical studies have shown altered endocannabinoid signaling in patients with chronic pain (43,44) as well as in psychiatric patients (45,46). Certain genetic polymorphisms in cb1 and cb2 receptors have been found to be associated with major depression and bipolar disorder (47,48) and resistance to treatment was observed in depression patients having a single nucleotide polymorphism in the cb1 receptor (49). Elevated components of the endocannabinoid system, including plasma 2-ag levels and cb1 and cb2 mrna levels were observed in the lymphocytes in osteoarthritic patients, who also exhibited a positive correlation between 2-ag levels, pain and depression (50). However, whether these changes are compensatory to tackle the pain in osteoarthritis patients, is not known . Additional studies are necessary to better understand the association of endocannabinoid system and pain and depression . Although, to the best of our knowledge, relatively few clinical studies have directly addressed the importance of endocannabinoids in pain - depression interactions, improved muscle and nerve pain by the intake of cannabis has been reported in hiv patients, who exhibited improved symptoms of depression and anxiety (51). In cancer patients, daily adjunctive administration of cesamet (nabilone, a -thc analogue) for 30 days the therapeutic efficacy of nabilone for pain management and quality of life improvement was demonstrated in a randomized, double - blind, placebo - controlled trial in patients with fibromyalgia (53) (table i). Similar results were obtained in studies using -thc (dronabinol) in patients with chronic central neuropathic pain or fibromyalgia (54). The above and other studies (5557) together indicate that depression / anxiety and pain, when present together in a variety of patients, respond to exogenously administered cannabinoids, although the underlying mechanism remains to be elucidated . It has been demonstrated that -thc - mediated reductions in pain are associated with enhanced amygdala activity and reduced functional connectivity between the amygdala and somatosensory cortex (58). Thus, the amygdala likely forms the common neural circuit and connecting link between emotional responding and pain . The precise mechanism(s) by which the endocannabinoids influence behavioral / emotional and nociceptive processing remains to be determined . At present, there is considerable evidence involving the endocannabinoid system in eliciting potent effects on neurotransmission, neuroendocrine, and inflammatory processes, which are all known to be deranged in depression and chronic pain . Depression and pain co - exist in the majority of patients and often contribute to high mortality . Most patients who suffer from the comorbid depression and pain are not responsive to pharmacological treatments that address either the pain or depression, exacerbating this comorbidity disorder . Cannabinoids present in marijuana are well - known to contain pain and depression, and -thc, the active ingredient of marijuana, exerts its activity by activating cb1 and cb2 receptors . These receptors are activated by naturally present endocannabinoids, anandamide and 2-ag, which exert cannabinomimetic effects . The endocannabinoid system is involved in eliciting potent effects on neurotransmission, neuroendocrine, and inflammatory processes, which are known to be deranged in depression and chronic pain . However, the precise mode of action of endocannabinoids on different targets in the body and whether their effects on pain and depression follow the same or different pathways, remains to be determined in future studies.
Obesity has been declared a global epidemic by world health organization (who) which has not only crossed geographical boundaries but has spread across all ages . Rising prevalence of obesity among children and adolescents and the fact that two - third of childhood obesity persists into adulthood, is increasingly contributing to the escalating pool of this noncommunicable disease . Both developed and developing countries have witnessed a steep rise in the prevalence of overweight and obesity among children and adolescents . The prevalence of overweight and obesity has shown increasing trends in india also . More so, the prevalence reported among children and adolescents of some metro cities in india are comparable to that in some developed countries westernization of culture, rapid mushrooming of fast food joints, lack of open spaces for physical activity, and increasing sedentary pursuits in the metro cities are some of the reasons implicated for increased overweight and obesity . Are the nutritional changes in small town school children following the same pattern of larger cities? Keeping these research questions in mind, a study was undertaken among the school - going adolescents of aligarh, a small town located in the central india, about 132 km from new delhi, with the following objectives: (1) to study the prevalence of overweight and obesity among school - going adolescents of aligarh and (2) to study the sociodemographic and behavioral correlates of the same . Part of this development includes opening up of fast food chains, increasing social norm of eating out, more access to internet, video games and television (tv) viewing and school children of affluent families having more spending money . Although in large sections of the population the age - old customs of eating home food still persists, multinational companies are luring the young to new eating habits . A cross - sectional study was conducted from august 2009 to july 2010, in two affluent (having tuition fees more than rs . 10,000/annum) and two nonaffluent schools (having tuition fees <rs . 10,000/annum) in aligarh . Taking estimated prevalence of overweight as 3.23%, alpha error of 5%, 2% absolute allowable error and 10% nonresponse rate, sample size calculated was 321 and rounded off to 330 . Thus, 330 adolescents were covered in both types of schools (affluent and nonaffluent group), making the total sample size 660 . Purposive selection of two affluent and two nonaffluent schools was done to allow for practical feasibility . Probability proportionate to size of the population technique was used and systematic random sampling done . Apparently healthy school children of v x standard, who had completed 10 years of age on the date of interview and were not more than 16 years of age (as per school records) were interviewed after taking informed consent from the school authorities and the parents . Children having any chronic illness, severe malnutrition, endocrinal problems, physical, and mental defects, those with apparent obesity - induced or associated with any syndrome and those found to be smokers (defined as any amount of smoking or tobacco chewing at any time during past 6 months) and those not cooperating for anthropometric measurements were excluded . A predesigned and pretested questionnaire was used to collect data for sociodemographic and behavioral factors . Information regarding parent's education and occupation and family history of obesity were collected from the child's parents . Deficient, adequate, and excess calorie intakes per day were defined as per the total calorie requirements of adolescents, age and sex wise, as recommended by indian council of medical research . A pretested food frequency questionnaire was used to assess the frequency of fruit and fast food intake during the past 1 month . Fast foods were defined as the foods sold in a restaurant or store which are rapidly prepared and quickly served in a packaged form for take away and included burgers, pizzas, fries, patties, nuggets, and indian foods such as pakora, samosa, and namkeen . Students were interviewed about duration of watching tv and time spent in other sedentary activities per day during the past 1 month, which were then converted into categorized variables . Total physical activity level (pal) of the adolescents and the total sedentary time per day was assessed using global physical activity questionnaire . Anthropometric measurements of weight (to the nearest 0.1 kg) and standing height (to the nearest 0.1 cm) were taken according to standard methodology . Body mass index (bmi) was calculated as the ratio of body weight to body height squared expressed as kg / m . Nutritional status was defined using bmi for age and sex percentiles given by who growth reference 2007 . For the purpose of studying determinants of overweight and obesity, all the students were grouped into (a) overweight (including obese) and (b) nonoverweight / nonobese . The strength of association of determinants of overweight (including obesity) variables having significant association were subjected to stepwise multiple logistic regression model to determine the significant independent risk factors of overweight and obesity . Data analysis was done using ibm spss version 20 and p <0.05 was considered as statistically significant . The age group of 1013 years included 49.1% of adolescents and 50.9% were in the> 1316 years age group . The nutritional status of the study population according to bmi has been shown in table 1 . Nutritional status of the study population with respect to sex and type of school the overall prevalence of overweight and obesity among school - going adolescents was found to be 9.8% and 4.8%, respectively [table 1]. Although a higher prevalence of overweight and obesity was found among boys (11.3% overweight and 5.5% obesity) as compared to girls (7.9% overweight and 3.9% obesity), the difference was not statistically significant . The nutritional status was found to differ significantly (= 99.593, df = 3, p <0.05) between the affluent and nonaffluent group as shown in table 1 . In the nonaffluent schools, the proportion of underweight adolescents was significantly higher than in affluent schools . Furthermore, in the affluent schools, the proportion of overweight (14.8%) and obese (8.2%) adolescents were significantly higher compared to nonaffluent schools . Looking at the two ends of the spectrum, proportion of over - nutrition in affluent schools (14.8% overweight and 8.2% obesity) was much higher than under - nutrition (13.6%). The sociodemographic profile of the study population and various behavioral factors were studied according to type of school, by applying chi - square, and found to differ significantly between the affluent and nonaffluent group [tables 2 and 3, respectively]. Ninety percent of the adolescents of nonaffluent group were having a large family size as compared to about three - fourth adolescents in affluent group . Significantly higher proportions of adolescents from affluent group had higher paternal and maternal education . More than 55% of the affluent adolescents had fathers with business as their occupation as compared to 40% in case of adolescents of nonaffluent group . Parental history of obesity was also found to be significantly higher among the adolescents of affluent group . Vegetarian diet, use of ghee and vanaspati, excess total calories intake, eating out at least once a week and fast food intake 5 times or more per week was found to be significantly higher among the adolescents of affluent group than the nonaffluent group . Pal was found to be high among only 15.8% of affluent group as compared to 23% among the nonaffluent group adolescents (p <0.05). Significantly higher proportions of affluent adolescents were found to have more total sedentary time per day and tv viewing as compared to nonaffluent adolescents . On multiple logistic regression analysis, belonging to affluent group, mother's education more than or equal to graduate, parental history of obesity, frequent fast food intake, and tv viewing more than 2 h / day were found to be the independent risk factors for overweight and obesity [table 4]. Sociodemographic profile of the study subjects association between behavioral factors and type of school risk factors for overweight (including obesity) using stepwise logistic regression analysis the prevalence of overweight and obesity among the school - going adolescents of aligarh was found to be almost as high as reported in larger cities of the country . The double burden of nutritional disease faced by the asian countries is also the result of this transition . This double burden of nutritional disorders is also evident in this study, with the prevalence of overweight (including obesity) being 14.7% (97 out of 660) while about 30% of the adolescents were underweight . Rapid urbanization has created an obesogenic environment by promoting motorized transport, unsafe roads and traffic, eating up open spaces and playgrounds on one hand; and on another by providing more opportunities for sedentary leisure pursuits and fast food consumption outlets . This has been reported to be the cause of rising trend of obesity in the larger cities of india, especially the affluent section of society . Interestingly, even smaller but fast developing cities are also witnessing the problem of overweight and obesity, as shown by this study . The obesogenic environment of large metro cities is being duplicated in these cities, and unless timely action is taken to address these changes, the problem of overweight and obesity may escalate uncontrollably . In this study, it was found that adolescents of affluent schools were 2.4 times more at risk of having overweight and obesity . This trend of increased overweight and obesity among affluent section has been reported by many other researchers too . This may be explained by the fact that affluent group goes hand - in - hand with more spending money and more accessibility to fast foods, motorized transport, and sedentary pursuits such as computer and video games . The affluent group in this study had significantly higher use of ghee, eating out, fast food intake, tv viewing, and time spent in sedentary activities as compared to the nonaffluent group . A higher maternal education level was found to increase 3.1 times the odds of having overweight and obesity among school - going adolescents . It is expected that mothers who are educated should be planning better meals for their children but apparently they are not . This emphasizes the need for enriching and reinforcing individual awareness at family and community level . Educating parents of obese children interestingly, working status of mothers was not found to be a risk factor for overweight . Parental obesity has been implicated as a risk factor for overweight and obesity among children and adolescents by many authors and was found to be an independent risk factor in this study too . Family history of obesity in both the parents increased the odds of overweight and obesity by 6.7 times . Parental obesity may increase the risk of obesity through genetic mechanisms or by shared familial characteristics in the environment such as food preferences . The study has found fast - food intake to be a significant risk factor of overweight and obesity, and the risk increased with increased frequency of intake . Fast food typically incorporates all of the potentially adverse dietary factors, including saturated and trans fat, high glycemic index, high energy density, and increasingly, large portion size . Another indian study has also reported the prevalence of overweight to be higher among those adolescents who were fond of junk foods . The authors have found in their study that the children from private schools consumed more of fast food items and carbonated drinks due to easy availability in the school canteen . Fast food intake was found to be higher in affluent adolescents in this study also . The association between fast food consumption and obesity clearly indicates the need for improvements in family and school food environments . Tv viewing> 2 h daily was found to increase the odds of overweight and obesity among adolescents by 2.8 times . Furthermore, tv advertising could adversely affect dietary patterns at other times throughout the day . A randomized controlled trial has shown that reducing tv, videotape, and video game use may be a promising, population - based approach to help prevent childhood obesity . On univariate analysis, low pal was found to increase the risk of overweight and obesity by 2.6 times (ci 1.25.4, p <0.05), but no independent risk was found on multivariate analysis . In spite of proven benefits, levels of physical activity have been decreasing among urban children and adolescents . In this study, high pal was found to be significantly (p <0.5) lower among affluent adolescents (15.8%) than among the nonaffluent adolescents (23.0%). It can be concluded from this study that overweight and obesity among school - going adolescents is a crisis facing even smaller cities in india, and action to control it must begin now . Given the current trends in pediatric overweight and obesity, it is very crucial that preventive strategies should be implemented through schools and community - based programs involving both education and intervention . Prevention starting in early childhood (life course approach) is a critical area of work to prevent obesity . Limitations of the study include a purposive selection of schools and use of 24 h recall for assessment of dietary intake per day . Because most individuals diets vary greatly from day to day, data from a single 24 h recall might fail to characterize an individual's usual diet . As the findings of a school - based study like this cannot be generalized to the whole population, a larger study conducted in schools as well as the general adolescent population can provide more conclusive results about overweight and obesity and their risk factors.
Scattering data were acquired using the pump probe method at the biocars 14-id - b beamline at the advanced photon source (aps) while the storage ring was operated in the standard top - up mode . Aqueous solution samples of wild - type sperm whale mbco were prepared at a 8 mm concentration in 0.1 m sodium phosphate buffer at ph 7.0 using a previously established protocol . Photodissociation of mbco was initiated by exciting the mbco solution contained in a capillary of 1 mm thickness with a 35 ps long laser pulse at 532 nm . Subsequently, a 100 ps long x - ray pulse probed the sample, and the scattered x - ray photons were recorded as a function of the time delay between the laser pump pulse and the x - ray probe pulse . Probe time delays in the range from 100 ps to 10 ms (four time points per decade). Taking the difference between the scattering curve measured at each time delay point and the reference scattering curve measured at 5 s yielded the difference scattering curve, s(q, t). The contribution from laser - induced solvent heating was removed from the measured scattering curves . The details of the experiment and the data processing are provided in the si.
The combination therapy with pegylated interferon (peg - ifn) and ribavirin (rbv) has come to be established as the standard treatment for chronic hepatitis c. a sustained virological response (svr) has been reported with this treatment in 30%50% of patients with hcv genotype 1b infection, which accounts for 70% of all japanese patients with chronic hepatitis c . However, the treatment often needs to be discontinued, or the dose of rbv changed, in these patients due to the development of hemolytic anemia . On the other hand, continuous treatment is important to obtain svr with the treatment . Rbv is incorporated into the cells via the equilibrative nucleoside transporter (ent) and converted to phosphates within the cells . Rbv monophosphate (rmp) and rbv triphosphate (rtp) are considered to have antiviral activity [3, 4]. In nucleated cells, the phosphorylated rbv is subsequently dephosphorylated by the dephosphorylating enzyme, and rbv is eliminated from the cells via the ent . However, in akaryocytes such as erythrocytes, which lack the dephosphorylating enzyme, accumulation of phosphorylated rbv occurs, which diminishes the cellular atp and alters the cellular characteristics; these changes in the characteristics of the erythrocytes activate the cell - elimination activity of the reticuloendothelial system, resulting in hemolysis [5, 6]. In this study, with the objective of reducing the adverse effects and improving the treatment completion rate in patients receiving combined peg - ifn and rbv therapy, we attempted to evaluate whether the erythrocyte phosphorylated rbv level might be useful as an index for the rate of early virological response (evr) and svr, the risk of anemia . Among the patients with chronic hepatitis c caused by genotype 1b infection in whom combined peg - ifn2b and rbv therapy was started, 24 patients who provided written informed consent for participation in this study were enrolled . The dosage regimen for the combined peg - ifn and rbv therapy was determined in accordance with the standard dosing recommended for japanese hcv patients . Rbv was started at the initial dose of 600 mg / day in patients with a body weight of 60 kg, 800 mg / day in those with a body weight of> 60 kg and 80 kg, and 1,000 mg / day in those with a body weight of> 80 kg . In patients with no cardiovascular disease, the rbv dosage of 600 mg / day was decreased to 400 mg / day, and 800 mg or 1000 mg / day was reduced to 600 mg / day, if hemoglobin (hb) level decreased to less than 10 g / dl; permanently discontinued the drug if hb decreased to less than 8.5 g / dl . In those with history of stable cardiovascular disease, the dosage of rbv was decreased to 400 mg or 600 mg if hb decreased by 2 g / dl or more during any 4-week period; permanently discontinued the drug if hb was less than 12 g / dl after 4weeks of a reduced rbv dosage . The patients were followed up until 48 weeks after the start of the combined ifn and rbv therapy, and hb level and hcv rna level were examined at week 12 after the start of treatment . Additionally, serum hcv rna negativity until 24 weeks after the therapy was completed and was defined as svr . This study was performed with the approval of the hospital research committee, in compliance with the ethical principles laid out in the declaration of helsinki . Ten - ml samples of venous blood were obtained at 2, 4, and 8 weeks after the start of the therapy, and the erythrocyte level of phosphorylated rbv was measured by the hplc method described by homma et al . . In this method, all phosphorylated rbv (rmp, rdp, and rtp) is converted back to rbv by treatment with an erythrocyte dephosphorylating enzyme, and the erythrocyte level of phosphorylated rbv is calculated as the difference in the rbv levels measured before and after the enzyme treatment . The changes in the hb level were analyzed by repeated - measures anova and dunnett's test . The relationships of the rbv level to the risk of anemia and the drug efficacy were examined by student's t - test, pearson's correlation coefficient, -test, and fisher's exact probability test . The subjects comprised 24 patients, and their demographic characteristics are indicated in table 1 . The combined peg - ifn and rbv therapy needed to be discontinued in 3 of the 24 patients (12.5%), and the rbv dose needed to be reduced in 7 of the patients (29.2%) due to the development of anemia (hb 10 g / dl). However, the conditions of rbv administration for the initial 4 weeks were not changed . There were no significant difference in the evr rates between the subjects in whom the rbv dose was reduced (72.7%) and those in whom the therapy continued at the initial dose (64.0%). The dosage of peg - ifn was 1.5 0.2 g / kg in accordance with a standard regimen, and the conditions of ifn administration have not changed for 48 weeks after the combination therapy was started in the subjects except for 4 patients who discontinued the therapy . The hb levels (11.0 1.3 g / dl) were significantly lower at week 4 of therapy as compared with 13.6 1.3 g / dl at the start . In addition, the rate of hb reduction [(hb level - hb level before administration)/hb level before administration] at week 4 of therapy was 12.4% in those aged less than 50 years, whereas it was 21.0% in those aged 50 years or over (p <.05). The phosphorylated rbv and nonphosphorylated rbv levels in the erythrocytes were 749.3 244.3 and 6.9 3.6 m at week 2, 1039.9 239.6 and 8.4 7.0 m at week 4, and 907.9 292.1 and 8.9 8.0 m at week 8 of therapy, respectively; thus, about 99% of the rbv in the erythrocytes was phosphorylated (figure 1). The relationship between the rbv level at week 2 and the rate of reduction of the hb level was examined in the 19 patients aged 50 years or over . There was a negative correlation (r = 0.548, p <.05) between the rbv level at week 2 and the rate of hb reduction (hb) at week 4 in the subjects in whom the dose of rbv did not reduce or the combination therapy did not discontinue until week 4 (figure 2). As shown in figure 3, the hg at week 4 was significantly higher (p <.05) in those with rbv level of 800 m (25.5 10.1%) than in those with the level of <800 m (15.6 7.7%). The relationship between the phosphorylated rbv level at week 2 and the evr was evaluated in 20 of the 24 patients (four cases were excluded because of the discontinuation of the therapy). The mean rbv level at week 2 was significantly lower (p <.05) in the non - evr patients (634.6 236.6 m) than the evr patients (889.7 210.6 m). As shown in table 2, 3 cases with the phosphorylated rbv level in erythrocytes 800 m discontinued the combination therapy prematurely due to anemia, whereas none of 14 cases with a levels <800 m discontinued prematurely . There were no evr or svr cases (0 of 7 cases) in patients with erythrocyte phosphorylated rbv levels <600 m at week 2, whereas, in those with levels 600 m, 11 of 17 cases (64.7%) had evr and 6 of 17 cases (35.3%) had svr (p <.05). Five of 7 cases (71.4%) with erythrocyte phosphorylated rbv level at week 2 of 600800 m achieved evr and 3 cases (42.9%) achieved svr without development of marked anemia . In this study, combined peg - ifn and rbv therapy needed to be discontinued, or the rbv dose needed to be reduced, in about 40% of the study subjects due to the development of hemolytic anemia (hb 10 g / dl). None of the patients in whom the combined peg - ifn and rbv therapy was discontinued by week 12 showed svr . However, no difference in the rate of svr was noted between the subjects in whom the rbv dose was reduced and those in whom the treatment could be continued at the initial dose . This suggests that continuation of the combination therapy was the most important factor for achieving the desired clinical outcome . However, it has been noted that such dose adjustment does not effectively prevent the progression of anemia; that is, once a decrease of the hb level has occurred, it is too late to stop the decline through rbv dose reduction, presumably because of erythropoietic delay . In our subjects, the phosphorylated rbv level reached a steady - state by 4 weeks of rbv therapy (1040 240 m), implying gradual accumulation of phosphorylated rbv . Inoue et al . Reported that the erythrocyte phosphorylated rbv level at the steady - state at week 4 was 1218 234 m and was well correlated with hb reduction . However, as the hb level had already decreased significantly by week 4, the rbv level at week 4 does not predict anemia . Therefore, we decided to evaluate whether the phosphorylated rbv level at week 2 might be useful for prediction of the subsequent development of anemia . A close negative correlation was observed between the erythrocyte phosphorylated rbv level at week 2 and the hb at week 4 in patients aged 50 years or over . In general, in elderly people, the percentage of fat cells in the bone marrow increases, and the reserve of marrow stem cells decreases with reduction of the hematopoietic mass and reduction in the ability for formation of erythroid colony - forming units [9, 10]. Additionally, rbv is known to be substantially excreted by kidney, and renal function decreases in elderly patients . Therefore, rbv accumulation is considered to be more likely to cause anemia in the elderly, especially due to the erythropoietic delay and the delay of rbv excretion . Reported that one of the higher risk of severe anemia was age higher than about 60 years . In patients with rbv level of 800 m at week 2, the hb at week 4 was significantly higher, and a higher percentage of patients needed discontinuation of the rbv due to the development of anemia . Thus, we recommend that the erythrocyte phosphorylated rbv level be maintained at a level of less than 800 m at week 2 for treatment safety . The plasma rbv level has been reported not to be correlated with the svr, but there have been few reports on the relationship between the erythrocyte phosphorylated rbv level and the treatment efficacy . The erythrocyte rbv level is about 150 times higher than the plasma rbv concentration, and most of the administered rbv is considered to be secreted into the urine . Of the proportion that remains in the body, furthermore, homma et al . Reported that little phosphorylated rbv existed in the plasma . Since rbv taken up by cells is considered to be phosphorylated, and rmp and rtp are considered to have antiviral activities, the erythrocyte phosphorylated rbv level is a useful index for the antiviral effect of the drug . Since evr, defined as a decrease of the hcv rna level to 1/100 or zero at week 12 after the start of therapy, has been reported to be useful as a prognostic factor, we focused on not only svr but also evr . In this study, moreover, none of the patients in whom the phosphorylated rbv level at week 2 was <600 m showed both evr and svr . Adjustment of the rbv dose to obtain an erythrocyte phosphorylated rbv level of 600 m at week 2 is considered to be required to obtain an evr . It would be necessary to have a large sample size, study of quality of life, and demonstration of better evr and svr in a prospective randomized trial . Reported that genetic variants leading to inosine triphosphatase (itpa) deficiency protects against clinically significant decline in hb level induced by hcv antiviral treatment . We should examine the relationships between itpa gene variants and rbv - induced anemia in japanese populations, and evaluate the usefulness as an index to reduce the risk of anemia with erythrocyte rbv level . In this study, the erythrocyte phosphorylated rbv level at week 2 is proposed as a useful indicator to determine an optimal dosage of ribavirin in patients with chronic hepatitis c under treatment with combination therapy with pegylated interferon and rbv.
Increases in the concentration of intracellular ca ([ca]i) control diverse functions in virtually all cell types . These ca signals differ spatially, temporally and in magnitude and are mediated by numerous channels, transporters and ca - sensing proteins . Opening of ca channels in either the plasma membrane or the membrane of intracellular ca stores directly elevates [ca]i . Ca entry from extracellular space and ca release from endoplasmic reticulum (er) operate independently or together to regulate different ca - dependent processes, such as exocytosis of neurotransmitters, contraction of skeletal muscle, activation of t lymphocytes or contraction of smooth muscle cells . Signaling via many cell surface receptors involves the activation of ca release from er via inositol-1,4,5-triphosphate (ip3). Because a second phase of ip3-mediated ca mobilization was frequently observed and identified as ca influx from extracellular space, the hypothesis of capacitative ca entry was formulated by putney in 1986 . He proposed a receptor - regulated pathway for ca that enters the depleted ca pool directly via plasma and er membranes and is controlled by ip3 and the ca content of the er . The capacitative or store - operated ca entry (soce), has been recognized as a an essential route for ca uptake in a wide variety of cell types to replenish intracellular ca stores and to regulate secretion, gene transcription and cell cycle progression . Several candidate molecules and ion channels mediating soce were discussed in recent years . In particular, some members of the transient receptor potential (trp) cation channel family were found to generate receptor - activated ca entry . However, electrophysiological characterization of soce in mast cells revealed a ca - release activated ca (crac) current, whose properties can be clearly distinguished from those of trp channels . In 2005 and 2006, different groups identified stromal interaction molecules (stim) and orai proteins (named after the keepers of heaven's gate in greek mythology) as essential components of soce . Stromal interaction molecule 1 (stim1) was originally identified as a potential tumor suppressor gene coding for a transmembrane protein . In vertebrates, one additional stim1-related gene, stim2, both stim homologues are ubiquitously expressed in different cell types with higher stim1 levels in most tissues but a predominant expression of stim2 in brain . Stim1 and stim2 each contain highly conserved domains, including a single transmembrane segment, an ef - hand ca - binding domain, a sterile -motif (sam) domain and coiled - coil regions (for review see refs . Stim1 and stim2 are primarily located to the er; stim1 was also detected in the plasma membrane . The ef - hand ca - binding domains (together with the sam domains) of stims are responsible for sensing [ca] in the lumen of the er . A decrease in er luminal ca concentration results in dissociation of ca from the ef - hand domain which, in turn, triggers oligomerization and activation of stim1, a process that is reversed when luminal [ca] returns to resting level (that is, 250600 m). The luminal ef - hand domain of stim1 binds ca with an apparent dissociation constant (kd) of 250 m, whereas the kd of stim2 for ca is 500 m . Hence, stim2 is more sensitive to small changes in luminal [ca] and partially active at resting er ca levels . Stim2 shows a slower oligomerization kinetics and is less effective than stim1 in orai1 binding and activation, which might be critical for limiting excessive soce . Orai1 and its homologues orai2 and orai3 were discovered by genome wide rnai screens for soce inhibition and by positional cloning in patients with immune deficiency and crac channel dysfunction . Co - expression with stim1 showed that orai1 is the pore - forming subunit of the crac channel . Orai1 channels (therefore, also named crac modulator 1; cracm1) are activated upon ca store depletion, translocation of activated stim1 to er - plasma membrane junctions and interaction of cytosolic stim1 domains with the c - terminal domains of orai1 tetramers . Heterologously expressed orai2 and orai3 channels (together with stim1) conduct crac - like currents but exhibit distinct inactivation and permeability properties . Store - depletion signaling via stim2 also activates all 3 orai channels . Over - expression of orai1 or orai2 (but not orai3) alone although trp channels do not account for crac currents, a contribution of some trp family members to stim1-induced and store - dependent ca entry has been shown . In this context, a soce - signaling complex consisting of trpc1, orai1, and stim1 has been proposed . The principal components stim1 and orai1 essentially or nearly exclusively contribute to soce in different cell types . The function of the innate and adaptive immune system requires stim1/orai1-dependent soce . In mouse t lymphocytes, orai2 and/or orai3 appear to have redundant functions in mouse t cells, whereas human t cells lacking functional orai1 failed to proliferate in vitro and to produce cytokines . Stim1 and orai1 regulate mast cell activation, and stim1 is essential for fc receptor - dependent ca signaling and phagocytosis in macrophages and neutrophils . Roles of both stim1 and orai1 in other cell types include growth and contractility of skeletal muscle, proliferation of vascular smooth muscle cells, and aggregation of platelets . Soce in the nervous system has been reviewed with respect to ca signaling and homeostasis in neurons and neuroglia . The present review is mostly based on studies regarding expression analyses or using gene silencing or knockout of stims and orais and gives a current overview on the putative functions of these soce components in both neurons and glial cells, i.e., astrocytes and microglia . Stim and orai isoforms are broadly expressed in murine and human tissues . At the rna level, stim1 was consistently detected in skeletal muscle and brain, whereas stim2 was preferentially found in brain and heart from both species . Both stim proteins were detected in murine and human brain . Within the murine brain, stim1 and stim2 are distributed in the cerebral cortex and can be assigned to hippocampal and cerebellar structures . Whereas stim1 is most prominent in the cerebellum, stim2 dominates in hippocampus and cortex . In human brain, stim1 protein expression is high in cerebellum, medium in cerebral cortex, and low in hippocampus . Stim2 protein levels are high in hippocampus and cerebral cortex, and medium in cerebellum, indicating that the differential distribution of stim1 and stim2 in cerebrum and cerebellum is similar in human and murine brain . Expression of stim mrnas at the cellular level was analyzed using cell isolation or separation by laser capture, cell soma harvest and cell culture . In primary hippocampal and cortical neurons, stim2 is the predominant stim isoform . In hippocampal cultures, stim2 was detected in both neuronal soma and dendrites, whereas stim1 protein is restricted to the soma . Furthermore, the expression of stim1 and stim2 increases during development of hippocampal neurons in vitro . In purkinje neurons, the principal neurons of the cerebellar cortex, stim1 levels were higher than those of stim2 . The second most important neuron within the cerebellum is the cerebellar granule cell, which is located in the nuclear layer and whose parallel fibers project to purkinje neurons in the upper molecular layer . Interestingly, orai1 mrna levels in both species appeared to be lower than those of orai3 . A high abundance of orai2 was found in hippocampal neurons, cerebellar tissue and purkinje neurons . The origin of the strong orai3 expression, particularly in human brain, is presently unclear and might come from glial cells . In mouse cerebellum, the role of orai2 in brain as well as in other tissues is still unclear . Store - operated ca entry has been implicated in neuronal ca signaling even before the discovery of stim and orai . Ca imaging experiments revealed soce in cultured and freshly dissociated cortical and hippocampal neurons . A role for soce in spontaneous synaptic activity and in synaptic plasticity of hippocampal neurons was suggested from studies using the common soce inhibitors 2-aminoethoxydiphenyl borate (2-apb), skf96365 and la . First molecular evidence for neuronal soce provided a study using stim and orai knockout mice . Have shown that stim2-deficient mice were protected from cerebral damage after ischemic stroke . The reduced infarct size and the improved neurological outcome of stim2 animals were independent of functional changes within the haematopoietic system . In cultured cortical neurons, soce was significantly decreased in the absence of stim2 but not of stim1 or orai1 . Hypoxia and hypoglycemia induced an increase in [ca]i which was markedly reduced in stim2 neurons . Hypoxia / hypoglycemia inhibits atp - dependent ca transport into the er and might, therefore, trigger persistent stim2 activity and soce - induced accumulation of cytosolic ca (fig . Glutamate - induced excitotoxicity is a process triggered by and contributing to neuronal ca accumulation during ischemia . Cholesterol, a major component of the plasma membrane, plays a key role in regulating neuronal functions . Glutamate - induced cholesterol loss required high intracellular [ca] and functional stim2 in hippocampal neurons . Apart from the role of stim2 in ischemic stroke, behavioral tests revealed an impairment of hippocampus - dependent spatial learning in stim2-deficient mice . (a) proposed model for the involvement of stim2 in ischemic ca accumulation in hippocampal and cortical neurons . A disturbed refilling of intracellular ca stores during ischemia may be induced by inhibition of sarco - endoplasmic reticulum ca pumps . The reduced [ca] in the endoplasmic reticulum (er), [ca]er, leads to the activation of stim2 and, possibly, of orai2 channels in the plasma membrane . Opening of orai channels results in soce, which contributes to deleterious ca accumulation in the cytosol . (b) role of stim2 in maintenance of postsynaptic mushroom spines in hippocampal neurons . Activation of stim2 due to reduced [ca]er induces continuous soce via orai (supposedly, orai2) channels . Increased cytosolic [ca] supports constant levels of ca / calmodulin - dependent protein kinase ii (camkii) and long - term stability of mushroom spines . Activation of metabotropic glutamate receptor type 1 (mglur1) induces ca release from er, a decrease in [ca]er, and the activation of stim1 . Soce results in ca store refilling and supports ca - dependent activation of the transient receptor potential channel trpc3 . Trpc3 mediates slow excitatory postsynaptic currents (epsc) which are important for purkinje neuron function and cerebellar motor behavior . (a) proposed model for the involvement of stim2 in ischemic ca accumulation in hippocampal and cortical neurons . A disturbed refilling of intracellular ca stores during ischemia may be induced by inhibition of sarco - endoplasmic reticulum ca pumps . The reduced [ca] in the endoplasmic reticulum (er), [ca]er, leads to the activation of stim2 and, possibly, of orai2 channels in the plasma membrane . Opening of orai channels results in soce, which contributes to deleterious ca accumulation in the cytosol . (b) role of stim2 in maintenance of postsynaptic mushroom spines in hippocampal neurons . Activation of stim2 due to reduced [ca]er induces continuous soce via orai (supposedly, orai2) channels . Increased cytosolic [ca] supports constant levels of ca / calmodulin - dependent protein kinase ii (camkii) and long - term stability of mushroom spines . Activation of metabotropic glutamate receptor type 1 (mglur1) induces ca release from er, a decrease in [ca]er, and the activation of stim1 . Soce results in ca store refilling and supports ca - dependent activation of the transient receptor potential channel trpc3 . Trpc3 mediates slow excitatory postsynaptic currents (epsc) which are important for purkinje neuron function and cerebellar motor behavior . Whereas no obvious abnormalities were reported for brain structures of stim2 mice, sun et al . Showed that conditional knockout of the stim2 gene in the hippocampus of 26 month old mice induced a massive neuronal loss in this brain region . Deletion of stim2 in hippocampal neurons caused a moderate reduction of somatic soce but a dramatic decrease of soce in dendritic spines . The absence of stim2 also reduced the number of spines and changed their morphology by reducing the fraction of mushroom spines which play an important role in the storage of memories . A loss of mushroom spines, concurrent with the decrease in synaptic soce and the downregulation of stim2 was observed in hippocampal neurons from the presenilin-1 m146v knockin mouse model of alzheimer's disease . Overexpression of stim2 (but not of stim1) rescued synaptic soce and increased the expression of phosphorylated ca / calmodulin - dependent protein kinase ii (camkii) suggesting that stim2-dependent soce is required for camkii activity and stabilization of mushroom spines in healthy neurons (fig . It is not clear why stim2 but not stim1 regulates synaptic soce in hippocampal neurons . Stim2 has a higher kd for ca than stim1 and induces soce near resting er ca levels . Thus, stim2 stabilizes basal [ca]i even at incomplete store depletion and, in turn, maintains steady - state camkii activity and integrity of mushroom spines . Another recent study supports the role of stim2 in regulating dendritic spine density and morphology in hippocampal neurons . Showed that stim2 preferentially localizes to large dendritic spines and is enriched in the postsynaptic density . Stim2 is required for regular synaptic activity and mediates camp - dependent phosphorylation and trafficking of the ampa receptor subunit glua1 to plasma membrane - er junctions . Analogous to stim interaction with orai1, the authors suggest that stim2 binds via its cytosolic domain to glua1 and couples glua1 to camp - dependent protein kinase (pka). Signaling of stim proteins through other pathways than soce has also been shown for the interaction of stim1 with the voltage - gated ca channel (vgcc) subtype cav1.2 . Stim1 inhibits vgcc activation by binding to cav1.2 via the cytosolic stim - orai activating domain . It has been suggested that stim1-mediated suppression of vgccs is involved in the differentiation of neurons from embryonic stem cells . Knockdown of stim1 or stim2 reduced soce and inhibited entry of mouse embryonic stem cells into neural lineage . Stim1 knockdown induced an increase in voltage - dependent ca entry and treatment of cells with the vgcc blocker nifedipine facilitated neural differentiation . Found a markedly reduced soce after knockdown / knockout of stim1 or orai1 in embryonic and neonatal neural precursor cells . Suppression of stim1 or orai1 diminished proliferation of neural precursors and inhibited activation of the transcription factor nfat (nuclear factor of activated t cells). Whereas stim2 regulates soce in hippocampal and cortical neurons, stim1 appears to be the primary stim isoform in cerebellar granule cells and purkinje neurons . Lalonde et al . Have shown that changes in the extracellular k concentration rapidly induce redistribution of overexpressed stim1 together with overexpressed orai1 and orai2 in cerebellar granule neurons . Because a switch of extracellular [k] from a high (2565 mm) to a low level (5 mm; near resting extracellular [k]) induces plasma membrane hyperpolarization (repolarization), the authors suggest that changes in the membrane potential induce soce in granule neurons . Furthermore, ryanodine receptor - dependent ca stores were depleted by k - induced hyperpolarization and a sustained soce signal was observed at resting extracellular [k]. Knockdown of stim1 in a neuroblastoma cell line and pharmacological inhibition of soce in granule neurons inhibited hyperpolarization - induced degradation of the neuron - specific transcription factor sp4 . Sp4 has been implicated in dendrite patterning in cerebellar and hippocampal neurons as well as in memory and synaptic plasticity . The activation of soce at resting (hyperpolarized) membrane potential suggests a homeostatic function of stims in neurons, i.e. The regulation of ca - dependent gene transcription by controlling resting intracellular ca levels . Reported that stim1 controls glutamate receptor - dependent synaptic transmission in purkinje neurons and motor learning in mice . The metabotropic glutamate receptor type 1 (mglur1), that is highly expressed in purkinje neurons, plays an important role in cerebellar functions like motor coordination . Signal transduction downstream of mglur1 involves the ip3-dependent ca release from er and the activation of a slow excitatory postsynaptic current which is mediated by the trp channel trpc3 . In the absence of stim1, the ip3-dependent ca release from dendritic er ca stores and the trpc3-mediated currents were abolished . Interestingly, stim1 was only required for ca store refilling and slow synaptic currents when purkinje neurons were held at resting membrane potential . Depolarization - induced ca entry via vgccs, however, induced a stim1-independent recovery of er ca release and trpc3 activity . The latter observation leads to the conclusion that activation of trpc3 channels depends on intracellular [ca] but not on interaction with stim1 or the mglur1-induced activation of phospholipase c (fig . . The essential role of stim1 in resting purkinje neurons suggests that neuronal soce replenishes intracellular and er ca levels when vgcc activity is low . Astrocytes are glial cells which are derived, like neurons and oligodendrocytes, from neuroepithelial progenitors . They perform a variety of homeostatic functions within the nervous system and communicate with neighboring glial cells and neurons by generating (intercellular) ca signals and by releasing and binding of transmitter molecules . Cultured astrocytes and tumor cells of astroglial origin, i.e., glioblastoma cells, exhibit soce in response to metabotropic receptor agonists like atp, histamine, and glutamate . Stim1 and stim2 proteins are expressed in cultured astrocytes while stim1 appeared to be the dominant isoform . Reported that knockdown of stim1 and orai1 or of orai1 alone diminishes thrombin - induced ca signals and soce in cultured rat astrocytes . . Showed a reduction of soce and of crac currents by silencing of stim1 or orai1 in human glioblastoma cells . Knockdown of stim1 and orai1 slightly affected proliferation but clearly inhibited invasive glioblastoma cell migration . Surprisingly, the authors were unable to detect orai1 but found a dominant expression of orai3 which was 6-fold more abundant than orai2 . Furthermore, previous studies suggest a role of trp channels in astrocytic soce and, therefore, challenge a sole contribution of orai channels . Golovina reported that antisense oligonucleotides targeted to the trp channel gene trpc1 inhibited soce in murine astrocytes . However, in a later study the same group showed a profound inhibition of astrocytic soce by knockdown of orai1 . Reported that an antibody against trpc1 reduces soce and abolishes atp - mediated ca entry in cultured rat astrocytes . Astrocytes also express further trp channel subtypes, including trpc4 and trpc5, which can form heteromeric channels with trpc1 . A concordant regulation of trpc1, trpc4, orai3, and of receptor - induced ca entry has been observed in astrocytes treated with different pro - inflammatory agents and amyloid- protein . Thrombin treatment of astrocytes induced the up - regulation of a further trp homolog, trpc3, and an increase in soce . In contrast to crac channels, which are highly selective for ca, trpc3 is permeable for some other divalent cations, including sr . Have shown that store depletion induces only a negligible sr entry in astrocytes suggesting a minor role of trpc3 in astrocytic soce . Microglia are derived from myeloid precursors and constitute up to 20% of the total glia population . Microglia sense disturbances or loss of brain homeostasis and undergo morphological and functional changes in response to brain injury and infection . This microglial activation includes shape changes toward an ameboid appearance, directed movement, proliferation, phagocytosis, and release of cytokines . Microglia are equipped with a variety of surface receptors for neurotransmitters, signaling molecules and pathogens . Extracellular nucleotides, i.e. Atp, adp and udp, accumulate during brain injury, activate purinergic p2y receptors and stimulate migration and phagocytosis in microglia . Showed that crac / orai channels rather than trp channels mediate soce in cultured rat microglia . Soce was inhibited by a high concentration of 2-apb (50 m) and by exchange of extracellular ca by sr or ba . From the high expression of orai1 and orai3 in microglia and the agonistic action of 50 m 2-apb on orai3, the authors suggested a mayor role of orai1 in microglial soce . The same group detected high levels of orai1 and stim1 in microglial podosomes, actin - rich structures involved in cell motility and invasion . Treatment of cultured rat microglia with soce inhibitors strongly reduced migration, i.e., transmigration across filters with defined pores, and invasion, i.e. Transmigration through pores coated with extracellular matrix molecules . Confirmed the expression of all orai isoforms and of stim1 in purified cultured mouse microglia and showed that downregulation of orai1 and stim1 reduces soce and udp - induced ca signals . Silencing of stim1 inhibited lipopolysaccharide - induced activation of nfat1 and production of interleukin 6 (il-6) but did not affect activation of nf-b . Stim1 knockdown slightly decreased release of tnf- and reduced the udp - stimulated phagocytosis of bacterial particles . The p2y6 receptor agonist udp was previously shown to induce activation of nfat1 and nfat2 as well as expression of the chemokines ccl2 and ccl3 in microglia . We investigated the role of stim1, stim2, and orai1 in microglia . To estimate the relative contribution of stim1 and stim2 to microglial responses and to evaluate the role of orai1 purity of microglial cultures was tested by staining with isolectin b4 and by using transgenic cx3cr1 mice, where gfp is exclusively expressed in microglia . In our primary cultures, which showed a purity of 99.5%, we analyzed the mrna levels of stim and orai isoforms by quantitative rt - pcr . Calcium imaging revealed a graded suppression of soce in the absence of stim1, stim2, or orai1 . Soce evoked by blockage of sarco - endoplasmic reticulum atpase (serca) was nearly absent in stim1 microglia (91% inhibition), whereas stim2 cells showed a less pronounced but significant inhibition (by 30%). Purinergic stimulation with the p2y6 receptor agonist udp or the p2y12 receptor agonist 2-methylthio - adp (2-mesadp) caused soce amplitudes which were significantly smaller than those evoked by serca inhibition . The p2y12 receptor - mediated soce was blocked by 40% in stim2 microglia and reduced by 77% in stim1 cells, suggesting a decreased effect of stim1 and an increased impact of stim2 on soce of lower magnitude . A role for stim2 in modulating the threshold for ca entry showed that stim2 exclusively contributed to ca signals evoked by tyrosine kinase receptors but not by g - protein coupled receptors in leukemia cells . Found a stim2-dependent soce signal after mild store depletion which was completely replaced by a stim1-dependent soce upon strong store depletion . Ong et al . Proposed that stim2 triggers soce by recruiting stim1 at low stimulus intensities when er ca stores are mildly depleted . Together, these data suggest that stim2 increases the sensitivity of intracellular ca signals to extracellular agonists . To evaluate the role of soce in microglial migration and phagocytosis we used stim and orai knockout mice, the soce blockers 2-apb, la, and the trp channel blocker n-(p - amylcinnamoyl)anthranilic acid (aca). We found that aca effectively blocks soce in microglia with an ic50 of 0.4 m . Transmigration of cultured microglia stimulated with atp was largely reduced by aca and soce inhibitors . Uptake of zymosan particles by udp - stimulated microglia was nearly abolished in the presence of 2-apb . P2y receptor - induced migration (induced by 2-mesadp, atp, and udp) was inhibited in stim1, stim2, and orai1 microglia . P2y6 receptor - dependent phagocytosis was nearly abolished in stim1 cells and largely decreased in stim2 and orai1 microglia . Basal migration and phagocytosis in the absence of purinergic stimuli were not affected by stim or orai knockout . From these data we concluded that stim1 and orai1 are the mayor constituents of microglial soce and that stim2 is an important component of this signaling pathway . Consequently, soce is essential for p2y receptor - dependent ca entry, migration, and phagocytosis in microglia (fig . 2). Possible downstream effectors of microglial soce involve protein kinase c (pkc) and the ca / calmodulin - activated myosin light chain kinase (mlck) which both activate phagocytosis in microglia . The ca - dependent phosphorylation of protein kinase b (akt) is required for adp - induced chemotaxis of microglia . The g protein - coupled receptors p2y6 and p2y12 are activated by udp and adp / atp, respectively . Activation of p2y receptors induces ca release from endoplasmic reticulum (er) and reduces [ca]er . In turn, the resulting soce promotes activation of different ca - dependent signaling molecules, including ca / calmodulin - activated myosin light chain kinase (mlck), protein kinase c (pkc), the serin / threonine specific kinase akt, and the transcription factor nfat . Remodeling of actin - myosin skeleton is probably involved in mlck / pkc - dependent phagocytosis and in akt - dependent cell migration . The g protein - coupled receptors p2y6 and p2y12 are activated by udp and adp / atp, respectively . Activation of p2y receptors induces ca release from endoplasmic reticulum (er) and reduces [ca]er . In turn, the resulting soce promotes activation of different ca - dependent signaling molecules, including ca / calmodulin - activated myosin light chain kinase (mlck), protein kinase c (pkc), the serin / threonine specific kinase akt, and the transcription factor nfat . Remodeling of actin - myosin skeleton is probably involved in mlck / pkc - dependent phagocytosis and in akt - dependent cell migration . Stim and orai proteins have been implicated in normal brain function but also in neurological disorders . Stim1, preferentially expressed in cerebellar neurons, and stim2, more abundant in hippocampal and cortical neurons, trigger neuronal soce . The versatile role of stims and, consequently, of soce in neuronal signaling was rather unanticipated because synaptic activity was thought to provide a sufficient supply for intracellular ca . However, recent studies suggest that soce is a mayor source for intracellular ca in resting neurons . Stim1 is required for slow glutamate receptor signals in purkinje neurons and for ca - dependent gene transcription in cerebellar granule neurons . Stim2 controls steady - state activity of camkii and thereby stabilizes mushroom spines in hippocampal neurons . Decreased stim2 activity and loss of mushroom spines may be responsible for memory loss in aging neurons and in alzheimer's disease . Stim2 is also a potential target for the treatment of glutamate - induced excitotoxicity during epilepsy, brain trauma and cerebral infarction . The role of orai proteins in neuronal ca signaling is still unresolved . In particular, consequences of the dominant orai2 expression, orai2-mediated currents show a smaller amplitude but undergo a decreased inactivation at higher [ca]i . In analogy to functional differences between stim1 and stim2, orai2 might, therefore, support a persistent and moderate less is known about the role of stims and orais in astrocytes . Here, a further orai isoform, orai3, might play a central role . Orai3 is, in contrast to orai1 and orai2, inhibited by reactive oxygen species (ros), and is required for store - independent crac channels activated by arachidonic acid, a phospholipase a2 (pla2) metabolite . Both, pla2 activity and ros production were implicated in neurodegenerative diseases, such as cerebral ischemia and alzheimer's disease, while astrocytes play diverse supportive roles in maintaining neuronal health . In a mouse model of alzheimer's disease, a deregulation of astrocytic ca homeostasis and signaling, including increased basal [ca]i and spontaneous network activity, was observed . Since soce is upregulated in astrocytes treated with amyloid-, future work on the role of stim and orai proteins might help to understand the pathophysiological regulation of astrocytic ca signaling and function . In microglia, stim1, stim2, and orai1 while stim1 is the primary er ca sensor, stim2 contributes to soce, particularly upon weak ca store depletion . Several microglial functions, i.e., release of cytokines, chemotaxis, and stimulated phagocytosis, are impaired in the absence or after downregulation of these soce components . Dysfunction and dystrophy of microglia has been recognized as a potential cause of neurodegeneration and impaired neuronal development . For example, engulfment of dysfunctional synapses during development and phagocytosis of cellular debris (such as amyloid- protein) are possibly regulated by intracellular ca release and soce in microglia . Therefore, stim1, stim2, and orai1 represent possible targets for the treatment of microglial dysfunction that may underlie neurodegenerative and neurodevelopmental disorders . The author was supported by the deutsche forschungsgemeinschaft (dfg; kr3408/2 - 1).
Acidic foods are consumed globally [1, 2] but universally assumed to be innocent as to their effects on the mouth . Acidic food and beverages can affect natural teeth, and chronic exposure often leads to the development of dental frangibles (attrition, erosion, abrasion, and decay). Not only is acid derived directly from colas, but also after imbibing the cola, acid is produced from biofilm bacteria metabolizing fermentable sugars in the drinks . Human saliva acts as a neutralizing and/or buffering solution on imbibed acid beverages [4, 5]. Intraoral ph (ph 6.8) decreases from, after drinking, an acidulated drink to below ph5 within 2 to 3 minutes . Oral acidogenic bacterial action on fermentable carbohydrates (monosaccharides like glucose and fructose; disaccharides like maltose and sucrose) also aggravates the ph reduction to below ph4 . Intraoral ph takes about 25 minutes to change the acid environment, as further stimulated saliva neutralises any residual acid . The critical ph, at which hydroxyapatite dissolves, is ph5.5, and because teeth are composed of calcium - deficient carbonated hydroxyapatite, they are vulnerable to decalcification in acid media . Tooth material in saliva as a saturated solution of calcium and phosphate will show dissolution or remineralisation, depending on incumbent acidity of the surrounding saliva . This is illustrated by the following formula: (1)ca10(po4)6 (oh)210ca2++6po43 + 2oh. Should the ph fall (increased h concentration and accordingly acidity), the 6po4 ions convert to h2po4 or h2po, and the oh ions are neutralized to water, driving the reaction to the right, that is, dissolution of calcium . If the h concentration diminishes (i.e., ph rises and acid activity falls), the reaction is driven to the left producing remineralization [5, 6]. Repeated exposure to acid drinks changes the intraoral ph to below the critical ph (ph5.5), when chemical dissolution of calcium from dental hydroxyapatite occurs . This sustained low ph, <ph5.5, allows mordant development of frangibles starting and deriving from decalcification . Besides ph and buffering, calcium, phosphate, and to a lesser extent fluoride chronic frequency of imbibing, method of drinking, timing of consumption, or type and quantity of beverage are known to influence the realization of frangibles . The formulae and contents of pop - cola beverages are closely guarded industrial secrets, and sparse data about acidity is provided on their consumer packages . In light of knowing the critical ph for hydroxyapatite, and also that acid drinks require a lot of alkaline salivary flow to neutralize dietary acid, it is most important to know the ph of pop - cola drinks, the strength of their acids, particularly their buffering capacities as to how much alkali is needed to change their ph . Various methods are used to assess ultramicroscopic effects of beverages on teeth; these include surface hardness measurements, surface profilometry, iodide permeability tests, chemical analysis of dissolved minerals, microradiography, confocal scanning microscopy, quantitative light - induced fluorescence, atomic force microscopy, element analysis of solid samples, nano - indentation, ultrasonic measurements, and scanning electron microscopy (sem) with environmental sem (esem). With esem, minimal sample preparation is required, is economical, and minimizes risks for artefacts . Also esem and sem permit progressive assessment of the identical test - tooth material without carbon or metal coating of native surfaces . Recently (2010), variable pressure scanning electron microscope (vp - sem) has refined this technique further . Aimthis study aims to (i) assess ph, and buffering capacities of six pop - cola beverages (initial rise of one ph unit to the critical ph and to neutral ph); (ii) measure calcium and phosphorous content of cola after rinsing from mouths with (dentate) and without teeth (edentulous); (iii) assess scanning electron microscopic (vp - sem) erosive effects on native dental hard tissues after exposure to pop colas; (iv) describe a clinical case of pop - cola erosion . This study aims to (i) assess ph, and buffering capacities of six pop - cola beverages (initial rise of one ph unit to the critical ph and to neutral ph); (ii) measure calcium and phosphorous content of cola after rinsing from mouths with (dentate) and without teeth (edentulous); (iii) assess scanning electron microscopic (vp - sem) erosive effects on native dental hard tissues after exposure to pop colas; (iv) describe a clinical case of pop - cola erosion . Six pop colas, namely, pepsi, diet pepsi, coca cola, diet coca cola, and selection cola and diet selection cola were analyzed . Standard aliquots of cola liquid were tested for ph and buffering capacity (batches of potable over - the - counter colas were purchased by chance at local retail outlets). The acidity as ph and buffering capacity was measured with an automated mettler dl25 titrator (at). Buffering capacities were assessed with 0.5 m naoh solution and titrated to raise the measured cola ph one ph unit, then to ph 5.5, and to ph 7 . Two fresh 60mls aliquots were collected directly from each of 12 cans and placed in separate polystyrene falcon test tubes for measurements . Titration measures were repeated for each sample from different cans in the same batch . For each of the 6 colas, automatic titrator (at) the at was used to determine the ph and buffering capacities of the various pop - cola liquids . For this analysis, the at was used according to described methods by larsen and nyvad 1998 method and measured initial ph's (figure 1) and buffering capacities at the three different stated levels: (i) ph change of one unit from the incipient ph (orange bars, figure 2), (ii) ph change up to the critical level ph 5.5 (red bars, figure 2), and (iii) ph change up to the neutral level ph 7.0 (green bars, figure 2). All measures were assessed at room temperature of 23c . To ensure measurement accuracy and establish baseline controls, the at was calibrated each time before use, at ph 2, 4, 7, and 10, using standard buffer solutions for all levels . A 60 ml volume of the tested sample was then titrated using a 0.5 m naoh solution . The at was used to determine the ph and buffering capacities of the various pop - cola liquids . For this analysis, the at was used according to described methods by larsen and nyvad 1998 method and measured initial ph's (figure 1) and buffering capacities at the three different stated levels: (i) ph change of one unit from the incipient ph (orange bars, figure 2), (ii) ph change up to the critical level ph 5.5 (red bars, figure 2), and (iii) ph change up to the neutral level ph 7.0 (green bars, figure 2). All measures were assessed at room temperature of 23c . To ensure measurement accuracy and establish baseline controls, the at was calibrated each time before use, at ph 2, 4, 7, and 10, using standard buffer solutions for all levels . A 60 ml volume of the tested sample was then titrated using a 0.5 m naoh solution . The six different pop - colas mentioned (coca cola, pepsi cola, selection cola, diet coke, diet pepsi, and diet selection cola) were tested . Two volunteer cohorts, one fully dentate (mean age 22, m: f 4: 2, n = 6) the second edentulous (mean age 52, m: f 3: 3, n = 6), were used to swish with 20 ml aliquots of deionized aquafina water as baseline and subsequently a 20 ml aliquots cola for 60 seconds for each volunteer . Every cola was sampled and analyzed twice, giving 12 analyses for swishes, using standardized analytical chemistry methods . The colas from source and postswish expectorates were analyzed for calcium, and phosphorous contents using inductively coupled plasma with optical emission spectroscopy (icp - oes) and ion chromatography (ic) were used to determine concentrations of anions (fluoride) present in the six colas direct from their cans . (see appendices a.2 and a.3) for our analysis, concentrations of calcium and the major ions of interest as phosphate and fluoride are reported here . To eliminate inter- and intraoperator bias, blindly by third - party technicians unaware of the source of procured samples . Results analysis reveals consistent significant (p <.01 student - t) increases in calcium leeched from dentate subjects after swishing with all the pop - colas tested . The icp - oes and ic measures were compared and there were no significant differences (p>.99) with regard to results obtained for phosphorous . Variable amounts of phosphorous were found in the various pop - colas tested, and this was reflected in the swish findings of phosphorous present after rinsing with these pop - colas . Hard tissue erosion, as chemical dissolution of calcium, may differ between colas, but is produced by all the colas tested . In none of the pop - colas is hard tissue erosive action absent from postswishing exposure, and calcium content in the swish expectorates with teeth all showed significant (p <.01 student - t) increases in dissolved calcium when compared to swishing with water, or swishing without teeth (see figure 3). <.01 student - t) increases in calcium leeched from dentate subjects after swishing with all the pop - colas tested . The icp - oes and ic measures were compared and there were no significant differences (p>.99) with regard to results obtained for phosphorous . Variable amounts of phosphorous were found in the various pop - colas tested, and this was reflected in the swish findings of phosphorous present after rinsing with these pop - colas . Hard tissue erosion, as chemical dissolution of calcium, may differ between colas, but is produced by all the colas tested . In none of the pop - colas is hard tissue erosive action absent from postswishing exposure, and calcium content in the swish expectorates with teeth all showed significant (p <.01 student - t) increases in dissolved calcium when compared to swishing with water, or swishing without teeth (see figure 3). Healthy teeth condemned for extraction (orthodontics or impactions) were used . Immediately after extraction, teeth were placed in deionized water refrigerated at 4c for a maximum of two hours . Each cola was tested at three selected sites on the same tooth; this was repeated on three teeth from the same set of wisdom teeth, which provided fresh native material for assessment . For enamel, flat tooth surfaces were selected; for dentine, samples were obtained by cutting horizontally through the middle of the crown, and the enamel - cemental junction on each tooth used was also tested and examined directly . After samples were cut, each specimen was rinsed with deionized water and air stream dried . All areas of focused interest were examined using scanning electron microscopy with vp - sem . Resolution was at 700x, with accelerating voltage 2025 kv, to assess fracture patterns and morphology . Immediately after scanning controls, each sample was immersed in a cola for 30 s, and subsequently the specimens were immersed for a further 30 s (for a total of 60 s), and the same location was scanned and recorded . Image analysis of morphology, properties and characteristics (cracks, openings, and changes of identifiable shapes) were done with microscopic scales and by micrometer measures using the same microscope magnification of the identical areas tested . After 60 seconds of cola exposure, the specimens were brushed with a standardized rotary tooth brush for 5 seconds under 60 grams pressure . Results ultrascaled measures (700x) reveal significant losses of hard tissue from erosion (p <.01, student's t - test paired). Initial erosion is seen after exposure to the colas, as the chemical loss of calcium widens cracks and causes surface crenellations to develop . After 60 s cola exposure, soft surface crenellations develop . After brushing, ultrascaled measures (700x) reveal significant losses of hard tissue from erosion (p <.01, student's t - test paired). Initial erosion is seen after exposure to the colas, as the chemical loss of calcium widens cracks and causes surface crenellations to develop . After 60 s cola exposure, soft surface crenellations develop . After brushing, evidence of tooth - brush abrasion enamel, ecj and dentin at 60 seconds exposure at 700 show effects after brushing . An 18-year - old male presented complaining of temperature sensitive teeth and that his teeth were getting smaller . His medical history revealed nothing significant to indicate that he may suffer from gord (gastro - oesophageal reflux disorder). The aetiology of gord is variable, but when the gastric contents flow from the stomach to the mouth, it is called gastro - oesophageal reflux disorder (gord). Common symptoms are heart burn along the oesophageal pathway; epigastric pain localised over the stomach; regurgitation into the mouth; dysphagia with or without pain; noncardiac retrosternal pain; chronic coughing and sore throat from laryngitis; vocal hoarseness; a throat globus [16, 17]. He attended his general dentist regularly and brushed and flossed his teeth regularly, morning and evening every day . He used fluoridated toothpaste and used a new soft nylon tufted tooth brush every month . His diet was derived from foods in all food groups (meat / fish, dairy, grain / cereals, and vegetables), but he specifically stressed that he did not drink fresh fruit juices and rarely ate fresh fruit because they hurt his teeth . However, he reported drinking copious amounts of cola solidly from my last two years of in primary school till now, the time which was calculated to be about ten years . He drank all brands of conventionally (carbohydrate) sweetened colas, until three years prior to presenting, the point from which he changed to drinking exclusively synthetically sweetened diet - colas . A fortnight of dietary analysis confirmed these predilections and showed that he consumed between at least one litre to one and a half litres of cola daily, from cans, or bottles depending on availability . This daily cola intake was a habitual part of his diet, and he would swish the cola over his teeth to reduce the gas and enhance the flavour . On presentation (figure 12(a)12(k)), his teeth appeared reduced in size apparently by erosion with reduced enamel and dentin exposure . The upper anteriors were more affected than the rest of the teeth, along with molar and premolar cuspal reduction and incisal attrition . Subgingival probing revealed no periodontal disease (no sulcus depth exceeding 3 mm), and further subgingival exploration showed only healthy tooth surfaces and no caries . All the standard cola drinks show a progressive increased requirement of base to neutral ph 7, as does all the diet cola beverages . The ph and buffering do not fully explain the erosive potential of colas, as the mineral content, concentrations of organic acids (phosphoric and citric), fluoride, and the ability of the mix to remove calcium from the mineral surface are all relevant . However, ph - expressing acid content, buffering ability, and acidic ions available for the overall general mordant effect of acid - cola beverages are more important in producing erosion, as without an acid environment the other stated ions are not active . Figure 1 shows how low the ph of the selected group of pop cola drinks is . All the cola drinks register ph values well below the critical ph 5.5, at which tooth decalcification occurs (figure 1). Among the tested drinks, pepsi cola is the most acidic (ph 2.53) while diet selection cola is the least (ph 3.4). From the results presented in figure 2, selection cola and diet pepsi - cola have the largest buffering capacities for a ph change of one unit . As for the ph change up to its critical value ph 5.5 (figure 2), pepsi cola requires the largest amount of sodium hydroxide; diet pepsi cola (figure 2) finally appears to be the most resistant drink to a ph change up to neutralization (ph 7.0). These results are noteworthy as the low ph values reflect that the pop - cola drinks are extremely acidic and consequently could all contribute to the decalcification of teeth (see figures 611). It is also important to say that an average of 5.86 ml of base is required to bring the ph back to the neutral level of ph 7.0 (figure 2). This indicates that not only are the cola drinks highly acidic when they are first exposed to teeth, but they would also require a large amount of alkaline - stimulated saliva to be neutralized . Whole saliva as a complex buffering solution may provide a definite protective buffering and neutralizing effect on acid dietary drinks [1921]. But with acid drinks, including pop colas, this buffering / neutralizing effect may be overwhelmed . The colas tested are not all chemically identical; yet they all leech calcium from teeth in vivo after contact for 60 seconds (figure 3). The phosphorous content varies (figure 4) because the phosphorous content of the colas from the cans varies . The dental and salivary calcium salts absorb some of the phosphorous from the cola; also the acidity from each cola varies as does the buffering, and reflex stimuli may vary in intensity of reaction . Other intraoral phosphate sources may derive from gingivo - crevicular fluid and calculus (tricalcium phosphate, octa - calcium phosphate, dicalcium diphosphate). Because the phosphorous composition of the colas is variable, the amount of phosphates produced after swishing is also variable (figure 4). Although the chemical composition of the colas tested varied, they all had high buffering capacities and acid activity (ph) well below the critical ph 5.5 . But even when accounting for any extra calcium released in stimulated saliva, the calcium found in swished expectorates from all these colas is consistently and significantly (p <.01 student - t) higher when compared to the calcium content in the can . Tooth erosion, as chemical dissolution of calcium, is derived from an intraoral source and is reflected by an increased content of calcium in all the swishes from the colas tested . The increase in calcium shown in the swishing experiment without teeth (figure 3) after rinsing with the colas may be derived from calcium ions secreted in stimulated saliva . Other sources of calcium may derive in miniscule aliquots from minor salivary glands and circulating oral glycoprotein . The calcium content in the expectorates varies, but the only confounding factor, as a variable that explains the increase of calcium in the swish expectorates, is the presence of teeth; this is particularly highlighted by comparisons in each cola of calcium content in the expectorate swish with teeth that shows significant (p <.01, student - t) increases in dissolved calcium when compared to swishing with water, or swishing without teeth . Some phosphates may derive from stimulated saliva, and some from the teeth or other intraoral phosphate sources, but the amounts of calcium contents measured in the swishes cannot be explained in this experiment, from sources other than from the natural teeth in vivo . Considering that the average rate of resting - saliva secretion is 0.78 ml / min, this would also mean that a long period of time is needed for the mouth to return to neutral ph 7 [4, 11]. Knowing that acids are the most potent stimuli for reflexive stimulated - salivary flow, the rate of stimulated secretion can increase to reach a maximum limit of 8ml saliva per minute . Even if this maximum rate is reached when consuming cola drinks, neutralisation to physiological stable oral ph levels would still need a long period of time to be achieved, and the calcium content from saliva is too low to account for calcium increases after rinsing . Typically after one test bolus in the mouth mg / l, f fails to reduce erosion of teeth in vitro, even with the mean fluoride content from the cans, it was 3.5 mg / l (ranging from 2 to 6 mg / l); this concentration does not stop calcium dissolution after swishing (see figure 5). Increasing the fluoride content is feasible, but higher concentrations would be undesirable, as it would be unacceptably toxic and also negatively affect organoleptic taste properties . The dietary acids from the colas over power any protective effect from saliva and the same applies to the fluoride content of the colas . Some other important biological factors may slightly affect decalcification and tooth erosion, such as the saliva flow rate, its composition, buffering capacity and stimulation capacity, and the acquired pellicle, which has diffusion - limiting properties by its composition, maturation, and thickness . The type of dental substrate and its density of composition also can effect erosion, as does the dental anatomy and occlusion influencing the flow of liquids over the tooth surfaces; and besides the anatomy and histology, the vigorous function of oral soft tissues in relationship to the teeth affects the development of erosion . None of these are as important as the acid composition and ph of the pop - cola drink in producing erosion . Keratosis on the tongue seems to act as a rasper that removes surface tooth material softened by decalcification . Decalcification caused by regurgitated gastric contents in bulimia often manifests first as palatal erosion because of tongue thrusting, removing softened tooth material [15, 23, 24]. The acidulated colas also act as a stimulus for stimulated saliva to flow which contains calcium . But the calcium content of saliva is negligible (mean 5.7 mg/100 ml; range: 210 mg/100 ml), and even with maximum secretion rate of saliva with ranges of 7 to 8 ml in one minute, comparisons between swish expectorates (with and without teeth) indicate that it is impossible for stimulated saliva to secrete calcium in amounts recorded after 60 seconds of swishing . Also it is inordinately difficult to procure age - matched (edentulous) controls without teeth below 35 (dentate controls with teeth had a mean age of 22 years old), or to find people aged 52 years (the mean age of the test edentulous group) without any dental restorative work . The recent increase of consumption of pop drinks is reflected in increased reporting of dental erosion [3, 27]. Data reported here shows that damage occurs at a microscopic level and corroborates information gleaned from epidemiology and marketing . Some consider a 30- or 60-second exposure unreasonably long; this is not valid criticism, as most reported data from other investigators uses target times in excess of 5 minutes, even hours or days of immersion, and 30 seconds and 60 seconds could well approximate the total time a 350 ml can of cola may be exposed to teeth, when people swallow a 60ml bolus and swish it for 5 to 10 seconds . Dental ravages from cola drinks have a high prevalence in children and young adults [7, 29]. Evidence presented here in this electron microscopic study re - enforces and confirms this theory . This study also demonstrates this interaction results in surface etching (figure 6) and cracks (figures 8 and 10). This sem evidence confirms observations about smear layer removal, opening of dentine tubules, and increasing of tubules diameter based on randomly selected sites [30, 31]. This is probably due to disruption of fluid dynamics in the tubules as well as by mechanical loss of tooth material [30, 31]. The results also indicate that with light brushing after 60 s exposure to pop colas, abrasion will be obtained . Tooth wear results for three main processes erosion, attrition, and abrasion, with chemical, physical, and physiological forces interacting to produce a clinical case of dental frangibles . Therapy ranges from eschewing acid drinks, avoiding brushing immediately after drinking pop - drinks, reducing frequency of drinking to fissure sealants and coating, occlusal build - up with overlays, or comprehensive oral rehabilitation with full - coverage crowns . (i) pop - cola drinks tested are acid and may decalcify tooth material . (ii)this study provides evidence that all these six common colas (pepsi cola, diet pepsi cola, coca cola, diet coke, selection cola and diet selection cola), leech calcium out of teeth, after rinsing with pop - colas . (iii) this sem experiment shows that acid pop - cola ignores the smear layer, softens and erodes dental hard tissues, and facilitates abrasion . (iv) the clinical case report shows erosion from chronic imbibing of acid pop colas . These data collectively provide more evidence as a proof that chemical dissolution by tooth decalcification is caused by drinking pop colas . This study demonstrates clear visual evidence of dental erosion with altered enamel, and dentine morphology changes due to short exposure to pop colas . The automatic titrator instrument was standardized with certified reference buffers at ph 2, 4, 7, and 10 . The icp - oes was used to detect the concentrations of calcium, phosphorus (and sodium, potassium, boron, tin, arsenic, iron, nickel, lead, copper, magnesium, zinc, sulphur, chrome, cobalt, and manganese not reported) present in the cola drinks being tested . In order to obtain accurate and precise results, twelve replicates for every drink being tested along with a deionized aquafina water blank were prepared with due diligence . Initial precleaned polypropylene digitubes (50 ml) were clearly labeled, and 20 ml samples were dispensed to each tube with a pipette . Because there is a high sugar content in the regular drinks, a matrix effect is formed, preventing the icp - oes from determining accurate concentrations of those elements being tested . To eliminate any potential matrix, 5 ml of trace metal grade nitric acid was added to each sample, and the samples were digested to 95c for 120 minutes using a 24-position digiprep equipped with a digiprobe . The probe was placed in a sample in order to maintain and monitor the digestion procedure . Once digestion was finished (1) the samples were cooled to room temperature, (2) volumes of twelve replicates and a blank were completed to 50 ml with the deionized water . A calibration curve was prepared from 0.5 to 50 mg / l with certified icp - oes standards diluted in a solution of 2% nitric acid . A total of four points including a calibration blank (consisting of 2% trace metal grade nitric acid) were prepared . Furthermore, two quality control standards were prepared from a different lot of certified icp - oes standards . They were prepared at a concentration of 5 and 25 mg / l . The ic is used to determine the concentration of different anions present in the six cola drinks being tested . For the purpose of our analysis, the concentrations of the major anions, which are fluoride, acetate, formate, chloride, phosphate, nitrate, and sulphate were studied . Other ions were also assessed but not reported here . A fixed volume (5 ml) of the cola is accurately delivered in a properly labeled digitubes (50 ml). The cola samples were then filtered using a very small filter (diameter of pores = 0.45 m) to remove all sediments and impurities present that would cause microbial alterations . A volume of 4 ml of every filtered sample the calibration curve covered a range from 0.01 to 10 mg / l for each anion . Furthermore, two quality control standards were used at concentration of 0.02 and 5 mg / l . The concentration of each ion can then be obtained by evaluating the area under each peak . The concentration of the anions in the cola drinks was determined by plotting calibration curves.
Uncorrected presbyopia impacts negatively on visual function (vf) and quality of life (qol) of affected persons.1 the symptoms of uncorrected presbyopia such as difficulty performing near work, fatigue, headache, and eyestrain, may adversely affect the qol of affected persons in varying degree depending on their occupation and near work habit . Modernization has made the use of devices requiring good near vision such as computers and mobile cell phones more widespread increasing the need for a good near vision . Globally, 419 million people are prevented from performing near tasks in the way they are supposed to, because of uncorrected presbyopia.2 based on the degree of near visual acuity (va), near visual impairment (nvi) have been categorized 3 as mild, moderate, severe, and near vision blindness using the presenting binocular near vision . Mild nvi is an acuity of <20/40 - 20/63 (<6/12 - 6/19); moderate nvi <20/63 - 20/200 (<6/19 - 6/60); severe nvi <20/200 - 20/400 (<6/60 - 3/60); and near vision blindness as a near acuity of <20/400 (<3/60). Studies 14 have reported increasing difficulty in reading, harvesting grains, sewing, and recognizing the small object as the main complaints of people with presbyopia . Presbyopes are also more likely than nonpresbyopes to report not being satisfied with near vision and general health.4 in a rural tanzania population,5 being presbyopic increased the odds of reporting some difficulty with near - vision tasks by two - fold and severe difficulty by more than eight - fold . A study from china 6 reported that difficulties with activities of daily living and resulting social impediments were commonly associated with presbyopia . Elderly people in ibadan, nigeria, were more likely to have significant decrease in qol due to problems with near vision than those of distance vision.7 there is a paucity of data on functional visual impairment and vision - related qol associated with presbyopia in nigeria . Zamfara state government with support from an international ngdo has been implementing a comprehensive eye care program since 20108 with a target of achieving the goals of vision 2020: the right to sight . The aim of this study was to provide data on the impact of presbyopia on vision - related qol in an adult population of northwestern nigeria . The specific objectives were to determine the mean vf scores of persons with presbyopia; to determine the near vision functions most impaired in the study population; to determine the relationship between the degrees of presbyopia and the mean vf score; and to determine the qol score of persons with presbyopia . The findings from this study will provide data to stakeholders for evidence - based planning; advocacy; and eye care services delivery in zamfara and nearby states in nigeria . Ethical approval was obtained from the human research and ethics committee of the national eye centre (nec), kaduna, nigeria . Approval was also given by zamfara state ministry of health and bungudu local government area (lga). This was a population - based cross - sectional survey conducted in april 2012 in bungudu lga of zamfara state, nigeria . Persons 40 years of age and older who have spent at least 6 months in the community were the study population . Person(s) whose presenting distance va is less than 6/60 on snellen chart and did not improve with pinhole (ph); and individuals with mental or other incapacitating illnesses whose vision cannot be tested were excluded from the study . A minimum sample size of 646 was calculated using the formula: where, n = required sample size, z = standard normal deviation, p = expected prevalence, q = (1 p), d = degree of accuracy and multiplied by the design effect, z = 1.96 (95%), p = 55%, d = 0.05 (5%), design effect = 1.7 . Thirteen clusters of 50 persons were selected using a two - stage random sampling with probability proportional to size . The selection of subjects in a sampling unit was by spin - the - bottle method at the center of the cluster, then random - walk process to identify households . All eligible persons in a selected household were included in the survey until the required numbers in a cluster were obtained . In situ ations where the required number of participants was not obtained in a cluster, a neighboring village was sampled for completion . The survey team comprised of an ophthalmologist, ophthalmic nurse (on), enumerator, and a village guide . The enumerator obtained demographic information comprising of age and sex of participants after the consent was signed . An on assessed the distance va of all subjects using the snellen tumbling e - chart at 6 m in ambient outdoor illumination under shade . Pinhole va was done on all subjects who had va <6/18 in either eye . Correct identification of 3 out of 5 optotypes in a line constituted success at reading that line . The ophthalmologist conducted objective and subjective refraction for subjects with va <6/18 after demonstrating improvement of at least one snellen acuity line when tested with a ph in either eye . Participants with the presenting vision of at least 6/60 but <6/18 without ph improvement also proceeded to have near vision test . Near vision was tested at 40 cm, with best distance correction where applicable, using logmar near e - chart under ambient indoor illumination . The distance was maintained using a rope string of 40 cm length attached to the chart at one end and on the forehead of the subject at the other end . Correct identification of 3 out of 5 characters constituted a success in reading a line . Any subject who could not correctly read the optotypes on n8 line had near refraction by addition of spherical plus lenses in increments of 0.25 d monocularly, and then binocularly until the subject read n8 or additional lenses yielded no further improvement in line reading . A person was diagnosed presbyopic if he or she cannot read the n8 optotype at 40 cm with the distance correction if required . Under - corrected presbyopia was present in a subject presenting with near vision spectacles but fails to read n8 . The interview questions included the impact of near vision on various activities; problems with near vision; spectacles use; and prior consultation by eye care professionals . Vfs covered in the interview comprised writing, recognizing small objects, cooking, farming (harvesting of grains), reading, sewing (threading a needle), using a mobile phone, and getting dressed up . If the answer was yes, they were then asked to rate the difficulty that they experience for their near - vision performance for each activity - based on a rating of 1 - 5 where: no difficulty, mild difficulty, moderate difficulty, severe difficulty, anddo not undertake the task (not applicable). Participants were instructed that this was a linear increase in severity, and other factors that did not relate to their near vision (e.g., mobility, distance vision) were not relevant to this question . They were asked how much satisfaction they had with their distance vision, near vision, and general health . Their level of satisfaction was recorded as: very good, good, moderate, bad, andvery bad . These ratings correspond to 100%, 75%, 50%, 25%, and 0% scores, respectively . The interview proceeded to questions on difficulty in carrying out daily tasks that were vision specific and graded as: never, sometimes, often, very often, andextreme / cannot do . The questions asked included difficulty in carrying out activities such as going down steps or stairs, noticing obstacles while walking alone, and recognition of the face of persons among several others . Questions were then asked on how their vision affected psychosocial functions and recorded as above . The questions were how often have you been hesitant to participate in social functions, how often have you found that you are ashamed or embarrassed, how often have you felt that you are a burden on others, and how often do you worry that you may lose your remaining sight . Finally, participants were asked to consider their vision in typical tasks of daily life such as, reading newspaper, recognizing objects on a postcard size (11 cm 16 cm) photograph, recognizing faces of people from across the room, reading road signs, and picking out details in pictures from a distance of 20 m. they were then asked how much difficulty they had with their vision and were recorded as: none, mild, moderate, severe, andextreme / cannot do . All collected data were entered into a modified vf-14 vision - related qol questionnaire for each participant . Visual functioning and qol scores were also calculated by allocating scores of 4, 3, 2, 1, and 0 to no difficulty, mild difficulty, moderate difficulty, and extreme difficulty / cannot in carrying out an activity, respectively . Multiplying the score for the individual by 25 provide the highest score of 100 and lowest of zero . The mean scores were compared for age, gender, and degree of presbyopia among participants . Ethical approval was obtained from the human research and ethics committee of the national eye centre (nec), kaduna, nigeria . Approval was also given by zamfara state ministry of health and bungudu local government area (lga). This was a population - based cross - sectional survey conducted in april 2012 in bungudu lga of zamfara state, nigeria . Persons 40 years of age and older who have spent at least 6 months in the community were the study population . Person(s) whose presenting distance va is less than 6/60 on snellen chart and did not improve with pinhole (ph); and individuals with mental or other incapacitating illnesses whose vision cannot be tested were excluded from the study . A minimum sample size of 646 was calculated using the formula: where, n = required sample size, z = standard normal deviation, p = expected prevalence, q = (1 p), d = degree of accuracy and multiplied by the design effect, z = 1.96 (95%), p = 55%, d = 0.05 (5%), design effect = 1.7 . Thirteen clusters of 50 persons were selected using a two - stage random sampling with probability proportional to size . The selection of subjects in a sampling unit was by spin - the - bottle method at the center of the cluster, then random - walk process to identify households . All eligible persons in a selected household were included in the survey until the required numbers in a cluster were obtained . In situ ations where the required number of participants was not obtained in a cluster, the survey team comprised of an ophthalmologist, ophthalmic nurse (on), enumerator, and a village guide . The enumerator obtained demographic information comprising of age and sex of participants after the consent was signed . An on assessed the distance va of all subjects using the snellen tumbling e - chart at 6 m in ambient outdoor illumination under shade . Pinhole va was done on all subjects who had va <6/18 in either eye . Correct identification of 3 out of 5 optotypes in a line constituted success at reading that line . The ophthalmologist conducted objective and subjective refraction for subjects with va <6/18 after demonstrating improvement of at least one snellen acuity line when tested with a ph in either eye . Participants with the presenting vision of at least 6/60 but <6/18 without ph improvement also proceeded to have near vision test . Near vision was tested at 40 cm, with best distance correction where applicable, using logmar near e - chart under ambient indoor illumination . The distance was maintained using a rope string of 40 cm length attached to the chart at one end and on the forehead of the subject at the other end . Correct identification of 3 out of 5 characters constituted a success in reading a line . Any subject who could not correctly read the optotypes on n8 line had near refraction by addition of spherical plus lenses in increments of 0.25 d monocularly, and then binocularly until the subject read n8 or additional lenses yielded no further improvement in line reading . A person was diagnosed presbyopic if he or she cannot read the n8 optotype at 40 cm with the distance correction if required . Under - corrected presbyopia was present in a subject presenting with near vision spectacles but fails to read n8 . The interview questions included the impact of near vision on various activities; problems with near vision; spectacles use; and prior consultation by eye care professionals . Vfs covered in the interview comprised writing, recognizing small objects, cooking, farming (harvesting of grains), reading, sewing (threading a needle), using a mobile phone, and getting dressed up . If the answer was yes, they were then asked to rate the difficulty that they experience for their near - vision performance for each activity - based on a rating of 1 - 5 where: no difficulty, mild difficulty, moderate difficulty, severe difficulty, anddo not undertake the task (not applicable). Participants were instructed that this was a linear increase in severity, and other factors that did not relate to their near vision (e.g., mobility, distance vision) were not relevant to this question . They were asked how much satisfaction they had with their distance vision, near vision, and general health . Their level of satisfaction was recorded as: very good, good, moderate, bad, andvery bad . These ratings correspond to 100%, 75%, 50%, 25%, and 0% scores, respectively . The interview proceeded to questions on difficulty in carrying out daily tasks that were vision specific and graded as: never, sometimes, often, very often, andextreme / cannot do . The questions asked included difficulty in carrying out activities such as going down steps or stairs, noticing obstacles while walking alone, and recognition of the face of persons among several others . Questions were then asked on how their vision affected psychosocial functions and recorded as above . The questions were how often have you been hesitant to participate in social functions, how often have you found that you are ashamed or embarrassed, how often have you felt that you are a burden on others, and how often do you worry that you may lose your remaining sight . Finally, participants were asked to consider their vision in typical tasks of daily life such as, reading newspaper, recognizing objects on a postcard size (11 cm 16 cm) photograph, recognizing faces of people from across the room, reading road signs, and picking out details in pictures from a distance of 20 m. they were then asked how much difficulty they had with their vision and were recorded as: none, mild, moderate, severe, andextreme / cannot do . All collected data were entered into a modified vf-14 vision - related qol questionnaire for each participant . Qol scores were also calculated by allocating scores of 4, 3, 2, 1, and 0 to no difficulty, mild difficulty, moderate difficulty, and extreme difficulty / cannot in carrying out an activity, respectively . Multiplying the score for the individual by 25 provide the highest score of 100 and lowest of zero . The mean scores were compared for age, gender, and degree of presbyopia among participants . A total of 635 subjects were examined out of 650 enumerated constituting a response rate of 97.7% as shown in table 1 . The mean age of participants was 53.59 years, (95% confidence interval [ci]: 52.75%-54.43%). Females constituted the majority of the subjects that were not examined (13/15 = 86%) due to absenteeism or refusal . One - hundred ninety - three persons were unable to read n8 optotype at 40 cm and thus diagnosed with presbyopia . Age and sex distribution of individuals examined the mean vf score of all the participants with presbyopia was (95% ci 83.09%-87.09%). The lowest mean score (82.36) was obtained in persons 70 years and above [table 2]. The mean vf score of the female participants was 87.95.for males, showing a strong association of high vf scores with being female (p = 0.003). The vfs that recorded the lowest scores were the use of mobile phones writing, the wind blowing of grains, reading, and threading a needle . None of the presbyopes aged 40 - 49 years had difficulty recognizing close objects or faces of people near - by . . The higher the degree of presbyopia, the lower the mean vf scores (p <0.005). Mean vf score by age group of presbyopes the relationship between the degree of presbyopia and mean visual function score the mean qol score of participants with presbyopia was (95% ci: 76.72%-79.52%). The mean score was higher for persons 40 - 49 years and lowest in the 70 years and above (p <0.0005) as shown in table 3 . The mean qol scores for male and female participants with presbyopia were 65.57 and 67.81, respectively (p = 0.237). The higher the degree of presbyopia, the lower the reported level of satisfaction with both distant and near vision [figure 2]. The mean qol score generally decreases with increasing age although this was only statistically significant in response to noticing obstacles while walking, going down stairs, and carrying out outdoor activities . Psychosocial activities such as hesitation to participate in social functions recorded higher mean scores [table 4]. Mean quality of life score of presbyopes by age group mean quality of life score of participants by the degree of presbyopia mean qol score per activity of daily living across age group this high response rate recorded in this study, attributable to adequate community mobilization, should be a reflection of qol and visual functional impairment associated with presbyopia in the target and possibly nearby population . The result of this population - based study on presbyopia will further add to the existing knowledge from few similar studies conducted in nigeria and other parts of africa . Since the amplitude of accommodation continues to recede with age,9 there is a need to take actions that will improve the negative impact of presbyopia on the qol . The mean age of participants in this study is similar to a study conducted in gwagwalada, nigeria.4 the number of females examined was less than their male counterparts; of the 15 persons that refused / absent during examination 13 were females . The high refusal rate (86% of refusals) among females contributed to the significant difference in the number examined, this was due to lack of consent by their dominant male spouses . This study revealed that the mean vf and qol scores of presbyopes were high (more than 75), and the higher the dioptric add required to read n8 at 40 cm, the lower the mean score, implying negative impact on vf . The lowest vf scores were in relation to ability to read, write, and use mobile cell phones . Similar studies 14 have reported increasing difficulty in reading, harvesting grains, sewing, and recognizing small objects as the main complaints of people with presbyopia . The visual impairment associated with the use of mobile cell phones among the rural populace with presbyopia in this study underscores how advancement in technology brings out new challenges in ophthalmic practice . The use of near vision spectacles will, therefore, allow easy use of mobile cell phones for communication and promote business in addition to other multiple benefits . Our study found that the higher the dioptric requirement the more difficulty in noticing obstacles, walking (p = 0.014), and recognizing faces of a person standing near - by other ocular and systemic conditions associated with increasing age might have influenced this finding since the older participants are more prone to other age - related diseases . A similar study 4 in nigeria has reported presbyopes to have less satisfaction with their distance and near vision . In rural tanzania,5 being presbyopic increased the odds of reporting some difficulty with near - vision tasks by two - fold and severe difficulty by more than eight - fold . The fact that presbyopia mainly affect near vision may explain why severity of presbyopia had minimal impact on psychosocial activities such as hesitant to participate in social functions, ashamed or embarrassed, and feels you are a burden on others . This finding contrast the findings of a study from china 6 that reported limitations in social functions although the authors were not specific as per the functions affected . The onset of presbyopia influences near work habits of individuals and as such no uniform method can accurately detect it among different persons . In this study, the inability to read n8 at 40 cm with logmar near chart may have underdiagnosed participants with presbyopia whose habitual near work distance is <40 cm . The questions on vf and qol were subjective, and rating of the amount of difficulty in carrying out an activity may be difficult considering the low literacy level of participants in this study.10 also being a descriptive study, other confounders not isolated, may have contributed to decrease vf and qol scores . Uncorrected presbyopia is associated with functional visual impairment and reduces qol especially in the ability to read, write, and use of mobile phones . The study populations have a need for awareness creation on presbyopia and provision of accessible and affordable near vision spectacles services to improve the qol of affected persons.
The fourth diabetes atlas, published by the international diabetes federation (idf), revealed that there were an estimated 50.8 million cases of diabetes in india in 2010 . Data on hypertension, available from several regional studies, indicate an increase of about 30 times among urban indians and 10 times among rural indians, within a span of three and six decades, respectively . To date, no nation - wide epidemiological study aimed at determining the prevalence of hypertension has been reported . Hypertension is 1.52 times more prevalent in diabetic as in non - diabetic individuals and about 3035% of hypertensives are detected to have diabetes . The combination of diabetes and hypertension imparts an additive risk and accelerates progression of both microvascular and macrovascular complications . Hence, these two conditions contribute significantly to the growing load of cardiovascular diseases and lead to a high economic burden that accounts for 520% of the total indian healthcare expenditure . This rapid increase in diabetes and hypertension in india is most likely to result in a twin epidemic . The screening india's twin epidemic (site) study was aimed at determining the number of diagnosed and undiagnosed cases of diabetes and hypertension in india and reporting the measures employed to manage and control these diseases in an outpatient setting . The study was conducted in eight states maharashtra, delhi, tamil nadu, west bengal, karnataka, andhra pradesh, madhya pradesh, and gujarat . Although site is not a population - based study, it is one of the largest studies of its kind . Also, in lieu of poor existing information, site serves as a good proxy for providing valuable insights on the national prevalence and management of both diabetes and hypertension . We present herewith the site study design, methodologies and highlight the amendments made as the study progressed . Site was a cross - sectional, national, multicentric, non - randomized, observational study . The primary objective of the study was to estimate the prevalence of diagnosed and undiagnosed diabetes and hypertension in outpatient settings in major indian states . The secondary objectives included estimation of the prevalence of other cardiovascular risk factors such as dyslipidemia, obesity [body mass index (bmi), waist circumference (wc), and waist - hip ratio (whr)], metabolic syndrome, urine microalbuminuria, arrhythmia, diet, smoking and alcohol consumption; estimation of the prevalence of abdominal obesity in sub - groups of patients (hypertension, diabetes, dyslipidemia, smokers and alcoholics); and understanding the management and the level of control of hypertension and diabetes . The study was conducted in accordance with the guidelines for good epidemiology practices . The protocol complied with recommendations of the 18 world health congress (helsinki, 1964) and the local laws and regulations of india . The sample size depended on the expected prevalence of diabetes / hypertension (whichever was lower). Based on the conservative expected prevalence of 15% for diabetes, it was determined that a sample size of 1225 patients was required per state . Assuming a dropout rate of 39%, it was estimated that 2000 patients were required to ensure 95% confidence such that the prevalence of diabetes is between 13% and 17%, that is, 15% 2% in each state . It was initially planned to conduct the study in 10 states across five zones, north, south, east, west and central, in waves one state at a time and recruit 2000 patients from 100 centers per wave . Physician selection: the study was conducted in outpatient settings and the data were collected from general practitioners rather than specialists . Also, the source of any potential bias was much reduced, as the number of people presenting the disease would evidently be higher at a specialist clinic rather than a general practitioner's clinic . Patient selection: patients 18 years of age and those ready to sign the data release consent form along with an acceptance to take the screening tests were included in the study . Pregnant women are predisposed to gestational diabetes and weight gain, and their exclusion decreased eventual confounding effects on the results . The first 10 patients visiting the physicians clinic on two consecutive days, irrespective of the purpose of the visit, and satisfying the inclusion criteria, were selected for the study . During 20092010, site was conducted in 8 states including 7 of the top 10 most populous indian states and the national capital territory [figure 1]. For the remaining two planned states, on the basis of the fact that the sample size was calculated per state and that a low dropout rate was observed among the patients enrolled, the national coordinators deemed the data adequate to pan out substantial results . So, by the end of the 8 wave, the recruitment was declared completed . Because of a national multicentric approach of the study, the data obtained were representative of diverse demographic populations within india . The states in which the screening india's twin epidemic study was conducted the study was sponsored by sanofi - aventis (india). Site management organization (smo) and data management services were provided by max neeman ltd . The sponsor ensured that the central laboratory, smo, and data management personnel dedicated to study, as well as the participating physicians, received adequate training on various aspects of the study . Thus, the complete study team was well versed with the data collection form (dcf) and the study procedures were executed uniformly . The data collection process was carried out in two steps [figure 2]. Data collection process of the screening india's twin epidemic study the dcf used was designed in line with the study objectives . It captured details on the following aspects: demographics (age and gender), anthropometrics (height, weight, waist and hip circumference), behavioral (smoking and alcohol use), medical history (diabetes and its treatment, hypertension and its treatment, and cardiovascular diseases like ischemic heart disease, myocardial infarction and stroke), type of diet (vegetarian / non - vegetarian), and family history of diabetes and hypertension . Details on the patient's socioeconomic, educational, psychosocial, and physical activity status were not collected . Anthropometric measurements: height was measured without shoes, in centimeters, using a standard stadiometer . Weight was measured with light clothes and without shoes, in kilograms, using a professional calibrated weighing scale, and rounded off to the nearest number . Since no standard weighing machine was employed, inter - instrument variations cannot be ruled out . Body mass index (bmi) was calculated using the formula: weight (kg)/height (m). The hip and waist measurements were taken using a standard non - stretchable anthropometric measure tape across all study sites . The hip circumference (hc) was measured over light clothing . The patient was made to stand erect with arms at the sides, feet placed together with weight equally distributed on each leg, and the gluteal muscles relaxed . The tape was placed horizontally around the point of the maximum posterior extension of the buttocks . The patient's measured hc was calculated as the mean of the two observations or the mean of the two closest measurements . The patient was made to stand erect with arms at the sides and feet placed about 2530 cm apart with weight equally distributed on each leg . The wc was measured at the part of the trunk located midway between the lower costal margin and the iliac crest . The measurer stood beside the patient and made the tape fit snugly, without compressing any underlying soft tissues . The circumference was measured in centimeters to the nearest 0.5 cm at the end of a normal expiration . The method and angles of measurement were adequately detailed to ensure limited variance in the technique . Whr was calculated by dividing the wc (cm) by the hc (cm). Blood pressure (bp) measurement: a mercury sphygmomanometer was used to measure the bp, preferably from the right arm of the patient . An average of two readings, recorded at 5-minutes interval, was taken to ensure accuracy . At the end of visit 1, patients received a coupon of identification and were required to visit any of the nearest metropolis clinical diagnostic centers, within 2 - 5 days . Fasting lipid profile, microalbuminuria, fasting plasma glucose (fpg), and hba1c were determined for each patient presenting a valid coupon . Hba1c was measured by the bio - rad d10 high - pressure liquid chromatography method . Urine microalbumin was measured by a turbidimetric inhibition immunoassay technique and enzymatic reactions were applied to quantify total cholesterol (cholesterol esterase), high - density lipoproteins (hdl; cholesterol oxidase, esterase, and peroxidase), and triglycerides (tg; lipoprotein lipase, glycerol kinase, glycerol-3-phosphate oxidase, and peroxidase). Low - density lipoprotein (ldl) was also calculated by using friedewald's equation for tg <300 mg / dl . Diabetes was diagnosed according to the american diabetes association (ada) guidelines, and hypertension was classified according to the recommendations of the seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure (jnc 7). Metabolic syndrome was diagnosed on the basis of national cholesterol education program (ncep). Bmi was categorized into normal (<23 kg / m), overweight (2325 kg / m) and obese (> 25 kg / m), according to the guidelines jointly suggested by the indian health ministry, diabetes foundation of india, all india institute of medical sciences, indian council of medical research, and national institute of nutrition and 20 other institutions . Normal and truncal obesity was categorized according to the whr, as suggested by the ncep . Fasting lipid profile revealed the total cholesterol, tg, hdl - cholesterol, and ldl - cholesterol levels . Of these, elevated tg (150 mg / dl) and concomitant low hdl - cholesterol (<40 mg / dl) were used to identify dyslipidemia . The criteria used are (1) primarily attributed to atherogenic dyslipidemia, (2) linked to insulin resistance, (3) measurable during the initial phase of development of metabolic syndrome, (4) individually extremely inheritable, (5) simple to measure, and (6) determinants of the atherogenic index of plasma (aip), a key prognostic marker of cardiovascular disease, calculated as log (tg / hdl - c). Urine microalbumin was defined according to the albumin creatinine ratio (acr) as normoalbuminuria (acr <30 g / mg), microalbuminuria (30300 g / mg), and macroalbuminuria (> 300 g / mg). Monitoring visits were randomly conducted for at least 20% of the active sites (enrolled at least one patient) and the reports documented . Phlebotomy services, testing, processing, shipment of blood and urine samples, and preparation and distribution of reports were undertaken by the central laboratory (metropolis healthcare ltd . ). Data entry, verification, and validation were carried out using pheedit database system, version 3.1 (sas institute inc ., cary, nc, usa). A double - entry method was used to ensure that the data (except comments) were transferred accurately from the dcfs to the database . Moreover, every modification in the database could be traced using an audit trail . A data checking plan was established to define all automatic validation checks, as well as the supplemental manual checks . All discrepancies were researched until resolved . Additionally, a national steering committee comprising india's leading endocrinologists and cardiologists was established to provide scientific direction for the study . The total sample number, mean, standard deviation, and range (minimum and maximum) were calculated for all continuous variables (age, height, wc, hc, and lipid values), and frequency and percentage were calculated for all categorical variables (gender, presence of hypertension and diabetes and their treatment, smoking status, and urine microalbuminuria). Student's t - test and chi - square test were used to make comparisons between groups, with p value <0.05 considered statistically significant . A multivariate analysis was performed to estimate the association of hypertension and diabetes with dyslipidemia, obesity, metabolic syndrome, microalbuminuria, arrhythmia, smoking, alcohol consumption, and diet . A simple linear regression analysis was performed to estimate the contribution of individual variables to systolic and diastolic bp . Relative risk for developing diabetes and hypertension was calculated by assessing the following variables: age, gender, bmi, whr, family history of diabetes and hypertension, and smoking status . Site was a cross - sectional, national, multicentric, non - randomized, observational study . The primary objective of the study was to estimate the prevalence of diagnosed and undiagnosed diabetes and hypertension in outpatient settings in major indian states . The secondary objectives included estimation of the prevalence of other cardiovascular risk factors such as dyslipidemia, obesity [body mass index (bmi), waist circumference (wc), and waist - hip ratio (whr)], metabolic syndrome, urine microalbuminuria, arrhythmia, diet, smoking and alcohol consumption; estimation of the prevalence of abdominal obesity in sub - groups of patients (hypertension, diabetes, dyslipidemia, smokers and alcoholics); and understanding the management and the level of control of hypertension and diabetes . The study was conducted in accordance with the guidelines for good epidemiology practices . The protocol complied with recommendations of the 18 world health congress (helsinki, 1964) and the local laws and regulations of india . The sample size depended on the expected prevalence of diabetes / hypertension (whichever was lower). Based on the conservative expected prevalence of 15% for diabetes, it was determined that a sample size of 1225 patients was required per state . Assuming a dropout rate of 39%, it was estimated that 2000 patients were required to ensure 95% confidence such that the prevalence of diabetes is between 13% and 17%, that is, 15% 2% in each state . It was initially planned to conduct the study in 10 states across five zones, north, south, east, west and central, in waves one state at a time and recruit 2000 patients from 100 centers per wave . Physician selection: the study was conducted in outpatient settings and the data were collected from general practitioners rather than specialists . Also, the source of any potential bias was much reduced, as the number of people presenting the disease would evidently be higher at a specialist clinic rather than a general practitioner's clinic . Patient selection: patients 18 years of age and those ready to sign the data release consent form along with an acceptance to take the screening tests were included in the study . Pregnant women are predisposed to gestational diabetes and weight gain, and their exclusion decreased eventual confounding effects on the results . The first 10 patients visiting the physicians clinic on two consecutive days, irrespective of the purpose of the visit, and satisfying the inclusion criteria, were selected for the study . During 20092010, site was conducted in 8 states including 7 of the top 10 most populous indian states and the national capital territory [figure 1]. For the remaining two planned states, on the basis of the fact that the sample size was calculated per state and that a low dropout rate was observed among the patients enrolled, the national coordinators deemed the data adequate to pan out substantial results . So, by the end of the 8 wave, the recruitment was declared completed . Because of a national multicentric approach of the study, the data obtained were representative of diverse demographic populations within india . Site management organization (smo) and data management services were provided by max neeman ltd . The sponsor ensured that the central laboratory, smo, and data management personnel dedicated to study, as well as the participating physicians, received adequate training on various aspects of the study . Thus, the complete study team was well versed with the data collection form (dcf) and the study procedures were executed uniformly . The data collection process was carried out in two steps [figure 2]. Data collection process of the screening india's twin epidemic study the dcf used was designed in line with the study objectives . It captured details on the following aspects: demographics (age and gender), anthropometrics (height, weight, waist and hip circumference), behavioral (smoking and alcohol use), medical history (diabetes and its treatment, hypertension and its treatment, and cardiovascular diseases like ischemic heart disease, myocardial infarction and stroke), type of diet (vegetarian / non - vegetarian), and family history of diabetes and hypertension . Details on the patient's socioeconomic, educational, psychosocial, and physical activity status were not collected . Anthropometric measurements: height was measured without shoes, in centimeters, using a standard stadiometer . Weight was measured with light clothes and without shoes, in kilograms, using a professional calibrated weighing scale, and rounded off to the nearest number . Since no standard weighing machine was employed, inter - instrument variations cannot be ruled out . Body mass index (bmi) was calculated using the formula: weight (kg)/height (m). The hip and waist measurements were taken using a standard non - stretchable anthropometric measure tape across all study sites . The hip circumference (hc) was measured over light clothing . The patient was made to stand erect with arms at the sides, feet placed together with weight equally distributed on each leg, and the gluteal muscles relaxed . The tape was placed horizontally around the point of the maximum posterior extension of the buttocks . The patient's measured hc was calculated as the mean of the two observations or the mean of the two closest measurements . The patient was made to stand erect with arms at the sides and feet placed about 2530 cm apart with weight equally distributed on each leg . The wc was measured at the part of the trunk located midway between the lower costal margin and the iliac crest . The measurer stood beside the patient and made the tape fit snugly, without compressing any underlying soft tissues . The circumference was measured in centimeters to the nearest 0.5 cm at the end of a normal expiration . The method and angles of measurement were adequately detailed to ensure limited variance in the technique . Whr was calculated by dividing the wc (cm) by the hc (cm). Blood pressure (bp) measurement: a mercury sphygmomanometer was used to measure the bp, preferably from the right arm of the patient . An average of two readings, recorded at 5-minutes interval, was taken to ensure accuracy . At the end of visit 1, patients received a coupon of identification and were required to visit any of the nearest metropolis clinical diagnostic centers, within 2 - 5 days . Fasting lipid profile, microalbuminuria, fasting plasma glucose (fpg), and hba1c were determined for each patient presenting a valid coupon . Hba1c was measured by the bio - rad d10 high - pressure liquid chromatography method . Urine microalbumin was measured by a turbidimetric inhibition immunoassay technique and enzymatic reactions were applied to quantify total cholesterol (cholesterol esterase), high - density lipoproteins (hdl; cholesterol oxidase, esterase, and peroxidase), and triglycerides (tg; lipoprotein lipase, glycerol kinase, glycerol-3-phosphate oxidase, and peroxidase). Low - density lipoprotein (ldl) was also calculated by using friedewald's equation for tg <300 mg / dl . It captured details on the following aspects: demographics (age and gender), anthropometrics (height, weight, waist and hip circumference), behavioral (smoking and alcohol use), medical history (diabetes and its treatment, hypertension and its treatment, and cardiovascular diseases like ischemic heart disease, myocardial infarction and stroke), type of diet (vegetarian / non - vegetarian), and family history of diabetes and hypertension . Details on the patient's socioeconomic, educational, psychosocial, and physical activity status were not collected . Anthropometric measurements: height was measured without shoes, in centimeters, using a standard stadiometer . Weight was measured with light clothes and without shoes, in kilograms, using a professional calibrated weighing scale, and rounded off to the nearest number . Since no standard weighing machine was employed, inter - instrument variations cannot be ruled out . Body mass index (bmi) was calculated using the formula: weight (kg)/height (m). The hip and waist measurements were taken using a standard non - stretchable anthropometric measure tape across all study sites . The hip circumference (hc) the patient was made to stand erect with arms at the sides, feet placed together with weight equally distributed on each leg, and the gluteal muscles relaxed . The tape was placed horizontally around the point of the maximum posterior extension of the buttocks . The patient's measured hc was calculated as the mean of the two observations or the mean of the two closest measurements . The patient was made to stand erect with arms at the sides and feet placed about 2530 cm apart with weight equally distributed on each leg . The wc was measured at the part of the trunk located midway between the lower costal margin and the iliac crest . The measurer stood beside the patient and made the tape fit snugly, without compressing any underlying soft tissues . The circumference was measured in centimeters to the nearest 0.5 cm at the end of a normal expiration . The method and angles of measurement were adequately detailed to ensure limited variance in the technique . Whr was calculated by dividing the wc (cm) by the hc (cm). Blood pressure (bp) measurement: a mercury sphygmomanometer was used to measure the bp, preferably from the right arm of the patient . An average of two readings, recorded at 5-minutes interval, was taken to ensure accuracy . At the end of visit 1, patients received a coupon of identification and were required to visit any of the nearest metropolis clinical diagnostic centers, within 2 - 5 days . Fasting lipid profile, microalbuminuria, fasting plasma glucose (fpg), and hba1c were determined for each patient presenting a valid coupon . Hba1c was measured by the bio - rad d10 high - pressure liquid chromatography method . Urine microalbumin was measured by a turbidimetric inhibition immunoassay technique and enzymatic reactions were applied to quantify total cholesterol (cholesterol esterase), high - density lipoproteins (hdl; cholesterol oxidase, esterase, and peroxidase), and triglycerides (tg; lipoprotein lipase, glycerol kinase, glycerol-3-phosphate oxidase, and peroxidase). Low - density lipoprotein (ldl) was also calculated by using friedewald's equation for tg <300 mg / dl . Diabetes was diagnosed according to the american diabetes association (ada) guidelines, and hypertension was classified according to the recommendations of the seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure (jnc 7). Metabolic syndrome was diagnosed on the basis of national cholesterol education program (ncep). Bmi was categorized into normal (<23 kg / m), overweight (2325 kg / m) and obese (> 25 kg / m), according to the guidelines jointly suggested by the indian health ministry, diabetes foundation of india, all india institute of medical sciences, indian council of medical research, and national institute of nutrition and 20 other institutions . Normal and truncal obesity was categorized according to the whr, as suggested by the ncep . Fasting lipid profile revealed the total cholesterol, tg, hdl - cholesterol, and ldl - cholesterol levels . Of these, elevated tg (150 mg / dl) and concomitant low hdl - cholesterol (<40 mg / dl) were used to identify dyslipidemia . The criteria used are (1) primarily attributed to atherogenic dyslipidemia, (2) linked to insulin resistance, (3) measurable during the initial phase of development of metabolic syndrome, (4) individually extremely inheritable, (5) simple to measure, and (6) determinants of the atherogenic index of plasma (aip), a key prognostic marker of cardiovascular disease, calculated as log (tg / hdl - c). Urine microalbumin was defined according to the albumin creatinine ratio (acr) as normoalbuminuria (acr <30 g / mg), microalbuminuria (30300 g / mg), and macroalbuminuria (> 300 g / mg). Monitoring visits were randomly conducted for at least 20% of the active sites (enrolled at least one patient) and the reports documented . Phlebotomy services, testing, processing, shipment of blood and urine samples, and preparation and distribution of reports were undertaken by the central laboratory (metropolis healthcare ltd . ). Data entry, verification, and validation were carried out using pheedit database system, version 3.1 (sas institute inc ., a double - entry method was used to ensure that the data (except comments) were transferred accurately from the dcfs to the database . Moreover a data checking plan was established to define all automatic validation checks, as well as the supplemental manual checks . All discrepancies were researched until resolved . Additionally, a national steering committee comprising india's leading endocrinologists and cardiologists was established to provide scientific direction for the study . The total sample number, mean, standard deviation, and range (minimum and maximum) were calculated for all continuous variables (age, height, wc, hc, and lipid values), and frequency and percentage were calculated for all categorical variables (gender, presence of hypertension and diabetes and their treatment, smoking status, and urine microalbuminuria). Student's t - test and chi - square test were used to make comparisons between groups, with p value <0.05 considered statistically significant . A multivariate analysis was performed to estimate the association of hypertension and diabetes with dyslipidemia, obesity, metabolic syndrome, microalbuminuria, arrhythmia, smoking, alcohol consumption, and diet . A simple linear regression analysis was performed to estimate the contribution of individual variables to systolic and diastolic bp . Relative risk for developing diabetes and hypertension was calculated by assessing the following variables: age, gender, bmi, whr, family history of diabetes and hypertension, and smoking status . Screening studies play a very important role in determining the trends of disease prevalence; such trends can provide valuable insight regarding necessary corrective measures that can be implemented in a given population . India has witnessed a constant increase in the prevalence of diseases such as diabetes and hypertension . However, most previous reports on the prevalence of both diabetes and hypertension are either regional or vary in methodologies and sampling frames . Site aims not only to indicate the most accurate prevalence data of diabetes and hypertension in an outpatient setting in india at the state, regional, and national levels, but also to raise awareness on the need for early diagnosis and on the management of these diseases to reduce complications . After wave-1 and wave-2, the protocol was refined and the laboratory investigations simplified and then employed for all other subsequent waves . During the first wave conducted in maharashtra, the investigations included: random blood sugar (rbs; in all patients), electrocardiogram (ecg; in patients 40 years of age), and hba1c (in known cases of diabetes) at visit-1; and the oral glucose tolerance test (ogtt; in patients with rbs 140 mg / dl) and microalbuminuria test (in patients with rbs 140 mg / dl and in patients with hypertension) at visit-2 . The ogtt involved a lengthy procedure that was logistically difficult to perform in general clinics . Going forward, this attrition could have potentially introduced a bias in the study results . Thus, based on the experiences of the pilot study in maharashtra, certain protocol improvements were suggested for the second wave conducted in the national capital territory, delhi . The ada recommends either ogtt or fpg and disease - related symptoms (polydipsia, polyuria, and unexplained weight loss) for the confirmation of diabetes . During wave-2, the ogtt was not performed; fpg was measured for all unknown cases of diabetes, as opposed to rbs in the pilot study . Though signs and symptoms of diabetes were recorded in this amended protocol, they did not play a decisive role in diagnosing new cases of diabetes . Additionally, specifications with regard to alcohol consumption and diet of patients were included in the dcf . Alcohol consumption and diet are established high - risk factors leading to the development and progression of diabetes and hypertension . Since significant regional differences exist in the indian diet, it would be interesting to assess if a correlation exists between the regional prevalence of these diseases and diet . To establish a connection between lifestyle factors (diet and alcohol consumption) and prevalence of diabetes and hypertension this inclusion also broadened the scope of secondary analyses to be performed with the results of this study . During wave-2, difficulties in maintaining uniformity in the ecg recordings and in dealing with logistical hurdles of organizing the ecg test at the sites surfaced . The inability to standardize ecg testing in a study of this magnitude would lead to inaccurate data generation . Additionally, as an effort to further simplify the protocol, the fpg and hba1c testing was conducted in all patients from wave-3 onward . However, by employing hba1c as a screening tool as per the newly proposed guidelines released in july 2009, our approach to diagnosing diabetes was further simplified . Additionally, the correlation between fpg and hba1c for the diagnosis of diabetes could also be studied . An important limitation of this study is the reliance on a single - day fpg and bp measurement to detect new cases of diabetes and hypertension, respectively . Also, owing to the cross - sectional nature of our study, the possibility of residual confounding cannot be ruled out . Nonetheless, site is one of the largest prevalence studies on diabetes and hypertension conducted in outpatient settings in india . Due to the national multicentric approach of the site study, the amendments to the site protocol were efforts made to maintain consistency and accuracy of the data collected as the study progressed through each subsequent wave . The wave-3 protocol (conducted in tamil nadu) reflected the most adequate data collection process for this study and was employed for all other waves, permitting robust analyses . In conclusion, site will provide a real - world national perspective on diabetes and hypertension and their contributing risk factors . It will identify practice variations across states and evaluate compliance to international guidelines for the management of diabetes and hypertension . The standardization of the data collection process and the data analyses will justify various comparisons . The study will raise awareness on the need for early diagnosis and management of disease to reduce complications . Site data will be useful for national recommendations, policies and guidelines, and will also support future exploratory researches.
Asthma control is characterised by the management of symptoms and reduction of predicted future risk, particularly exacerbations . Some asthma exacerbations can be caused by a range of risk factors and irritants, including exercise, allergens, pollutants and occupational exposures, viral infections, medications and other factors such as emotional stress or co - existing medical conditions . Triggers. Ongoing exposure to asthma triggers is often a factor in poorly controlled asthma [1,37]. Asthma management guidelines and educational materials recommend that healthcare professionals educate patients to identify those triggers that worsen asthma symptoms and lead to loss of control, and take steps to avoid these triggers [1,811]. However, there is mixed evidence whether the monitoring and provision of advice, and discussion of environmental control strategies with asthma patients is adequate or suboptimal . The reasons why education on trigger identification and avoidance may be suboptimal are unclear but may relate to a perception that little can be done about asthma triggers or that consultations focus on direct clinical management . Another potential reason could be a lack of understanding of what an individual patient considers to be a trigger and the total trigger burden for that patient . Therefore, there is a clear need to improve understanding of the types of triggers that affect asthma patients, how often triggers are encountered, and how the overall burden of asthma triggers impacts disease control . The current epidemiological study was conducted to identify the types, frequency and impact of asthma triggers, and to investigate the relationship between asthma trigger burden (number and frequency) and asthma control among adults with asthma across five european countries . This was a questionnaire- and diary - based study designed to investigate the type, frequency and impact of asthma triggers among adults with asthma . The study was conducted in five european countries (france, germany, italy, spain and the uk) between november and december 2010 . Members of the panel were sent an invitation to take part in the survey via an email containing a link to the online questionnaire . The questionnaire contained a series of screening questions and only eligible participants completed the main questionnaire (approximately 25 min in duration). A random subset of participants who completed the questionnaire and who reported that specific events or activities triggered a worsening of their asthma were then asked to complete an online diary at least every 2 d over 34 weeks . Eligible participants were adults with a physician diagnosis of asthma (self - reported) and currently receiving maintenance asthma treatment and self - reported exposure to known asthma triggers . Eligibility was determined using a series of online screening questions and only those meeting the inclusion criteria proceeded to the online questionnaire . Patients were advised in the email invitation that accessing the online questionnaire provided their consent to partake in the study . The survey was developed based on qualitative research and expert opinion and was designed to describe and quantify common triggers for patients with asthma, understand and quantify coping behaviours in the presence of asthma triggers, and to understand and quantify how patients modify their lifestyles in response to triggers, and the impact this lifestyle modification has on their everyday quality of life . Participants were also asked about the frequency of severe asthma attacks, asthma worsening and use of reliever medication . Participants were then asked to indicate how well controlled they perceived their asthma to be on a scale of 010 (0 = very poorly controlled and 10 = completely controlled). The previously defined cut - off score of 20 was used to identify participants as having well controlled (act score 20) or uncontrolled asthma (act score <20). Asthma control was also evaluated in relation to the global initiative for asthma (gina) guidelines . The gina guidelines recommend evaluation of asthma control in relation to five characteristics: daytime symptoms, limitation of activities, nocturnal symptoms / awakenings, need for reliever / rescue medication and lung function . Participants were asked to report on the occurrence of day- and night - time asthma symptoms and the frequency with which these affected their planned activities as well as the frequency of rescue medication use in the previous week . Lung function data was not available so the remaining four characteristics were used as a proxy to define the level of asthma control as follows: (1) controlled: daytime symptoms no more than twice a week, no night - time symptoms, no limitation to activities in the previous four weeks and rescue medication use no more than once a week; (2) poorly controlled: daytime symptoms more than twice a week or night - time symptoms or limitation to activities in the previous four weeks or rescue medication use more than once a week; and (3) uncontrolled: three or more features of poorly controlled asthma . Participants were presented with a list of 36 potential asthma triggers and asked to indicate which had ever resulted in a worsening of their asthma . The list of 36 potential asthma triggers was developed based on qualitative interviews conducted with patients with asthma . The perceived impact of asthma on the daily lives of participants was derived from the question overall, how much of an impact do you think asthma has had on your life and the things you are able to do?. Participants were asked to respond on a likert - type scale of 1 (very mild) to 10 (very severe). The purpose of the diary was to understand the impact of asthma triggers over a period of time . A random subset of participants were recruited after completing the main online survey . At the end of the survey participants were asked if they wanted to take part in the diary part of the research and an additional online link was then sent to them . The diaries specifically elicited information on daily exposure to asthma triggers, the impact of exposure to asthma triggers on asthma symptoms, coping strategies and behaviours in relation to exposure to asthma triggers, as well as the impact of these strategies and behaviours on work and home activities . To investigate the relationship between asthma trigger burden (number and frequency) and asthma control an asthma trigger frequency index (ranging from 0 to 1) was calculated using the annual self - reported trigger exposure for all asthma triggers for each individual participant . A cut off of 0.5 was used to stratify patients into high trigger frequency> 0.5 and low trigger frequency (0.5). Participants were also stratified according to the level of asthma control (act or gina) and their self - reported asthma trigger burden (number of triggers identified by the patient as causing asthma symptoms): very low (15), low (610), medium (1115) and high (16). Statistically significant differences between groups in relation to trigger burden, level of asthma control and behavioural changes in relation to asthma triggers were determined using chi - square testing and analysis of variance (kruskal wallis). Descriptive statistics and multivariate regression analyses are presented on risk factors associated with differences in the mean number of severe attacks in a lifetime, the annual number of times individuals reported hospitalisation in the last year and the annual number of days missed at work or study in the last year due to asthma . A post hoc, investigational regression analysis was also undertaken in order to examine the impact of a variety of potential covariates on socioeconomic outcomes (poisson regression), impact of daily life and activities (linear regression) and the number of behavioural changes patients made in order to manage their exposure to known asthma triggers (logistic regression). The following covariates of interest were included in all analyses: trigger burden: very low (reference), low, medium, highage group: 1830 years (reference), 3144 years and> 45 yearsdwelling: rural (reference), urbancountry: germany (reference), uk, spain, france, italygender: male (reference), femaleself - reported physician - defined asthma severity: mild (reference), moderate, severe, not informed trigger burden: very low (reference), low, medium, high age group: 1830 years (reference), 3144 years and> 45 years dwelling: rural (reference), urban country: germany (reference), uk, spain, france, italy gender: male (reference), female self - reported physician - defined asthma severity: mild (reference), moderate, severe, not informed results are reported with 95% confidence intervals . This was a questionnaire- and diary - based study designed to investigate the type, frequency and impact of asthma triggers among adults with asthma . The study was conducted in five european countries (france, germany, italy, spain and the uk) between november and december 2010 . Members of the panel were sent an invitation to take part in the survey via an email containing a link to the online questionnaire . The questionnaire contained a series of screening questions and only eligible participants completed the main questionnaire (approximately 25 min in duration). A random subset of participants who completed the questionnaire and who reported that specific events or activities triggered a worsening of their asthma were then asked to complete an online diary at least every 2 d over 34 weeks . Eligible participants were adults with a physician diagnosis of asthma (self - reported) and currently receiving maintenance asthma treatment and self - reported exposure to known asthma triggers . Eligibility was determined using a series of online screening questions and only those meeting the inclusion criteria proceeded to the online questionnaire . Patients were advised in the email invitation that accessing the online questionnaire provided their consent to partake in the study . The survey was developed based on qualitative research and expert opinion and was designed to describe and quantify common triggers for patients with asthma, understand and quantify coping behaviours in the presence of asthma triggers, and to understand and quantify how patients modify their lifestyles in response to triggers, and the impact this lifestyle modification has on their everyday quality of life . Participants were also asked about the frequency of severe asthma attacks, asthma worsening and use of reliever medication . Participants were then asked to indicate how well controlled they perceived their asthma to be on a scale of 010 (0 = very poorly controlled and 10 = completely controlled). As part of the survey participants the previously defined cut - off score of 20 was used to identify participants as having well controlled (act score 20) or uncontrolled asthma (act score <20). Asthma control was also evaluated in relation to the global initiative for asthma (gina) guidelines . The gina guidelines recommend evaluation of asthma control in relation to five characteristics: daytime symptoms, limitation of activities, nocturnal symptoms / awakenings, need for reliever / rescue medication and lung function . Participants were asked to report on the occurrence of day- and night - time asthma symptoms and the frequency with which these affected their planned activities as well as the frequency of rescue medication use in the previous week . Lung function data was not available so the remaining four characteristics were used as a proxy to define the level of asthma control as follows: (1) controlled: daytime symptoms no more than twice a week, no night - time symptoms, no limitation to activities in the previous four weeks and rescue medication use no more than once a week; (2) poorly controlled: daytime symptoms more than twice a week or night - time symptoms or limitation to activities in the previous four weeks or rescue medication use more than once a week; and (3) uncontrolled: three or more features of poorly controlled asthma . Participants were presented with a list of 36 potential asthma triggers and asked to indicate which had ever resulted in a worsening of their asthma . The list of 36 potential asthma triggers was developed based on qualitative interviews conducted with patients with asthma . The perceived impact of asthma on the daily lives of participants was derived from the question overall, how much of an impact do you think asthma has had on your life and the things you are able to do?. Participants were asked to respond on a likert - type scale of 1 (very mild) to 10 (very severe). The purpose of the diary was to understand the impact of asthma triggers over a period of time . A random subset of participants were recruited after completing the main online survey . At the end of the survey participants were asked if they wanted to take part in the diary part of the research and an additional online link was then sent to them . The diaries specifically elicited information on daily exposure to asthma triggers, the impact of exposure to asthma triggers on asthma symptoms, coping strategies and behaviours in relation to exposure to asthma triggers, as well as the impact of these strategies and behaviours on work and home activities . To investigate the relationship between asthma trigger burden (number and frequency) and asthma control an asthma trigger frequency index (ranging from 0 to 1) was calculated using the annual self - reported trigger exposure for all asthma triggers for each individual participant . A cut off of 0.5 was used to stratify patients into high trigger frequency> 0.5 and low trigger frequency (0.5). Participants were also stratified according to the level of asthma control (act or gina) and their self - reported asthma trigger burden (number of triggers identified by the patient as causing asthma symptoms): very low (15), low (610), medium (1115) and high (16). Statistically significant differences between groups in relation to trigger burden, level of asthma control and behavioural changes in relation to asthma triggers were determined using chi - square testing and analysis of variance (kruskal wallis). Descriptive statistics and multivariate regression analyses are presented on risk factors associated with differences in the mean number of severe attacks in a lifetime, the annual number of times individuals reported hospitalisation in the last year and the annual number of days missed at work or study in the last year due to asthma . A post hoc, investigational regression analysis was also undertaken in order to examine the impact of a variety of potential covariates on socioeconomic outcomes (poisson regression), impact of daily life and activities (linear regression) and the number of behavioural changes patients made in order to manage their exposure to known asthma triggers (logistic regression). The following covariates of interest were included in all analyses: trigger burden: very low (reference), low, medium, highage group: 1830 years (reference), 3144 years and> 45 yearsdwelling: rural (reference), urbancountry: germany (reference), uk, spain, france, italygender: male (reference), femaleself - reported physician - defined asthma severity: mild (reference), moderate, severe, not informed trigger burden: very low (reference), low, medium, high age group: 1830 years (reference), 3144 years and> 45 years dwelling: rural (reference), urban country: germany (reference), uk, spain, france, italy gender: male (reference), female self - reported physician - defined asthma severity: mild (reference), moderate, severe, not informed results are reported with 95% confidence intervals . A total of 1202 adults with asthma were considered eligible to participate in this study and completed the online survey (table 1). Of these, 177 also completed the online diary and provided data on a total of 1947 d.table 1.demographics and disease characteristics.characteristicproportion of participants,% (n = 1202)gender male49 female51age range 1830 years35 3144 years37 4555 years27place of residence large town or city53 small town or suburbs31 village / hamlet16working status employed full time55 not currently working25 employed part - time10 self - employed10do not currently smoke72age at diagnosis 25 years of age68 2640 years24> 40 years of age8duration with asthma 5 years25 620 years45> 20 years31asthma control: self - reported daytime symptoms in the last 7 d85 night - time symptoms in the last 7 d79 experienced a worsening of symptoms at least once a week53uncontrolled asthma (act score <20 points)76partly controlled or uncontrolled asthma (gina guidelines) 92 proxy measure for gina definition: daytime symptoms experienced more than twice a week, night - time symptoms, had to limit activities in the last four weeks, had to use rescue medication more than once a week . Proxy measure for gina definition: daytime symptoms experienced more than twice a week, night - time symptoms, had to limit activities in the last four weeks, had to use rescue medication more than once a week . Most (85%) reported experiencing daytime symptoms and 79% reported experiencing night - time symptoms at least once in the past 7 d. in all, 53% of participants indicated that they experienced a worsening of their asthma symptoms at least once a week . The majority of participants (76%) were classed as uncontrolled using the act score, while 92% of participants were described as partly controlled or uncontrolled according to the gina guidelines . The number of self - reported asthma triggers varied considerably between participants, ranging from 1 to 5 (13% of participants) to 16 (35%); 52% of participants reported exposure to between 6 and 15 triggers . The most common asthma triggers as reported by participants were dust or dusting (72%), colds, flu, infections or sinusitis (69%), coughing (68%), tobacco smoking (60%) and smog or air pollution (58%) (table 2).table 2.self-reported asthma triggers as ranked by the proportion of participants who have ever experienced the trigger and by how frequently the trigger was reported.trigger% who have ever experienced n = 1202triggerfrequency (weeks / year) n = 1202dust or dusting72dust or dusting18.6cold, flu, infections, sinusitis69smoking (e.g. Cigarettes or cigars)14.2coughing68coughing11.3smoking (e.g. Cigarettes or cigars)60exercise11.1smoke59vacuum cleaning10.1smog, air pollution58perfumes, hairspray or air fresheners10.0exercise54animals9.6strong odours54smog, air pollution9.5weather changes51smoke9.0mould and mould spores51emotions8.2animals50cleaning products8.2damp places48damp places8.1humidity48weather changes8.0perfumes, hairspray or air fresheners48humidity7.9grass, mowing the lawn, weeds47strong odours7.6cold air45mould and mould spores7.4emotions43cold air7.2cleaning products42grass, mowing the lawn, weeds6.9vacuum cleaning41lying flat6.8feathers40cold, flu, infections, sinusitis6.7air conditioning37air conditioning5.8lying flat29feathers5.4your work environment25your work environment4.8rain19rain3.4food, drinks or food colourings19food, drinks or food colourings3.1alcohol18indigestion or heart burn2.8other medications18alcohol2.4indigestion or heart burn17aspirin2.1eating out at particular restaurants15other medications2.0aspirin15eating out at particular restaurants1.8hormonal changes14hormonal changes1.8paracetamol12paracetamol1.6 self - reported asthma triggers as ranked by the proportion of participants who have ever experienced the trigger and by how frequently the trigger was reported . Dust and dusting were the most commonly reported triggers and the most frequently experienced (table 2). Colds / flu were commonly reported asthma triggers but were experienced less frequently during the year . A high asthma trigger burden was associated with a lack of asthma control, as measured by patient - reported control (5.4 for patients with very low and low trigger burdens, 5.8 for patients with medium trigger burden, and 6.5 for patients with high trigger burden) or using the act score (figure 1). Patients whose asthma was not well controlled (act score) made more behavioural modifications than those with controlled asthma . There were statistically significant differences between control groups in terms of the amount and type of housework undertaken, the amount of exercise taken and the choice to remain indoors due to weather conditions (p <0.05 across groups; chi - square test) (figure 2).figure 1.relationship between trigger burden (exposure and frequency) and asthma control as measured by the act score (total participants = 1202). Panel a depicts participants (n = 696 [58%]) with a low to medium asthma trigger burden (<16 self - reported known asthma triggers) and a low total frequency burden (annual frequency index <0.5). Panel b depicts participants (n = 107 [9%]) with a low to medium asthma trigger burden (<16 self - reported known asthma triggers) and a high total frequency burden (annual frequency index 0.5). Panel c depicts participants (n = 278 [23%]) with a high asthma trigger burden (16 self - reported known asthma triggers) and a low total frequency burden (annual frequency index <0.5). Panel d depicts participants (n = 121 [10%]) with a high asthma trigger burden (16 self - reported known asthma triggers) and a high total frequency burden (annual frequency index 0.5). The shaded area on each panel depicts well controlled asthma (act score> 20). Figure 2.asthma control (act score) and behavioural changes . This figure depicts the frequency with which participants stratified according to asthma control (act score) reported making behavioural changes to avoid or minimise exposure to known asthma triggers . Relationship between trigger burden (exposure and frequency) and asthma control as measured by the act score (total participants = 1202). Panel a depicts participants (n = 696 [58%]) with a low to medium asthma trigger burden (<16 self - reported known asthma triggers) and a low total frequency burden (annual frequency index <0.5). Panel b depicts participants (n = 107 [9%]) with a low to medium asthma trigger burden (<16 self - reported known asthma triggers) and a high total frequency burden (annual frequency index 0.5). Panel c depicts participants (n = 278 [23%]) with a high asthma trigger burden (16 self - reported known asthma triggers) and a low total frequency burden (annual frequency index <0.5). Panel d depicts participants (n = 121 [10%]) with a high asthma trigger burden (16 self - reported known asthma triggers) and a high total frequency burden (annual frequency index 0.5). The shaded area on each panel depicts well controlled asthma (act score> 20). Asthma control (act score) and behavioural changes . This figure depicts the frequency with which participants stratified according to asthma control (act score) reported making behavioural changes to avoid or minimise exposure to known asthma triggers . An analysis of the number of triggers to which participants reported being exposed to did not necessarily correspond to the frequency of asthma worsening, with a minority of participants (9%) experiencing few asthma triggers but frequent asthma worsening (figure 1). The majority of participants (99%) believed that exposure to known asthma triggers would have a long - term effect on their asthma . One in five participants reported that they made no modifications to their behaviour in response to known asthma triggers until prompted to do so by worsening asthma symptoms . Participants were asked to rate the severity of asthma worsening in relation to the most commonly reported asthma triggers; all scored 6 or above on a scale of 0 (very mild) to 10 (very severe). The perceived severity of asthma worsening increased as the number of triggers experienced increased . As the number of known asthma triggers increased, participants reported statistically significantly more severe asthma attacks during their lifetime, more hospitalisations in the previous year, more days missed at work or study in the last year and more symptomatic days and nights per week (table 3).table 3.burden of asthma triggers.patient groups by trigger numbers total n n = 1202very few (15) n = 156low (610) n = 293medium (1115) n = 335high (16 +) n = 418kruskal wallismean number of severe attacks in lifetime3.2 (median 2, range 099)2.0 (median 1, range 015)2.7 (median 2, range 099)2.6 (median 2, range 020)4.6 (median 2, range 099)<0.001mean number of times hospitalised in the last year0.7 (median 0, range 025)0.5 (median 0, range 07)0.5 (median 0, range 06)0.8 (median 0, range 025)0.8 (median 0, range 020)0.038mean days missed at work or study in the last year because of asthma7.9 (median 2, range 0365)2.9 (median 1, range 080)6.1 (median 2, range 0288)6.3 (median 2, range 0265)12.2 (median 2, range 0365)<0.001 burden of asthma triggers . A number of covariates were associated with an increase in the socioeconomic burden of asthma (table 4). Any increase in the level of known asthma triggers above very low was positively associated with a statistically significant increase in mean number of severe attacks in a lifetime (p 0.01), the number of times individuals were hospitalised in the last year (p <0.001) and the number of days missed at work or study in the last year (p <0.001) due to asthma . Female gender was associated with an increased number of severe attacks (p <0.001), hospitalisation (p <0.001) and missing work (p <older age (> 31 years) was associated with an increased likelihood of hospitalisations and days lost to work or study in the previous year compared with age <30 years . Moderate or severe asthma severity (physician informed and reported by patient) was associated with increased rates of all the three above outcomes (p <0.001). Patients who reported they had severe asthma had three times the mean number of hospitalisations and four times more days off work than patients who reported they had mild asthma.table 4.covariates associated with an increase in the socioeconomic burden of asthma (poisson regression analysis).mean severe attacks in a lifetime hospitalisations in the previous 12 months number of days of work or study missed in the previous 12 months variableestimate (confidence interval) p valueestimate (confidence interval) p valueestimate (confidence interval) p valuetrigger burden (vs very low) low1.38 (1.141.6)<0.011.13 (0.851.51)0.402.15 (1.942.39)<0.01 medium1.20 (1.051.37)0.011.90 (1.472.49)<0.011.93 (1.742.14)<0.01 high2.07 (1.832.34)<0.011.95 (1.512.55)<0.013.45 (3.133.81)<0.01age (vs <30 years) 3144 years0.92 (0.861.00)0.040.63 (0.540.73)<0.010.99 (0.941.04)0.73> 45 years1.06 (0.98115)0.170.47 (0.390.57)<0.011.48 (1.401.56)<0.01dwelling (vs rural) urban1.03 (0.971.1)0.360.74 (0.640.86)<0.010.56 (0.530.58)<0.01country (vs germany) uk1.24 (1.121.37)<0.010.87 (0.691.09)0.221.05 (1.001.11)0.07 spain1.21 (1.091.34)<0.011.02 (0.831.25)0.870.59 (0.550.63)<0.01 france1.22 (1.101.35)<0.010.75 (0.600.94)0.010.44 (0.410.47)<0.01 italy0.87 (0.780.98)0.020.81 (0.641.01)0.060.62 (0.590.66)<0.01gender (vs male) female0.83 (0.780.89)<0.010.60 (0.510.69)<0.011.05 (1.011.09)0.02asthma severity (vs mild) moderate1.43 (1.271.62)<0.012.04 (1.522.80)<0.012.00 (1.832.19)<0.01 severe2.44 (2.132.80)<0.013.41 (2.474.79)<0.014.09 (3.724.51)<0.01 not told1.82 (1.552.15)<0.010.87 (0.501.47)0.610.75 (0.650.87)<0.01 covariates associated with an increase in the socioeconomic burden of asthma (poisson regression analysis). Participants reporting a high number of known asthma triggers (16) reported that their asthma had a significantly greater impact on their overall daily life and job choice compared with those reporting very few, low or medium number of triggers . For overall daily life, asthma impact score was 5.9 compared with 4.5, 4.7 and 5.3, respectively (p <0.0001 across groups; kruskal - wallis test). For job choice, asthma symptom score was 5.2 compared with 3.9, 3.9 and 4.2, respectively (p = 0.002 across groups; kruskal wallis test). Linear regression analyses indicated that participants reporting a medium or high asthma trigger burden were significantly more likely to report an adverse impact on their overall daily life, as were participants in italy (versus those in germany; asthma impact score 0.61 points higher, p <0.001), females (versus males; 0.42 points lower, p <0.001) and those with moderate or severe asthma (versus mild; asthma impact score 1.33 and 2.36 points, respectively, p <0.001 for both). Participants who reported that their asthma had impacted their daily life over the previous four weeks also reported making considerable behaviour changes to manage their asthma symptoms, the frequency of which increased as the number of triggers to which participants were exposed increased . The proportion of patients making any behaviour changes ranged from 67% for those with very few triggers to 89% for those with a high trigger burden (p <0.001 across groups; chi - square test). A logistic regression analysis showed a significant positive association between the trigger burden and the number of times behavioural changes were made in order to manage exposure to known asthma triggers in the previous four weeks . The higher the burden, the greater the number of changes, with high trigger patients being 10 times more likely to change behaviour up to five times in a week than those with very low number of triggers . Most (85%) reported experiencing daytime symptoms and 79% reported experiencing night - time symptoms at least once in the past 7 d. in all, 53% of participants indicated that they experienced a worsening of their asthma symptoms at least once a week . The majority of participants (76%) were classed as uncontrolled using the act score, while 92% of participants were described as partly controlled or uncontrolled according to the gina guidelines . The number of self - reported asthma triggers varied considerably between participants, ranging from 1 to 5 (13% of participants) to 16 (35%); 52% of participants reported exposure to between 6 and 15 triggers . The most common asthma triggers as reported by participants were dust or dusting (72%), colds, flu, infections or sinusitis (69%), coughing (68%), tobacco smoking (60%) and smog or air pollution (58%) (table 2).table 2.self-reported asthma triggers as ranked by the proportion of participants who have ever experienced the trigger and by how frequently the trigger was reported.trigger% who have ever experienced n = 1202triggerfrequency (weeks / year) n = 1202dust or dusting72dust or dusting18.6cold, flu, infections, sinusitis69smoking (e.g. Cigarettes or cigars)14.2coughing68coughing11.3smoking (e.g. Cigarettes or cigars)60exercise11.1smoke59vacuum cleaning10.1smog, air pollution58perfumes, hairspray or air fresheners10.0exercise54animals9.6strong odours54smog, air pollution9.5weather changes51smoke9.0mould and mould spores51emotions8.2animals50cleaning products8.2damp places48damp places8.1humidity48weather changes8.0perfumes, hairspray or air fresheners48humidity7.9grass, mowing the lawn, weeds47strong odours7.6cold air45mould and mould spores7.4emotions43cold air7.2cleaning products42grass, mowing the lawn, weeds6.9vacuum cleaning41lying flat6.8feathers40cold, flu, infections, sinusitis6.7air conditioning37air conditioning5.8lying flat29feathers5.4your work environment25your work environment4.8rain19rain3.4food, drinks or food colourings19food, drinks or food colourings3.1alcohol18indigestion or heart burn2.8other medications18alcohol2.4indigestion or heart burn17aspirin2.1eating out at particular restaurants15other medications2.0aspirin15eating out at particular restaurants1.8hormonal changes14hormonal changes1.8paracetamol12paracetamol1.6 self - reported asthma triggers as ranked by the proportion of participants who have ever experienced the trigger and by how frequently the trigger was reported . Dust and dusting were the most commonly reported triggers and the most frequently experienced (table 2). Colds / flu were commonly reported asthma triggers but were experienced less frequently during the year . A high asthma trigger burden was associated with a lack of asthma control, as measured by patient - reported control (5.4 for patients with very low and low trigger burdens, 5.8 for patients with medium trigger burden, and 6.5 for patients with high trigger burden) or using the act score (figure 1). Patients whose asthma was not well controlled (act score) made more behavioural modifications than those with controlled asthma . There were statistically significant differences between control groups in terms of the amount and type of housework undertaken, the amount of exercise taken and the choice to remain indoors due to weather conditions (p <0.05 across groups; chi - square test) (figure 2).figure 1.relationship between trigger burden (exposure and frequency) and asthma control as measured by the act score (total participants = 1202). Panel a depicts participants (n = 696 [58%]) with a low to medium asthma trigger burden (<16 self - reported known asthma triggers) and a low total frequency burden (annual frequency index <0.5). Panel b depicts participants (n = 107 [9%]) with a low to medium asthma trigger burden (<16 self - reported known asthma triggers) and a high total frequency burden (annual frequency index 0.5). Panel c depicts participants (n = 278 [23%]) with a high asthma trigger burden (16 self - reported known asthma triggers) and a low total frequency burden (annual frequency index <0.5). Panel d depicts participants (n = 121 [10%]) with a high asthma trigger burden (16 self - reported known asthma triggers) and a high total frequency burden (annual frequency index 0.5). The shaded area on each panel depicts well controlled asthma (act score> 20). Figure 2.asthma control (act score) and behavioural changes . This figure depicts the frequency with which participants stratified according to asthma control (act score) reported making behavioural changes to avoid or minimise exposure to known asthma triggers . Relationship between trigger burden (exposure and frequency) and asthma control as measured by the act score (total participants = 1202). Panel a depicts participants (n = 696 [58%]) with a low to medium asthma trigger burden (<16 self - reported known asthma triggers) and a low total frequency burden (annual frequency index <0.5). Panel b depicts participants (n = 107 [9%]) with a low to medium asthma trigger burden (<16 self - reported known asthma triggers) and a high total frequency burden (annual frequency index 0.5). Panel c depicts participants (n = 278 [23%]) with a high asthma trigger burden (16 self - reported known asthma triggers) and a low total frequency burden (annual frequency index <0.5). Panel d depicts participants (n = 121 [10%]) with a high asthma trigger burden (16 self - reported known asthma triggers) and a high total frequency burden (annual frequency index 0.5). The shaded area on each panel depicts well controlled asthma (act score> 20). Asthma control (act score) and behavioural changes . This figure depicts the frequency with which participants stratified according to asthma control (act score) reported making behavioural changes to avoid or minimise exposure to known asthma triggers . An analysis of the number of triggers to which participants reported being exposed to did not necessarily correspond to the frequency of asthma worsening, with a minority of participants (9%) experiencing few asthma triggers but frequent asthma worsening (figure 1). The majority of participants (99%) believed that exposure to known asthma triggers would have a long - term effect on their asthma . One in five participants reported that they made no modifications to their behaviour in response to known asthma triggers until prompted to do so by worsening asthma symptoms . Participants were asked to rate the severity of asthma worsening in relation to the most commonly reported asthma triggers; all scored 6 or above on a scale of 0 (very mild) to 10 (very severe). The perceived severity of asthma worsening increased as the number of triggers experienced increased . As the number of known asthma triggers increased, participants reported statistically significantly more severe asthma attacks during their lifetime, more hospitalisations in the previous year, more days missed at work or study in the last year and more symptomatic days and nights per week (table 3).table 3.burden of asthma triggers.patient groups by trigger numbers total n n = 1202very few (15) n = 156low (610) n = 293medium (1115) n = 335high (16 +) n = 418kruskal wallismean number of severe attacks in lifetime3.2 (median 2, range 099)2.0 (median 1, range 015)2.7 (median 2, range 099)2.6 (median 2, range 020)4.6 (median 2, range 099)<0.001mean number of times hospitalised in the last year0.7 (median 0, range 025)0.5 (median 0, range 07)0.5 (median 0, range 06)0.8 (median 0, range 025)0.8 (median 0, range 020)0.038mean days missed at work or study in the last year because of asthma7.9 (median 2, range 0365)2.9 (median 1, range 080)6.1 (median 2, range 0288)6.3 (median 2, range 0265)12.2 (median 2, range 0365)<0.001 burden of asthma triggers . A number of covariates were associated with an increase in the socioeconomic burden of asthma (table 4). Any increase in the level of known asthma triggers above very low was positively associated with a statistically significant increase in mean number of severe attacks in a lifetime (p 0.01), the number of times individuals were hospitalised in the last year (p <0.001) and the number of days missed at work or study in the last year (p <0.001) due to asthma . Female gender was associated with an increased number of severe attacks (p <0.001), hospitalisation (p <0.001) and missing work (p <older age (> 31 years) was associated with an increased likelihood of hospitalisations and days lost to work or study in the previous year compared with age <30 years . Moderate or severe asthma severity (physician informed and reported by patient) was associated with increased rates of all the three above outcomes (p <0.001). Patients who reported they had severe asthma had three times the mean number of hospitalisations and four times more days off work than patients who reported they had mild asthma.table 4.covariates associated with an increase in the socioeconomic burden of asthma (poisson regression analysis).mean severe attacks in a lifetime hospitalisations in the previous 12 months number of days of work or study missed in the previous 12 months variableestimate (confidence interval) p valueestimate (confidence interval) p valueestimate (confidence interval) p valuetrigger burden (vs very low) low1.38 (1.141.6)<0.011.13 (0.851.51)0.402.15 (1.942.39)<0.01 medium1.20 (1.051.37)0.011.90 (1.472.49)<0.011.93 (1.742.14)<0.01 high2.07 (1.832.34)<0.011.95 (1.512.55)<0.013.45 (3.133.81)<0.01age (vs <30 years) 3144 years0.92 (0.861.00)0.040.63 (0.540.73)<0.010.99 (0.941.04)0.73> 45 years1.06 (0.98115)0.170.47 (0.390.57)<0.011.48 (1.401.56)<0.01dwelling (vs rural) urban1.03 (0.971.1)0.360.74 (0.640.86)<0.010.56 (0.530.58)<0.01country (vs germany) uk1.24 (1.121.37)<0.010.87 (0.691.09)0.221.05 (1.001.11)0.07 spain1.21 (1.091.34)<0.011.02 (0.831.25)0.870.59 (0.550.63)<0.01 france1.22 (1.101.35)<0.010.75 (0.600.94)0.010.44 (0.410.47)<0.01 italy0.87 (0.780.98)0.020.81 (0.641.01)0.060.62 (0.590.66)<0.01gender (vs male) female0.83 (0.780.89)<0.010.60 (0.510.69)<0.011.05 (1.011.09)0.02asthma severity (vs mild) moderate1.43 (1.271.62)<0.012.04 (1.522.80)<0.012.00 (1.832.19)<0.01 severe2.44 (2.132.80)<0.013.41 (2.474.79)<0.014.09 (3.724.51)<0.01 not told1.82 (1.552.15)<0.010.87 (0.501.47)0.610.75 (0.650.87)<0.01 covariates associated with an increase in the socioeconomic burden of asthma (poisson regression analysis). Participants reporting a high number of known asthma triggers (16) reported that their asthma had a significantly greater impact on their overall daily life and job choice compared with those reporting very few, low or medium number of triggers . For overall daily life, asthma impact score was 5.9 compared with 4.5, 4.7 and 5.3, respectively (p <0.0001 across groups; kruskal - wallis test). For job choice, asthma symptom score was 5.2 compared with 3.9, 3.9 and 4.2, respectively (p = 0.002 across groups; kruskal wallis test). Linear regression analyses indicated that participants reporting a medium or high asthma trigger burden were significantly more likely to report an adverse impact on their overall daily life, as were participants in italy (versus those in germany; asthma impact score 0.61 points higher, p <0.001), females (versus males; 0.42 points lower, p <0.001) and those with moderate or severe asthma (versus mild; asthma impact score 1.33 and 2.36 points, respectively, p <0.001 for both). Participants who reported that their asthma had impacted their daily life over the previous four weeks also reported making considerable behaviour changes to manage their asthma symptoms, the frequency of which increased as the number of triggers to which participants were exposed increased . The proportion of patients making any behaviour changes ranged from 67% for those with very few triggers to 89% for those with a high trigger burden (p <0.001 across groups; chi - square test). A logistic regression analysis showed a significant positive association between the trigger burden and the number of times behavioural changes were made in order to manage exposure to known asthma triggers in the previous four weeks . The higher the burden, the greater the number of changes, with high trigger patients being 10 times more likely to change behaviour up to five times in a week than those with very low number of triggers . The aim of this large - scale epidemiological questionnaire and diary - based study conducted among adults with asthma across five european countries was to identify the types, frequency and impact of asthma triggers, and to investigate the relationship between trigger burden and asthma control . The majority of participants in the study had uncontrolled asthma and reported 615 known asthma triggers . Dust and dusting, colds / flu, sinusitis and coughing were the most common known asthma triggers with dust and dusting, smoking and exercise being the most frequently experienced triggers . An increase in trigger burden was associated with an increased likelihood of uncontrolled asthma, previous severe asthma attacks during a lifetime, more hospitalisations, more missed days at work / study, and behavioural changes to manage trigger exposure . The number of known asthma triggers reported by participants in this survey varied considerably from just one to more than 16 . Many of these triggers have been reported in asthma guidelines, by a systematic literature review and documented in the literature [1524], but the identification of such triggers has typically been through studies focusing on single triggers, not through population - based approaches . However, studies that more fully characterise the wide range of triggers experienced by individual asthma patients, and how frequently they are encountered, have been limited . One such approach was the development of the asthma trigger inventory, a questionnaire designed to measure the main categories of triggers self - reported by asthma patients, with a specific focus on psychosocial triggers . The most common categories of triggers were climate, physical and air pollution (experienced by> 50% of patients), followed by infection, house dust, animal allergy, pollen allergy and psychology (> 20 to <50% of patients), then respiration, food, sleep, alcohol, mould and medication (all <20% of patients). With the exception of mould, which was a much more common trigger in the present survey than in the asthma trigger inventory (51% versus 2%), some of the triggers perceived by participants in this survey are not considered to be classic asthma triggers, for example coughing, strong odours and lying flat . Although coughing was the third most common trigger (reported by 68% of participants), it is a well - established symptom of asthma and is not listed as an asthma trigger by the gina guidelines . There is literature to suggest that cough can be a precursor to an asthma exacerbation, but this association could be secondary to the worsening of asthma via another trigger (leading to more symptoms, including coughing), rather than coughing being a true trigger . Strong smells are not listed as an asthma risk factor by gina, but a small - scale study showed that perfume can cause exacerbations and airway obstruction, particularly in patients with severe asthma . There are clearly discrepancies between what patients consider to be triggers and what physicians and guidelines class as triggers . Possible explanations for such discrepancies include the lack of a standardised definition of the term asthma trigger, a lack of distinction between a trigger and an irritant, and documented gaps between patient perception and probable actual role of a trigger . Irritants can make the patient feel worse but may not necessarily lead to an exacerbation, while triggers are often thought to cause exacerbations . The present research suggests that patients do not discriminate between an irritant and a trigger and they will categorise any factor that increases symptoms, leads to loss of control, or causes an exacerbation as an asthma trigger . This has important implications for how healthcare professionals listen to and communicate with their patients, and emphasises the need for healthcare professionals to seriously consider all complaints about triggers, irrespective of whether they are real or perceived . The frequency with which participants reported exposure to triggers varied considerably in this survey, with self - reported exposures ranging from every few months to every few days . These findings are important as there is little information in the literature about how frequently specific asthma triggers are encountered, and they help to characterise the full extent of trigger burden experienced by patients . Although the authors are not aware of comparable reports that assessed the frequency of exposure to a wide range of triggers, those triggers that were experienced most commonly by patients concord with other investigations that aimed to identify common trigger types . Those triggers experienced by patients for 8 weeks of the year (see table 2) generally fall into the most important trigger categories reported by a systematic literature review (allergic, physical, environmental) or during the development of the asthma trigger inventory (climate, physical, air pollution, infection, house dust, animal allergy). The key exception was coughing, which was not reported as a trigger in either the systematic literature review or asthma trigger inventory . Frequent exposure to a large number of triggers can worsen symptoms, cause exacerbations and lead to loss of asthma control [1,37], therefore reducing a patient s exposure to triggers forms part of the overall recommended therapeutic approach to asthma management . However, the relationship between trigger burden and asthma control has not been well documented extensively in the literature . In the present study, a higher trigger burden was associated with more severe attacks in a lifetime, more hospitalisations in the last year, more days absent from work / study in the last year, and larger proportions of patients with uncontrolled asthma, as defined using the act (see table 3 and figure 1). These findings align with those of a previous study in which patients were asked at the time of hospitalisation for an exacerbation what their usual triggers were and what trigger they felt caused their exacerbation . These patients reported a high trigger burden (mean of 12 triggers) and there were statistically significant correlations between more triggers and more exacerbations, worse quality of life, more hospitalisation due to asthma, and oral corticosteroid use . These collective findings strongly suggest that a greater trigger burden leads to adverse asthma outcomes and worse asthma control, and highlights the importance of patient education to eliminate or mitigate trigger exposure . Asthma guidelines and authoritative groups recommend patients with asthma work to identify triggers that worsen their symptoms and try to reduce exposure to those triggers with the aim of improving asthma control and reducing medication needs . However, it is recognised that a response to triggers is a marker of poorly controlled disease, therefore trigger avoidance should not be used in isolation but rather as part of the holistic approach required to improve disease control overall . In the current study the majority of participants did not report any behavioural avoidance strategies for known environmental triggers, while some did but only after being prompted to do so by worsening asthma symptoms . Further, there was no apparent consistent approach to how patients managed trigger exposure and a wide range of behavioural strategies were reported . Patient education of trigger avoidance and advice on trigger management strategies appear to be logical steps to reduce the impact of triggers, but there are conflicting reports whether these steps are routinely taken . Tools for use in the primary care setting that aim to capture specific information on trigger exposure and other reasons for poor asthma control, such as the asthma apgar and the active life with asthma tool, may help to improve the dialogue between healthcare professionals and patients with respect to triggers . Nonetheless, although some asthma patients do make temporary or permanent lifestyle changes to manage trigger exposure, literature reviews and task force recommendations have not yielded consistent and conclusive evidence that interventions and trigger avoidance strategies improve symptoms, reduce exacerbations, and increase quality of life and productivity, at least in adults . Although better understanding and identification of asthma triggers, more consistent provision of advice on asthma triggers, and the development of more effective trigger avoidance strategies appear to be needed, it should also be considered whether overall asthma control can be improved to the point where the avoidance of triggers is not necessary . The strengths of the study lie in the relatively large cohort of adults with asthma who took part (n = 1202) and in the validity of the act scale as a measure of asthma control both as a written and, as used in the current study, as an online questionnaire . The knowledge gained from the comprehensive characterisation of asthma triggers in this survey can be applied to broader epidemiological assessments of asthma triggers . The results should be considered within the limitations expected of the survey nature of the study design . The results apply to a large population of asthma patients from five european countries who were currently receiving maintenance treatment and who self - reported exposure to known asthma triggers; however, generalising the findings to other populations of asthma patients is not without its limitations . Furthermore, patient assessment of asthma control and trigger burden were based on self reporting with recall periods of up to one year . Finally, verification of self - reported asthma worsening or healthcare service utilisation was beyond the scope of the current study . The results of the current study support those of previous surveys that suggest asthma control among adults in europe is suboptimal . Individuals with asthma identify a wide range of environmental and behavioural triggers and often employ avoidance strategies or behavioural changes to manage their exposure . These behavioural changes often have a significant impact on daily life, social life and employment performance and choices . Individuals who perceive that their asthma is worsened on exposure to multiple triggers may be at particular risk for poorly controlled asthma . Physicians should seek to routinely assess the trigger burden for individual patients and offer education and support to enable individuals to manage their exposure while minimising the effect of behavioural changes on their daily lives.
This study is an experimental designed pre - test - post - test, with a control group . The population of the study included 98 mentally retarded male students in the third grade at guidance schools in tehran province 2009 - 2010 school year . Students' selection was done through a multistage cluster; a sample of 30 mentally retarded students was selected . In the sampling process, three cities of varamin, rey and karaj then a school was selected from each city, and 10 students with the age range of 13 to 17 years whose iq was between 60 to 70 were randomly selected from each school . This scale includes sub - tests that are conducted individually and offers three iq scores: 1) verbal iq; 2) non - verbal iq; 3) general iq . A persian version of scale was prepared for normal children aged 6 to 13 years (original version for ages 6 to 16 years and 11 months). Reliability coefficient was calculated by two half way methods for sub - tests of non - verbal and verbal (other than numerical memory, which is made up of two different parts, and encoding which is a speed test), using spearman brown corrected correlation coefficient, which were from 0.42 to 0.98 with a median coefficients of 0.69 . Reliability coefficient of the test was calculated through test retest that ranged from 0.44 to 0.94, and only two cases (account sub - test and encoding) were below these values . The obtained reliability coefficient median was 0.73 (20). To determine validity; shahim et al ., compared this scale with the wechsler preschool and primary scale intelligence and obtained correlation coefficients of verbal, non - verbal and global iq to be 0.84, 0.74 and 0.85 respectively (20). This chapter was selected, and parallel tests were designed for each lesson that the questions were descriptive, and each test included 6 questions . A total of 16 teacher- made tests that were similar for all the three groups were used to evaluate students' progress in learning chapter 3 of the science book . To evaluate the validity of the tests, the content validity criterion - related tests and opinion of several teachers who taught this level were used . The validity coefficients for the eight tests of the first forms were: 0.94, 0.96, 0.92, 0.95, 0.92, 0.90, 0.89, 0.92, respectively; and for the eight tests of the second form they were: 0.93, 0.93, 0.95, 0.97, 0.91, 0.91, 0.91, 0.94 . To determine the reliability criterion - related reliability coefficient tests, percent agreement method was used, because the tests used in this study were criterion - related . The reliability coefficient for the eight tests of the first form was: 0.89, 0.86, 0.91, 0.84, 0.87, 0.90, 0.89, 0.92; and the reliability coefficient for the eight tests of the second form was: 0.88, 0.94, 0 . 93, 0.93, 0.89, 0.90, 0.87, and 0.91 . To conduct the research, a referral was obtained from the management of special education department of districts of tehran . Then, using multistage cluster sampling, three cities from tehran province were chosen, and a school from each city was also selected . Finally, 10 students whose age range was from 13 to 17 and their iq was between 60 to 70 were randomly selected . Then, each student was assigned in one of the following three groups: token economy reinforcements; social reinforcements; and control group . Then, the third chapter of the science book for the ninth grade class that included eight lessons was selected . Then, the procedure of teaching the eight lessons of chapter3 was taught to the three selected teachers in two sessions . The three teachers were taught how to use the lecture method, talk to students about the course subjects, ask questions, conduct experiment for them, and to show them samples and models . In other words, integrated method was used for training (21). Before teachers start teaching the first evaluation of achievement in science course (it was mentioned in the instrument section) conducted in three days for all three groups of the students . The mean scores of the eight tests were calculated and considered as a pretest score . At the next step, it was explained to the students how and when they will receive reinforcement, and the conditions of receiving it . In addition, a list of reinforcements and how they will receive them was provided to students in the token economy and strengthen social reinforcements group . The type of token was determined for the token economy reinforcements group, and the procedure of exchanging the token to reinforcements was clearly explained . In order to determine which student and to what extent to be strengthened, an absolute criterion was used in which four grade of a, b, c, d, and e were given to students according to percentage of their correct answers . Grade " a " was given to those students who scored 90% or more; grade " b " to those who provided correct answers to 80 to 89% of the questions; grade " c " to students who provided correct answers to 70 to 79% of the questions; grade d to those who provided correct answers to 60 to 69% of the questions; and grade " e " was given to the students who provided correct answers to less than 60% of the test questions (4). In this study, reinforcement was provided only to students who have obtained grades a, b and c. in the next step of this research project, the lesson was taught and reinforcement was provided to the token economy and social reinforcement groups by trained teachers . The teachers in both groups of the token economy and social reinforcements taught each lesson in two sessions . At the beginning of the third session, the tests were checked and scores were given by three science teachers, and the mean of these scores was considered as the score of the student in that lesson . At the beginning of the next session, those students whose score was a, b or c were given a chance to choose a reinforcement from the list, and then the teaching continued . It should be mentioned that reinforcements were not provided to the control group . Of course, the token economy reinforcement group could keep their chips and replace them with their precious reinforcements (from their perspective). The teacher in the control group did not provide reinforcements to the students; however, the same method of teaching, number of training sessions and exams were provided for all the three groups of students . Table 1 and 2 provide more details about social and token economy reinforcements, respectively . List of social reinforcements list of token economy reinforcements finally, the content of the eight lessons were taught to students in 16 sessions . At the end, the mean score of the students in three groups in eight tests of academic achievement was considered as posttest . The difference between the scores of pretest and posttest was calculated; and using one - way analysis of variance and shefe prosecution test, the obtained data were analyzed . The population of the study included 98 mentally retarded male students in the third grade at guidance schools in tehran province 2009 - 2010 school year . Students' selection was done through a multistage cluster; a sample of 30 mentally retarded students was selected . In the sampling process, three cities of varamin, rey and karaj then a school was selected from each city, and 10 students with the age range of 13 to 17 years whose iq was between 60 to 70 were randomly selected from each school . This scale includes sub - tests that are conducted individually and offers three iq scores: 1) verbal iq; 2) non - verbal iq; 3) general iq . A persian version of scale was prepared for normal children aged 6 to 13 years (original version for ages 6 to 16 years and 11 months). Reliability coefficient was calculated by two half way methods for sub - tests of non - verbal and verbal (other than numerical memory, which is made up of two different parts, and encoding which is a speed test), using spearman brown corrected correlation coefficient, which were from 0.42 to 0.98 with a median coefficients of 0.69 . Reliability coefficient of the test was calculated through test retest that ranged from 0.44 to 0.94, and only two cases (account sub - test and encoding) were below these values . The obtained reliability coefficient median was 0.73 (20). To determine validity; shahim et al ., compared this scale with the wechsler preschool and primary scale intelligence and obtained correlation coefficients of verbal, non - verbal and global iq to be 0.84, 0.74 and 0.85 respectively (20). This chapter was selected, and parallel tests were designed for each lesson that the questions were descriptive, and each test included 6 questions . A total of 16 teacher- made tests that were similar for all the three groups were used to evaluate students' progress in learning chapter 3 of the science book . To evaluate the validity of the tests, the content validity criterion - related tests and opinion of several teachers who taught this level were used . The validity coefficients for the eight tests of the first forms were: 0.94, 0.96, 0.92, 0.95, 0.92, 0.90, 0.89, 0.92, respectively; and for the eight tests of the second form they were: 0.93, 0.93, 0.95, 0.97, 0.91, 0.91, 0.91, 0.94 . To determine the reliability criterion - related reliability coefficient tests, percent agreement method was used, because the tests used in this study were criterion - related . The reliability coefficient for the eight tests of the first form was: 0.89, 0.86, 0.91, 0.84, 0.87, 0.90, 0.89, 0.92; and the reliability coefficient for the eight tests of the second form was: 0.88, 0.94, 0 . This scale includes sub - tests that are conducted individually and offers three iq scores: 1) verbal iq; 2) non - verbal iq; 3) general iq . A persian version of scale was prepared for normal children aged 6 to 13 years (original version for ages 6 to 16 years and 11 months). Reliability coefficient was calculated by two half way methods for sub - tests of non - verbal and verbal (other than numerical memory, which is made up of two different parts, and encoding which is a speed test), using spearman brown corrected correlation coefficient, which were from 0.42 to 0.98 with a median coefficients of 0.69 . Reliability coefficient of the test was calculated through test retest that ranged from 0.44 to 0.94, and only two cases (account sub - test and encoding) were below these values . The obtained reliability coefficient median was 0.73 (20). To determine validity; shahim et al ., compared this scale with the wechsler preschool and primary scale intelligence and obtained correlation coefficients of verbal, non - verbal and global iq to be 0.84, 0.74 and 0.85 respectively (20). This chapter was selected, and parallel tests were designed for each lesson that the questions were descriptive, and each test included 6 questions . A total of 16 teacher- made tests that were similar for all the three groups were used to evaluate students' progress in learning chapter 3 of the science book . To evaluate the validity of the tests, the content validity criterion - related tests and opinion of several teachers who taught this level were used . The validity coefficients for the eight tests of the first forms were: 0.94, 0.96, 0.92, 0.95, 0.92, 0.90, 0.89, 0.92, respectively; and for the eight tests of the second form they were: 0.93, 0.93, 0.95, 0.97, 0.91, 0.91, 0.91, 0.94 . To determine the reliability criterion - related reliability coefficient tests, percent agreement method was used, because the tests used in this study were criterion - related . The reliability coefficient for the eight tests of the first form was: 0.89, 0.86, 0.91, 0.84, 0.87, 0.90, 0.89, 0.92; and the reliability coefficient for the eight tests of the second form was: 0.88, 0.94, 0 . To conduct the research, a referral was obtained from the management of special education department of districts of tehran . Then, using multistage cluster sampling, three cities from tehran province were chosen, and a school from each city was also selected . Finally, 10 students whose age range was from 13 to 17 and their iq was between 60 to 70 were randomly selected . Then, each student was assigned in one of the following three groups: token economy reinforcements; social reinforcements; and control group . Then, the third chapter of the science book for the ninth grade class that included eight lessons was selected . Then, the procedure of teaching the eight lessons of chapter3 was taught to the three selected teachers in two sessions . The three teachers were taught how to use the lecture method, talk to students about the course subjects, ask questions, conduct experiment for them, and to show them samples and models . In other words, integrated method was used for training (21). Before teachers start teaching the first evaluation of achievement in science course (it was mentioned in the instrument section) conducted in three days for all three groups of the students . The mean scores of the eight tests were calculated and considered as a pretest score . At the next step, it was explained to the students how and when they will receive reinforcement, and the conditions of receiving it . In addition, a list of reinforcements and how they will receive them was provided to students in the token economy and strengthen social reinforcements group . The type of token was determined for the token economy reinforcements group, and the procedure of exchanging the token to reinforcements was clearly explained . In order to determine which student and to what extent to be strengthened, an absolute criterion was used in which four grade of a, b, c, d, and e were given to students according to percentage of their correct answers . Grade " a " was given to those students who scored 90% or more; grade " b " to those who provided correct answers to 80 to 89% of the questions; grade " c " to students who provided correct answers to 70 to 79% of the questions; grade d to those who provided correct answers to 60 to 69% of the questions; and grade " e " was given to the students who provided correct answers to less than 60% of the test questions (4). In this study, reinforcement was provided only to students who have obtained grades a, b and c. in the next step of this research project, the lesson was taught and reinforcement was provided to the token economy and social reinforcement groups by trained teachers . The teachers in both groups of the token economy and social reinforcements taught each lesson in two sessions . At the beginning of the third session, the tests were checked and scores were given by three science teachers, and the mean of these scores was considered as the score of the student in that lesson . At the beginning of the next session, those students whose score was a, b or c were given a chance to choose a reinforcement from the list, and then the teaching continued . It should be mentioned that reinforcements were not provided to the control group . Of course, the token economy reinforcement group could keep their chips and replace them with their precious reinforcements (from their perspective). The teacher in the control group did not provide reinforcements to the students; however, the same method of teaching, number of training sessions and exams were provided for all the three groups of students . Table 1 and 2 provide more details about social and token economy reinforcements, respectively . List of social reinforcements list of token economy reinforcements finally, the content of the eight lessons were taught to students in 16 sessions . At the end, the mean score of the students in three groups in eight tests of academic achievement was considered as posttest . The difference between the scores of pretest and posttest was calculated; and using one - way analysis of variance and shefe prosecution test, the obtained data were analyzed . The comparison of the mean scores of achievement in science course between the three groups of the mentally retarded students (the token economy reinforcements, social reinforcements and control groups) are presented in table 3 . The age range of three groups comparison of pre - and post - test mean achievement test in science class experiment three groups; token, social reinforcements and control in addition, because of differences between the groups, shefe prosecution test was used, and the results are presented in the table 4 . According to table 3, there is a significant difference in academic achievement between the three groups of the token economy reinforcements, social reinforcements and control (p<0.001). Scheffe test results for comparison of mean achievement scores in science class experiment three groups; token, social reinforcements and control as demonstrated in table 4, the mean scores of achievement in science course was significantly higher in the token economy reinforcement group than the social reinforcement group (p = 0.002) and the control group (p<0.001). Furthermore, the mean scores of achievement in the science course was significantly higher in the social reinforcement group than the control group (p=0.021). This study aimed to compare the effectiveness of social reinforcements and token economy reinforcements on academic achievement of mentally retarded students in a science course . According to findings of this study, mentally retarded students in groups of token economy reinforcements and social reinforcements have indicated more academic achievement in the science course than the control group . Also, the token economy reinforcement group has indicated more academic achievement in science course than the social reinforcement group . The results of the first section of the present research (indicating token economy reinforcements group make more academic achievement) is consistent with previous research findings . Scott and porter have investigated the effect of token reinforcement on behavioral discipline, active participation and academic achievement of normal students and mental retarded students in regular primary and secondary levels in a sample of 750 students . Based on the findings of their research, academic achievement in normal students and mentally retarded students keller in a research found that food as a token economy reinforcement has the most impact on academic achievement (23). Hardman et al ., in a research study on 27 mentally retarded subjects used tokens that could be exchanged with sweets, whistles or costume jewellery . They found that the token economy reinforcement has a significant effect on academic skills (15). Kord investigated the effect of feedback reinforcements on academic achievement of a science course of 140 male students at the fifth grade of elementary level . He found that the feedback reinforcements in various forms, including verbal, written or a combination are more effective on academic achievement (16). Abarqhuie found that token economy reinforcements were effective to decrease the educational failure and increase motivation of academic achievement of 20 boy and 20 girl students (17). Soltani in a study found that sport rewards were effective on academic achievement of 32 students of the fourth level of elementary school (24). Haji ali mohammadi, found that the token economy reinforcements were effective on academic achievement of a social science course; also he found that the token economy reinforcement method increased motivation of academic achievement of failed students (18). The more effectiveness of token reinforcements on academic achievement in a science course of mentally retarded students may be justified by noting that mentally retarded students consider material reinforcements more important, and social reinforcements are less attractive to them . Future research could provide further enquieries regarding the relationship between different reinforces and academic achievement, particularly in mentally retarded boys and girls students . Token economy and social reinforcements increase the academic achievement of students with intellectual disabilities in the science class; and also the effect of token economy reinforcements is more than social reinforcements on students . Appreciation and thanks we sincerely thank the respected management of exceptional training department of city of tehran province and respected management of noor - e islam in varamin, taher in ray; and the parsi arbabi in karaj exceptional schools helped us in implementation of the research . Conclusion
Gastrointestinal manifestations of brucellosis in humans are relatively uncommon and may manifest as anorexia, nausea, vomiting, abdominal pain, diarrhea, or constipation, but systemic symptoms, such as artharlgia and myalgia, are more common than localized gastrointestinal symptoms . This report presents a case with gastroenteritis caused by brucella species . A previously healthy 21-year - old male presented with a history of 3 days of fever, vomiting, and diarrhea without other symptoms . He had eaten food at a local restaurant a few days prior to his presentation . A physical examination revealed a temperature of 38.4 c, respiratory rate of 20/minutes, pulse of 62/minutes, blood pressure of 110/50 mmhg, and no other findings . Laboratory investigations showed leucopenia: white blood cell count of 2,800/mm (64% polymor - phonuclear cells, 25% lymphocytes, 6% monocytes, 2% bands, 3% atypical lymphocytes), hemoglobin of 12.7 g / dl, platelet count of 69,000/mm and esr of 52 mm / hour . Other biochemical findings showed na+ 132, alt 73 u / l, ast 112 u / l, cpk 674 u / l, ldh 417 u / l, amylase, prothrombin time, and partial thromboplastin time were normal, as were other electrolytes and liver function tests . A blood culture was positive for brucella species, which displayed sensitivity to streptomycin, rifampin, tetracycline, and bactrim . The patient was started on oral doxycycline 100 mg twice daily for 6 weeks and streptomycin 1 g intramuscular injection daily for 3 weeks, and started to improve after 57 days of therapy . The patient was seen at follow - up in the clinic, was doing well, and was discharged after completing his antibiotic course in a very good condition . Brucellosis is a zoonotic infection that is endemic in the middle east.1 in saudi arabia, the known incidence rate is 40 cases per 100,000 inhabitants per year.2 gastrointestinal manifestation of brucellosis are relatively uncommon.3 brucellosis presenting as gastroenteritis is rare in adults and has been reported in sporadic case reports from as early as 1934.4 petrella and young described a case of acute brucella ileitis in 1988.5 labrune et al reported recurrent enterocolitis - like symptoms as the possible presenting manifestations of neonatal brucella mellitensis infection in 1990.6 stermer et al reported a case of brucellosis as the cause of severe colitis in 1991.7 locutura et al reported a case of diarrhea as the first manifestation of brucellosis in 1998.8 in children, brucellosis can present as gastroenteritis more commonly than in adults . In one iranian study it was reported that 11% of cases of brucellosis in children manifested as gastroenteritis.9 acute brucellosis as a cause of infective colitis was reported by mazokopakis et al in 2008.10 erbay et al reported brucellosis presenting as enteric fever in 2009.1 our case was very interesting as the presenting symptoms suggested gastroenteritis without musculoskeletal symptoms, and the laboratory findings were similar to that of gastroenteritis; only blood culture and brucella titer led to the diagnosis . We would recommend that, in any case suggestive of gastroenteritis, the differential diagnosis of acute brucellosis should be considered in countries where brucellosis is endemic.
The genitourinary syndrome of menopause (gsm) is the new definition for the variety of menopausal symptoms associated with physical changes of the vulva, vagina, and lower urinary tract, related with estrogen deficiency . Gsm is chronic and is likely to worsen over time, affecting up to 50% of postmenopausal women . The symptoms related to gsm include genital symptoms of dryness, burning, irritation, but also sexual symptoms of lack of lubrication, discomfort or pain, and impaired function, as well as urinary symptoms of urgency, dysuria and recurrent urinary tract infections . Most moisturizers and lubricants are available without prescription, at a non - negligible cost, and may provide only a temporary relief . Conversely, hormone replacement therapy (hrt) can provide quick and long - term relief, while urinary symptoms often require additional, effective therapies . When hrt is considered solely for the treatment of vaginal atrophy, local vaginal estrogen administration is the treatment of choice . Although systemic risks have not been identified with local low - potency / low - dose estrogens, long - term efficacy and safety data are lacking . In addition, many women do not accept protracted hrt, or may present absolute contraindications, such as a personal history of estrogen - dependent tumors, particularly endometrial and breast cancer . New management strategies for gsm can increase our armamentarium in order to offer a wide range of options to enable women to choose, considering the benefits and risks associated with each strategy . Recently, a seminal paper has clearly demonstrated that a treatment with the microablative carbon dioxide (co2) laser induced a significant improvement of vaginal health in postmenopausal women . Indeed, we have to consider that various lasers possess diverse properties that can be usefully applied in different conditions . The non - ablative erbium laser technology may provide a non - invasive treatment option and it is widely used . The aim of the present study was to evaluate the short - term effectiveness and acceptability of the vaginal erbium laser (vel) as a new, second - generation, non - ablative photothermal therapy for the treatment of gsm . This is a pilot prospective, longitudinal study performed in postmenopausal women suffering from gsm, attending the outpatient menopause clinic of pisa university hospital . All women gave written informed consent and an independent national advisory board reviewed this protocol approved by the division ethics committee . Inclusion criteria were the presence of gsm in healthy postmenopausal women (at least 12 months since last menstrual period or bilateral oophorectomy) with plasma gonadotropin and estradiol levels in the postmenopausal range (follicle stimulating hormone> 40 u / l; estradiol <25 pg / ml) and negative pap smear . Exclusion criteria were vaginal lesions, scars, active or recent (30 days) of the genitourinary tract infections; abnormal uterine bleeding; use of lubricants or any other local preparations, within the 30 days prior to the study; history of photosensitivity disorder or use of photosensitizing drugs; genital prolapse (grade ii iii according to the pelvic organ prolapse quantification, pop - q, system classification); serious or chronic condition that could interfere with study compliance; treatment with hormones or other medicines to relieve menopausal symptoms in the 12 months before the study . All the participants (n = 45) were treated with the vaginal erbium laser (vel), a non - ablative, solid - state erbium in yttrium aluminum - garnet crystal (er: yag) laser (fotona smooth xs, fotona, ljubljana, slovenia) with a wavelength of 2940 nm . The spot size (diameter of the laser beam on the target) is 7 mm, with a pulse according to the technique smooth, at a frequency of 1.6 hz, and a fluence (laser energy delivered per unit area) of 6.0 j / cm . The parameters were selected based on extensive preclinical and clinical studies performed in different experimental conditions . Briefly, variable square pulse (vsp) technology controls the energy and time duration (or pulse width) simultaneously, reducing the power and increasing the pulse duration . The smooth mode, with the sequence of low - fluence longer - shaped erbium pulses, distributes the heat approximately 100 microns deep into the mucosa surface, achieving a controlled deep thermal effect, without ablation . The thickness of mucosa varies, and normally its height is several hundred microns . Therefore, the erbium smooth mode pulses allow controlled tissue heating, in a safe and harmless ambulatory procedure without ablation and carbonization of the tissues, practically avoiding the risk of perforation with accidental lesions of the urethra, bladder or rectum . The vel procedures were performed in an outpatient clinical setting, without any specific preparation, anesthesia, or post - treatment medications . Before the procedures, patients were treated with three laser applications (l1, l2, l3) every 30 days, with a screening visit 24 weeks prior to the first laser treatment (baseline) and follow - up visits after 4 (t + 4), 12 (t + 12) and 24 (t + 24) weeks from the last laser application . Briefly, the laser parameters (renovalase phase 1) were settled with a fluence of 5.5 j / cm, with the smooth mode at 1.6 hz; the spot size was 7 mm non - fractional . After inserting a specifically designed vaginal speculum, the probe is inserted into the speculum, with no direct contact with the vaginal mucosa . Thus, circular irradiation of the vaginal wall is performed, with four pulses given every 5 mm, retracting the probe by 5 mm each time (using the graduated scale on the probe). Finally, after removing the speculum and using a different probe (ps03 handpiece), the vestibule and introit are irradiated with a spot size of 7 mm, fluence of 10 j / cm, with the smooth mode at 1.6 hz (renovalase phase 2). In addition, in 19 postmenopausal women suffering also from stress urinary incontinence (sui), the degree of incontinence was assessed with the international consultation on incontinence questionnaire urinary incontinence short form (iciq - ui sf), where a maximum score of 21 represents permanent incontinence . None of these patients presented a pelvic organ prolapse greater than stage ii, according to the pelvic organ prolapse quantification (pop - q) system classification . These patients during the vel procedures were submitted also to additional laser treatment of the anterior vaginal wall (incontilase phase 1 procedure) specifically designed for urinary incontinence . As the active control group, we selected a group of 25 postmenopausal women treated with an established treatment for gsm (1 g of vaginal gel containing 50 g of estriol twice weekly, for 3 months). This formulation provides an ultra - low dose of estriol per application, shown to be safe and effective in the treatment of postmenopausal vaginal atrophy . At the first visit, the eligibility of the patient was verified, the written informed consent was obtained, and the sociodemographic and clinical characteristics were collected . Subjective symptoms (vaginal dryness and dyspareunia) were evaluated by a visual analog scale (vas) at every visit (range 010 cm, 0 = total absence of the symptom and 10 cm = the worse possible symptom). In addition, at each visit during the gynecologic examination, the vaginal health score index (vhis) was measured . The vhis evaluates the appearance of vaginal mucosa (elasticity, ph, vaginal discharge, mucosal integrity and moisture). The women were asked to evaluate the general acceptability and efficacy of the finalized therapy as excellent, good, acceptable, bad, or unacceptable . Being an exploratory study, the sample size was not based on a statistical rationale . Two - way analysis of variance for repeated measures and factorial analysis of variance were used to test the differences within and between the groups, respectively . The post - hoc comparison was made by the scheffe f - test, using sigma stat view software (spss science, chicago, il, usa). There were no significant differences in age, age at menopause and years since menopause, body mass index and basal hormone levels in the two treatment groups before the study (table 1). In the vel group, 43 patients completed the study; one patient left the study for personal reasons and one left because of the discomfort related to the first application . In the estriol group, 19 women completed the study, while the others dropped out because of personal problems or because they needed other pharmacological or surgical interventions . Fsh, follicle stimulating hormone the basal vas and vhis scores were similar in the two groups (figures 1 and 2). In both groups, the vas scores for vaginal dryness and dyspareunia, from basal values (t0) of 8.3 1.3, and 8.2 1.3 cm, respectively, showed significant (p <0.01) decreases to 5.1 1.4 and 4.5 1.6 cm, respectively, 4 weeks after the first vel treatment or 4 weeks of estriol cream (l1), to 3.0 1.1 and 2.8 1.5 cm, respectively, after the second vel treatment or 8 weeks of estriol cream (l2), to 2.7 0.7 and 2.8 1 cm, respectively, after the third vel treatment or 12 weeks of estriol cream (l3) (figure 1). In the follow - up period, the dryness and dyspareunia values were 2.9 0.6 and 2.8 1.0 cm, respectively, 4 weeks after the last vel or last estriol cream application (t + 4), 3.0 0.6 and 3.1 0.9 cm, respectively, after 12 weeks (t + 12), and 3.5 0.9 and 3.5 1.1 cm, respectively, 24 weeks after the last vel or last estriol cream application (t + 24). The difference from baseline values was statistically significant (p <0.01) after the first laser treatment and the values remained significantly lower after the second and third laser applications, as well as during the follow - up period up to the 24 weeks of observation (figure 1). The vas values in the estriol group showed a similar decrease during the treatment period, with a comparable pattern, and no significant difference was evident between the vel and estriol groups during the treatment period . Conversely, the vas scores for both vaginal dryness and dyspareunia in the estriol group showed a small but significant increase after the end of the treatment period (figure 1). The values measured after 24 weeks (t + 24) in the estriol group were significantly (p <0.05) different from corresponding values measured in the vel group (figure 1). The vhis increased significantly (p <0.01) in both the vel and estriol groups, from basal values (t0) of 10.6 3.6 and 11.2 2.8, respectively, to 16.6 2.1 and 18.2 3.2, respectively, after the first vel treatment (l1); 20.1 1.8 and 21.2 2.9, respectively, at l2; 20.1 1.8 and 22.4 4.0, respectively, at l3; the values were 20.0 1.4 and 20.1 3.1, respectively, after 4 weeks of follow - up (t + 4); 19.8 1.3 and 15.3 1.5, respectively, after 12 weeks of follow - up (t + 12); and 19.0 1.4 and 16.2 1.7, respectively, after 24 weeks of follow - up (t + 24) (figure 2). The values measured after 12 and 24 weeks (t + 12 and t + 24) in the estriol group were significantly (p <0.05) different from corresponding values measured in the vel group (figure 2). In the 19 patients suffering from sui, the vel treatment induced a significant (p <0.01) decrease in the iciq - sf scores from basal values of 12.0 1.8, to 7.5 2.4 at t1, to 5.8 2.6 at t2, and 5.6 2.6 at t3 . The iciq - sf scores remained significantly (p <0.01) lower than basal values at t + 4 (5.5 2.6), at t + 12 (5.5 2.9) as well as at t + 24 (5.0 2.6) (figure 3). Vel was well tolerated, with less than 3% of patients discontinuing treatment due to adverse events: one patient defined the procedure as unacceptable, reporting a burning sensation starting 36 h after the first application and lasting for a couple of days . One patient left the study for personal reasons . In the 43 valid completers in the vel group, 34 patients (79.5%) defined the procedure as excellent good, seven patients (16.3%) as acceptable, and two patients (4.2%) reported their experience as bad . In the estriol group, 16 patients (84%) defined the treatment as excellent good, while three patients defined the vaginal treatment, respectively, as acceptable, bad or unacceptable . To our knowledge, this is the first study designed to evaluate the effects of vel as second - generation laser thermotherapy on gsm . Our data show that vel is well tolerated by women who subjectively perceived a clinical benefit that was confirmed by the objective improvement of the vaginal milieu, as measured by the vhis . These results indicate that vel treatment is able to induce a rapid and long - lasting improvement in the signs and symptoms of gsm . A significant subjective and objective improvement was evident after the first laser application; a more pronounced effect was apparent after the second and third laser applications, although the difference did not reach statistical significance . No anesthesia was necessary and the effects were long lasting, at least up to the 6-month follow - up after the last vel application . Our study is a pilot prospective, longitudinal study and has the limitation of sample size . However, the data obtained in the vel group were compared with those obtained in a group of similar women treated with a standard estrogen treatment for gsm . Present results show comparable effects of vel and estriol treatment on gsm parameters . In the estriol group, conversely, the vel group maintained the same positive results throughout all the study period up to the 6-month follow - up . Salvatore and colleagues, in a recent noteworthy paper, clearly demonstrated that microablative co2 laser energy is effective in improving vaginal dryness as well as dyspareunia and vhis, in a 12-week follow - up study . This paper clearly indicates that laser may be considered a new opportunity for non - hormonal treatment of gsm . The procedure was easy to perform, particularly after the first laser application, and the insertion of the probe into the vaginal canal was well tolerated . The procedure used in our study is slightly different for that used with the microablative co2 laser . The vel procedure is performed using a special vaginal speculum introduced as a guide for the handpiece laser beam delivery system . Thus, the patients do not feel the several longitudinal passes performed using a step - by - step retraction of the handpiece . In a two - step protocol, the laser irradiation was applied first into the vaginal canal and after at the introitus area . The innovative techniques used in the vel procedures can guarantee not only the efficacy, but also, mainly, an intrinsic safety, since the erbium beam cannot damage the tissues in depth, eliminating the risk of tissue necrosis, in a non - ablative form, without abrasion or bleeding . This characteristic makes the erbium laser an ideal candidate for the thermal treatment of the vaginal walls . The smooth technique releases precise impulses, leading to a controlled rise in tissue temperature for vasodilation and up to the optimum for the collagen remodeling and neocollagenesis, comprised in a range between 45 and 60c . The collagen exposed to an appropriate temperature is contracted and this leads to the temporary shrinkage, which stimulates a subsequent remodeling, with the consequent generation of new collagen and an overall improvement of the elasticity of the treated tissue . In the treatment of superficial tissues, the laser can be provided in a fractionated manner, producing a matrix of small the fractionated technique is comfortable for the patient, allowing the use of higher fluences in the irradiated points . For the treatment of sui, the vaginal anterior wall is treated with five passes in addition to the passes irradiating the entire vaginal canal at 360. taken together, these data clearly suggest that laser energy can be used for the treatment of postmenopausal women suffering from gsm . Our study confirms and extends previous data reported with vel in abstract form by gaspar and colleagues . Vel can determine an increase of epithelial thickness and glycogen content, associated with changes in lamina propria, increased angiogenesis, collagenesis, papillomatosis and cellularity of the extracellular matrix . All these changes are long lasting and can be observed 6 months after the last vel treatment . These results are confirmed by our findings, showing persistent effects of vel 24 weeks after the end of treatment . At variance from gaspar and colleagues, in our study the vel treatment was performed in postmenopausal women suffering from gsm without any previous or concomitant treatment with estrogens or even non - hormonal vaginal creams . Therefore, our study suggests that the effects of vel are independent from any pretreatment, suggesting that vel can be proposed in postmenopausal women who cannot be treated with hormones, as in breast cancer survivors . The possible difference in the outcomes with or without estrogen pretreatment or the current use of estrogenic or non - hormonal therapies is a matter of future studies . In addition, our data suggest that vel treatments can be of help in postmenopausal women suffering from mild to moderate sui . In fact, in postmenopausal women suffering from gsm and also mild to moderate sui, the vel improved the iciq - sf scores . Urinary incontinence is a common and important health - care problem that is underreported, underdiagnosed, and therefore undertreated in women . The non - surgical treatment of sui is a major challenge for women's health . Non - pharmacologic management for sui, such as pelvic floor muscle training, is effective, can improve sui and may provide complete continence, but many patients discontinue the treatment . As previously reported by fistonic and colleagues, our preliminary data indicate that vel might be useful for the non - invasive treatment of sui . Obviously, future well - designed and controlled studies are needed to validate the use of vel for sui . In our study further studies are needed to evaluate the effects of vel on sexuality in postmenopausal women suffering from gsm . In conclusion, this pilot study suggests that the treatment with vel is reasonable, efficacious and safe as a new, second - generation, non - ablative photothermal therapy for the treatment of gsm . Further larger, long - term and well - controlled studies are required to explore the use of vel in comparison with different therapeutic options, in order to offer a procedure as an alternative or in association with proven therapies, as a new safe and effective option to treat gsm symptoms in menopausal practice.
Blood samples: peripheral blood samples were obtained from several farmers and veterinarians for diagnosis of blv infection at the hokkaido university veterinary teaching hospital . Informed consent was obtained from each owner, and approval for all procedures was obtained from the institutional animal care and use committee of hokkaido university . Dna was purified from 500 l blood samples using the wizard genomic dna purification kit (promega, madison, wi, u.s.a .) According to the manufacturer s protocol and finally suspended in 50 l buffer . Cells: ku-1 cells infected with blv were maintained at 37c in roswell park memorial institute (rpmi)-1640 medium (sigma - aldrich, st . Louis, mo, u.s.a .) Supplemented with 10% fetal bovine serum (cell culture technologies, gravesano, switzerland) and penicillin / streptomycin / l - glutamine (life technologies, carlsbad, ca, u.s.a . ). Blood samples not infected with blv that contained various numbers of ku-1 cells (1010 cells/1,000 l of blood; 1010 cells/l) were used as pcr - db templates, and dna purified from those blood samples was used as nested pcr templates . Pcr - db: amplification of blv provirus from whole blood used kod fx neo (toyobo, osaka, japan), which is superior for amplification from crude samples, and a specific primer pair (pv2-f 5-act ttc aga ccc cct tga ctg aca-3 and pv2-r 5-aaa cct ctg ccc tgg tga tta agg-3). This primer pair was designed by primer3 to amplify blv provirus (genebank accession number: both k02120 and af033818), and the intron region of the provirus (33083580 in k02120) was amplified by the primer pair . Briefly, each 30 l reaction mixture contained 0.4 mm dntps, 0.5 m of primers, 1 u of kod fx neo and 1 l of whole blood (10-, 50- or 100-fold dilutions in double distilled water; fig . 2.comparison of nested pcr and pcr - db sensitivity . To assess pcr method sensitivity, dna samples purified from blv - uninfected blood containing ku-1 cells (1010 cells/l) were used as nested pcr templates, and the blood samples were used as pcr - db templates (1-, 10-, 50- or 100-fold diluted in double distilled water). (a) representative images of electrophoresis of amplicons generated using each pcr condition . (b, c) the results from detectable samples using each pcr condition are presented . White bars indicate ku-1 cell counts which were undetectable by electrophoresis, while the gray (dna) and black (blood) bars indicate the templates used for each pcr condition . Amplifications were performed under the following conditions: one lysis cycle at 94c for 2 min and then 45 cycles of template denaturation at 94c for 15 sec followed by annealing and extension at 68c for 50 sec . The -globin gene was amplified as an internal control using the following primer pairs: 5-tgc tga ctg ctg agg aga agg ctg-3 and 5-gtc ctc aca cgc cca ggt gca ttt c-3. Comparison of nested pcr and pcr - db sensitivity . To assess pcr method sensitivity, dna samples purified from blv - uninfected blood containing ku-1 cells (1010 cells/l) were used as nested pcr templates, and the blood samples were used as pcr - db templates (1-, 10-, 50- or 100-fold diluted in double distilled water). (a) representative images of electrophoresis of amplicons generated using each pcr condition . (b, c) the results from detectable samples using each pcr condition are presented . White bars indicate ku-1 cell counts which were undetectable by electrophoresis, while the gray (dna) and black (blood) bars indicate the templates used for each pcr condition . Nested pcr: to amplify the long terminal repeat (ltr) in the blv provirus, nested pcr was performed using rtaq (takara - bio, otsu, japan) as previously described . Briefly, the blv ltr was amplified using primer pairs, blv - ltr1 and blv - ltr533, for the first pcr reaction, and 1.5 l of dna from whole blood or blv - uninfected blood with ku-1 cells were used as templates . And then, 1.5 l of the first pcr products were reamplified using blv - ltr256 and blv - ltr453 for the second pcr reaction . Real - time pcr: to confirm the provirus loads of blv - infected cattle diagnosed by nested pcr, we used a real - time pcr system (lightcycler 480 system ii; roche diagnostics, mannheim, germany), sybr premix dimer - eraser (takara - bio), and the primers pv2-f and pv2-r for blv and 5-aca caa ctg tgt tca cta gc-3 and 5-caa ctt cat cca cgt tca cc-3 for -globin . Amplification of dna samples from whole blood was performed as follows: one cycle at 95c for 30 sec, followed by a 3-step pcr procedure consisting of 5 sec at 95c, 30 sec at 60c and 30 sec at 72c for 45 cycles . To obtain a standard curve, serial dilutions of the standard plasmid containing from 10 to 10 copies tumor samples: to diagnose bovine leukemia by using pcr - db, tumor cells from three cattle with clinically diagnosed lymphoma were collected: case no . 1 (holstein - friesian, 4 months old, enzootic bovine leukosis), case no . 2 (japanese black, 5 years old, enzootic bovine leukosis) and case no . The tumor cells were stained with an antibody specific to b cell markers and analyzed by flow cytometry as described previously . In brief, double staining was performed using anti - bovine igm (il - a30; abd serotec, oxford, u.k .) Pre - labeled with zenon alexa fluor 488 (life technologies) and the following antibodies: anti - wc4 (cc55; cd19-like; abd serotec); anti - b - b7 (gb25a; cd21-like; vmrd, pullman, wa, u.s.a . ); and anti - bovine cd5 (cact105a; vmrd). Alexa fluor 647-conjugated anti - mouse igg (life technologies) was used for bound antibody detection (anti - wc4, anti - b - b7 and anti - cd5). Tumor cells were incubated with anti - wc4, anti - b - b7 and anti - cd5 as the first antibody, anti - mouse igg as the second antibody and anti - igm as the third antibody . Blv infection was diagnosed from blood and tumor cells by nested pcr as described above . Approximately 12 mm of tumor tissue suspended in 1 ml of phosphate buffered saline was used as a template for pcr - db . Pcr - db amplification of blv provirus from whole blood: first of all, we tried to use blv - ltr 256 and blv - ltr 453 for pcr - db amplification of blv provirus . But, the tm value of this primer pair was not suitable for the polymerase we used for pcr - db, resulted in appearance of many extra bands (data not shown). Because of that, we designed an optimal primer pair for the polymerase of pcr - db following the manufacturer s protocol . The pv2 primer pair was determined from seven ones based on their ability to show the most sensitive and reproducible results (data not shown). To confirm the amplification of blv provirus from whole blood, pcr - db was performed using blood samples for which blv infection had been previously diagnosed by nested pcr . We found single bands approximately 272 bp using the pv2 primer pair, with the results from pcr - db amplification completely consistent with those from nested pcr reactions (fig . Blv provirus amplification was performed by (a) nested pcr and (b) pcr - db . Blood samples were collected from cattle with (n=4) and without (n=4) diagnosed blv infections . Nested pcr and pcr - db, dna samples purified from blv - positive cattle were used as a positive control (pc), and double distilled water was used as a negative control (nc).). The amplification of -globin was observed as bands of approximately 100 bp in all samples, although a second band of approximately 400 bp was also produced in pcr - db using the -globin primer pair (data not shown). Blv provirus amplification was performed by (a) nested pcr and (b) pcr - db . Blood samples were collected from cattle with (n=4) and without (n=4) diagnosed blv infections . The -globin gene was amplified as an internal control . For both of nested pcr and pcr - db, dna samples purified from blv - positive cattle were used as a positive control (pc), and double distilled water was used as a negative control (nc). Pcr - db results are reproducible with sample dilution: to compare the sensitivity of nested pcr and pcr - db methods, blv provirus was amplified from ku-1 cells mixed in blood from healthy cattle (fig . The provirus was detected by nested pcr even from samples containing 0.1 cells or 1 cell per 1 l of blood (average level of undetectable cell numbers: 0.083 cells/l; fig . Pcr - db amplification using dna samples as templates was also performed to confirm the effects of its primer pair and polymerase on assay sensitivity . Results showed a similar sensitivity to that of nested pcr (0.067 cells/l; fig . However, the results of pcr - db used undiluted blood samples indicated less sensitivity and reproducibility . On the other hand, pcr - db performed under variable conditions, with blood samples diluted 10-, 50- and 100-fold with double distilled water, showed improved reproducibility, even though the sensitivity was lower than for undiluted samples (0.55, 5.5 and 25 cells/l, respectively; fig . Pcr - db diagnosis of most of blv - infected cattle that showed detectable provirus loads: in total, 225 bovine blood samples were tested by nested pcr and pcr - db . Whole blood was diluted 50-fold with double distilled water for pcr - db, representing the middle condition tested in fig . 2c . Using both methods, 37 samples were positive, and 176 samples were negative, with no samples that were positive by pcr - db and negative by nested pcr, thus indicating a pcr - db specificity of 100% (table 1table 1.amplification of blv provirus in clinical blood samplespcr resultnested pcr positivenested pcr negativetotalpcr - db positive37037pcr - db negative12176188total49176225). The provirus in 12 samples was detected only using nested pcr and not with pcr - db, indicating a pcr - db sensitivity of 75.51% . Because we considered that those false - negative results are responsible for poor infection levels, provirus loads of clinical samples that were positive by nested pcr were measured by real - time pcr . The results showed that the provirus loads of these 12 samples were quite low, suggesting that the animals were asymptomatic carriers at the aleukemic stage (fig . Dna samples from blv - infected cattle diagnosed by nested pcr were evaluated to determine provirus loads using real - time pcr (n=49). The y - axis indicates the rates of blv genome copies in 100 cells as determined by -globin amplicon copy numbers . The dot colors depict the pcr - db results (white: negative; black: positive). Dna samples from blv - infected cattle diagnosed by nested pcr were evaluated to determine provirus loads using real - time pcr (n=49). The y - axis indicates the rates of blv genome copies in 100 cells as determined by -globin amplicon copy numbers . The dot colors depict the pcr - db results (white: negative; black: positive). Pcr - db directly detected blv provirus in tumor samples: to investigate whether pcr - db can be used to diagnose blv infection from tumor tissues, the provirus in tumor samples was amplified using pcr - db . In the three sample cases, two out of three cases were blv - positive tumor tissues, and all pcr - db results were entirely consistent with those of nested pcr (fig . Amplification of the blv provirus in blood and tumor samples isolated from three cattle with bovine leukemia was performed using pcr - db, with 50-fold diluted blood and tumor suspensions used as templates . Ln: lymph node . (a) case no . 1 (holstein - friesian, 4 months old): 1, blood; 2, thymus; 3, spleen; 4, gastric ln; 5, mesenteric ln; and 6, inguinal ln . (b) 2 (japanese black, 5 years old): 1, blood; 2, spleen; 3, heart; 4, superficial cervical ln; 5, mesenteric ln; 6, mediastinal ln; and 7, renal ln . (c) 3 (holstein - friesian, 2 years old): 1, blood; 2, cervical thymus; 3, thoracic thymus; 4, superficial cervical ln; and 5, bronchial ln . ). 1, a blv - positive tumor, expressed several b - cell markers, such as igm, wc4 (cd19-like) and b - b7 (cd21-like) on the tumor cell membrane, and on the other hand, case no . 3, which was blv - negative, did not show b - cell phenotypes (table 2table 2.phenotyping and diagnosis of tumor samples from cattlecase no.blv infectionlymphocytes (10/l)cell surface markerstumor cell typesdiagnosislymphocytesb cellscd5wc4b - b7igm1 + 3,881++/++b cellsenzootic bovine leukosis2+n.t.n.t.unknownenzootic bovine leukosis3 - 23 + ---t cellsthymic lymphosarcoma). These results strongly supported the clinical diagnosis, cases no.1 and no.2 as enzootic bovine leukosis and case no.3 as thymic lymphosarcoma of sporadic bovine leukosis . Amplification of the blv provirus in blood and tumor samples isolated from three cattle with bovine leukemia was performed using pcr - db, with 50-fold diluted blood and tumor suspensions used as templates . 1 (holstein - friesian, 4 months old): 1, blood; 2, thymus; 3, spleen; 4, gastric ln; 5, mesenteric ln; and 6, inguinal ln . 2 (japanese black, 5 years old): 1, blood; 2, spleen; 3, heart; 4, superficial cervical ln; 5, mesenteric ln; 6, mediastinal ln; and 7, renal ln . 3 (holstein - friesian, 2 years old): 1, blood; 2, cervical thymus; 3, thoracic thymus; 4, superficial cervical ln; and 5, bronchial ln . In this study, we developed novel diagnosis method named pcr - db for amplifying the blv provirus directly from whole blood . Our method showed high specificity and reproducibility with diluted blood samples, while undiluted ones resulted in less reproducibly probably because of endogenous pcr inhibitors contained in the blood samples and blood viscosity, which made it difficult to measure the sample volume accurately . Although the sensitivity of pcr - db was lower than that of nested pcr, all clinical samples detected only using nested pcr and not with pcr - db showed slight provirus loads, suggesting that those were from carriers at early stage . So, we believe that our study demonstrates the utility of pcr - db for rapid diagnosis of blv infection . In previous studies, some researchers have reported the amplification of dna directly from whole blood using pcr methods for diagnosing bacterial or viral infections, including mycoplasma haemofelis, bartonella quintana and hepatitis b virus . Moreover, in another report, pcr - db was used for mutation screening of gm1 gangliosidosis in dogs, demonstrating that this technique can also be a good method for hereditary disease screening and not only for detecting infectious diseases . These reports suggest that pcr - db has the potential to be applied in multiple clinical situations as a novel, rapid and viable method of diagnosis . One of the primary methods used to identify blv - infected cattle is a serological test (agid, elisa, etc .) Those tests are appropriate for screening thousands of cattle, because of easy sample preparation compared with pcr tests which require dna extraction and availability of a rapid and cost - effective diagnosis kit . However, in some cases, these serological tests show several problems, including poor sensitivity compared with pcr testing, false positive samples, detection of maternal antibodies and inability to perform a diagnosis using tissue or semen samples [3, 5, 11, 15]. Although sweden achieved eradication of blv - infected cattle and elimination of blv using only an elisa test, pcr testing would enable a more definitive surveillance program to eradicate blv infection . There are many advantages of using pcr - db to detect the blv provirus: i) pcr - db specificity as calculated by diagnosing clinical samples was 100%; ii) the technique does not require a special and expensive thermal cycler like real - time pcr; iii) it is not labor intensive or time - consuming and is cost - effective; iv) because it is a rapid and straightforward procedure, there is less possibility of contamination; and v) less than 10 l of blood is enough to run the pcr - db assay, even in duplicate . The biggest challenge with pcr - db is the existence of pcr inhibitors in whole blood, which contains igg, hemoglobin and lactoferrin [1, 2]. Our results demonstrate that endogenous pcr inhibitors do not impact the reproducibility of the pcr reaction by sample dilution . Thus, pcr - db is a suitable method for use by clinical veterinarians to perform blv diagnosis in a typical veterinarian s office . Nested pcr using purified genomic dna is likely the best method for detecting blv infection, because provirus in the blood from several cattle showing slight provirus loads was not detected by pcr - db . Although the amplification of low - copy provirus may be difficult using pcr - db, the sensitivity of pcr - db can be improved by simply increasing the template blood volume . As demonstrated in fig . 2c, pcr - db using 10-fold diluted blood samples showed higher sensitivity than other dilutions and adequate reproducibility . Therefore, we consider pcr - db to be appropriate in first screening diagnosis in an individual farm or region to systematically eradicate blv - infected cattle . This technique is preferred, because the use of nested pcr for diagnosis in a large number of cattle is both labor intensive and time - consuming . Pcr - db represents the best practical way for eliminating blv infection through periodic screening and isolation or culling of all infected animals at intervals of several months or a few years . Our preliminary data showed that the cell number in the pcr reaction buffer influenced the stability of the results and that excessive cell numbers inhibited the pcr reaction (data not shown). Thus, suspending tumor cells in phosphate buffered saline or other suitable solutions are important in order to adjust the templates to the appropriate pcr conditions . In tumors, pcr - db may be a better method for diagnosing blv infection than nested pcr or other methods, because the most rapid and definitive method for diagnosing blv infection in cattle with lymphoma is required by clinical veterinarians and because serological tests are not suitable for tissue diagnosis . Incidentally, calf lymphoma (case no.1) was diagnosed with enzootic bovine leukosis which is caused by blv infection, because the lymphoma was constructed with clonal cd5 igm b cells highly expressing viral protein gp51 (data not shown). However, denmark did so by peripheral blood cell counts without serological tests . In our opinion, other countries can eliminate the infection more definitively than denmark using pcr - db . Countries with a much higher prevalence can work to eradicate all infected cattle through pcr - db screening and other strategies, such as isolating blv - positive cattle and reducing the incidence of bovine leukemia . We conclude that this pcr - db assay is a highly simplified, cost - effective and rapid method (results obtained in less than 3 hr) that serves as a new alternative way to diagnosis blv infection without dna purification.
Drh rats were established by the selective mating of donryu rat progeny that exhibited resistance to hepatocarcinogenesis induced by the feeding of a 3-methyl-4-dimethylaminoazobenzene (3-me - dab) diet [1, 2]. Drh rats exhibit hepatocarcinogenesis resistance to 3-me - dab as well as other hepatocarcinogens . In drh rats, preneoplastic hepatocytic lesions are smaller in size and fewer in number than those of ordinary rats [4, 5]. Genetic analysis has demonstrated that resistance is a dominant trait in a cross of drh and donryu rats and that the number and size of preneoplastic hepatocytic lesions and the expression levels of glutathione - s - transferase - placental form (gst - p) mrna and protein, a marker for preneoplastic hepatocytes, are strongly affected by highly significant quantitative trait loci (qtl) on rat chromosomes 1q (drh1) and 4q (drh2) [5, 6]. It has also been shown that the small size of preneoplastic hepatocytic lesions might be dependent mainly on the tissue environment; that is, transplanted drh preneoplastic hepatocytes were observed to form large colonies in the livers of donryu rats treated with dietary 2-acetylaminofluorene (2-aaf) plus partial hepatectomy (ph), which enables the selective growth of preneoplastic hepatocytes, and transplanted donryu preneoplastic hepatocytes formed small colonies within the livers of drh rats treated with the same protocol . Drh hepatocytes have a number of unique properties compared to hepatocytes of ordinary rats . First, drh hepatocytes exhibit less proliferation after treatment with lead nitrate (ln) [4, 9], a nonnecrogenic hepatocyte proliferating agent, which is thought to be mediated, at least in part, by the cytokines generated by activated kupffer cells [11, 12]. Second, drh hepatocytes are resistant to various hepatotoxic chemicals, including carbon tetrachloride (ccl4), diethylnitrosamine (den), thioacetamide, 2-aaf, and 3-me - dab . Third, in primary culture, drh hepatocytes exhibited less apoptosis and lower activation of p38 and jun terminal kinase (jnk) than donryu hepatocytes, although the expression levels of 8-hydroxyguanine and p53/p21 proteins that reflect dna damage [13, 14] were comparable to those of donryu hepatocytes . Fourth, drh hepatocytes are smaller in size than donryu hepatocytes . If some of these properties of drh hepatocytes are linked to the drh1 or drh2 locus, then the properties might also be related to the mechanism(s) of hepatocarcinogenesis resistance in drh rats . Drh.f344-drh1 rats represent the congenic drh strain in which the chromosome 1 segment that includes the drh1 locus is substituted with that of f344 rats . These rats exhibit a high incidence of gst - p(+) foci in response to treatment with 3-me - dab / ph and high liver cell proliferation after ln treatment, comparable to that in f344 rats, but formed intermediate - sized gst - p(+) foci and exhibited intermediate gst - p mrna expression levels between the levels of f344 and those of drh rats after treatment with den followed by dietary 3-me - dab / ph . This finding indicates that the drh1 locus contributes to the low incidence of gst - p(+) focus formation and the low level of liver cell proliferation in response to ln treatment, but it does not fully explain the smaller size of the gst - p(+) foci . In the present study, we investigated which properties of drh hepatocytes are linked to the drh1 locus by comparing the hepatocytes of drh.f344-drh1 rats with those of drh, donryu and f344 rats in vivo and in vitro . Hiai, medical center of the shiga prefecture, japan), drh (seak yoshitomi, fukuoka, japan), donryu (charles river, tokyo, japan), and f344 rats (charles river) were used . The drh1 locus on chromosome 1 of these rats is illustrated in figure 1 . The animals were maintained in 12 h light / dark cycles and given laboratory rodent pellet chow (oriental, tokyo, japan) and water ad libitum . One group of rats was intravenously administered ln at a dose of 100 mmole / kg body weight, given bromodeoxyuridine (brdu) on day 1, 2, or 3 after ln treatment, and sacrificed 1 h after brdu treatment . The liver tissues were perfusion - fixed with a 10% buffered - formalin solution, immersed in fixative solution for 24 h, and then processed for paraffin embedding . The tissue sections were immunostained using an anti - brdu antibody (becton dickinson, san jose, calif, usa), and the brdu labeling index (li) of hepatocytes was determined microscopically . Other groups of rats were administered den dissolved in physiological saline at a dose of 200 mg / kg body weight by intraperitoneal injection or ccl4 dissolved in olive oil at a dose of 5 ml / kg body weight by gastric tube and sacrificed 24 h later . Blood samples were taken from each rat, and serum was isolated to measure alanine aminotransferase (alt) and aspartate aminotransferase (ast) levels . All animal procedures were approved by the asahikawa medical college committee according to the guidelines for humane care of laboratory animals and conducted according to the asahikawa medical college committee of animal care and use . They were first cultured in williams' e medium with 10% fetal bovine serum (fbs) for 24 h and then cultured in hepatocyte growth medium (hgm) supplemented with 5 u / ml penicillin, 100 g / ml streptomycin, and various concentrations of egf (0, 1, 10, or 50 ng / ml) or hgf (0, 5, 20, or 100 ng / ml) for 3 days . The cultured hepatocytes were treated with [h]-thymidine for 3 days after beginning culture with hgm, and [h]-thymidine incorporation into dna was determined using a liquid scintillation counter (beckmann - coulter, palo alto, calif, usa). Freshly isolated hepatocytes were plated on a 15 mm diameter cover glass coated with cellmatrix type 1-a (nitta gelatin, tokyo, japan) in williams' e medium supplemented with 10% fbs, cultured for 2 h to allow attachment to the cover glass, fixed in 1% buffered - formalin solution and stained by dapi . The cells were observed under a fluorescence microscope (nikon, tokyo, japan) equipped with a cool - scanning digital camera (hamamatsu photonics, hamamatsu, japan). Nonfluorescent and dapi - stained nuclear images of hepatocytes were saved as photoshop files, and each cell was divided into small single- or double-, medium single- or double- or large single- or double - nuclear cells using imagej software (national institute of health, bethesda, md, usa). At least 500 hepatocytes were measured for each rat, using three rats from each strain . Protein lysates were prepared from liver tissue samples, run on 813% polyacrylamide gels containing 0.1% sodium dodecyl sulfate and transferred to pvdf membranes (amersham, uppsala, sweden). The membranes were probed with primary antibodies and then hybridized with horseradish peroxidase- (hrp-) conjugated anti - rabbit immunoglobulin (amersham). The bound antibodies were detected using an ecl - plus kit (amersham). Emsa was performed for nuclear extracts of ln - treated liver tissues, and the dna - protein complexes were resolved on a 6% polyacrylamide gel . A biotin - labeled oligonucleotide representing an nfb consensus site (5-agttgaggggactttcccaggc-3) and a stat3 consensus site (5-ttct ggg aat t-3) was used as a probe . Fresh liver samples were isolated at 0, 3, 6, 12, and 24 h after ln treatment, frozen in liquid nitrogen, and stored at 80c until further use . Rna was isolated with sepazol and reverse - transcribed using the generic oligo dt primer and reverse transcriptase (invitrogen, carlsbad, calif, usa). The following pcr primers were used: for tnf-, 5-cccatttgggaacttctcct-3 (forward) and 5-agatgtggaactggcagagg-3 (reverse); for il-6, 5-gcccttcaggaacagctatg-3 (forward) and 5-tcagtcccaagaaggcaact-3 (reverse); for cox2, 5-gagatacgtgttgacgtcc-3 (forward) and 5-actgatgagtgaagtgctgg-3 (reverse). Real - time rt - pcr was performed using a lightcycler (roche molecular biochemicals, basel, switzerland) with lightcycler3 run software (version 5.32). Fluorescence was generated by a platinum sybr green qpcr supermix udg, and data were collected with lightcycler3 data analysis software (version 3.5.28). Each experiment was performed at least three times, and representative data are shown . The means were analyzed using student's t - test or anova, and p values less than 0.05 were considered statistically significant . When donryu, drh, drh.f344-drh1, and f344 rats were treated with ln, the brdu li markedly increased in drh.f344-drh1 hepatocytes on day 2 as reported by liu et al ., similarly to the increases observed in donryu and f344 hepatocytes, but the brdu li did not increase in drh hepatocytes (figures 2(a) and 2(b)), indicating that the drh1 locus is linked to the low response of drh hepatocytes to ln . It has been suggested that hepatocyte proliferation induced by ln is mediated at least partly by cytokines such as tnf- and il-6, which are generated by activated kupffer cells [11, 12]. In addition, cox2 protein expression in activated kupffer cells, which leads to the overproduction of prostaglandins, has been shown to play an important role in hepatocyte proliferation . Furthermore, when donryu rats were treated with indomethacin, a cox1/2 inhibitor, prior to and after ln treatment, the brdu li of hepatocytes decreased to approximately 50% of the values after ln treatment alone on day 2, indicating that prostaglandins at least partly mediate hepatocyte proliferation after ln treatment (unpublished data by m. yamamoto). We therefore investigated whether the drh1 locus plays a role in cytokine and cox2 activation after ln treatment . As shown in figure 3(a), tnf-, il-6, and cox2 mrna expression levels were increased to the same extent from 3 to 12 h after ln treatment, peaking at 6 h in drh.f344-drh1, donryu, and drh livers, suggesting that the drh1 locus is not related to cytokine and cox2 activation after ln treatment . Because tnf- and il-6 can activate nfb and stat3, respectively, which are transcription factors that play an important role in hepatocyte proliferation, we investigated nfb / stat3 activation by emsa using nuclear lysates prepared from ln - treated livers and nfb / stat3-specific oligonucleotide probes . Figure 3(b) illustrates that nfb / stat3 activation occurred 616 h after ln treatment, with a peak at 6 h, in donryu, drh, and drh.f344-drh1 livers . It has been reported that when the liver is exposed to toxic chemicals, the phosphorylation status of mapk pathway proteins, including erk1/2, jnk, and p38, fluctuates shortly after exposure [2022]. We therefore investigated whether there was any difference in the phosphorylation status of mapks after ln treatment in the livers of donryu, drh, drh.f344-drh1, and f344 rats . Erk1/2, which was hypophosphorylated in normal livers, became hyperphosphorylated 312 h after ln treatment, whereas jnk, which was also hypophosphorylated in normal livers, showed no remarkable change in phosphorylation status after ln treatment in all rats (data not shown). However, although p38 was hyperphosphorylated in the normal livers of all donryu, drh, drh.f344-drh1, and f344 rats, as reported previously [2022], it became hypophosphorylated after ln treatment in donryu, drh.f344-drh1, and f344 livers, peaking at 12 h, but such hypophosphorylation was at much less degree in drh livers (figure 4). Drh hepatocytes have been demonstrated to be resistant to various hepatotoxic chemicals, including ccl4, thioacetamide, den, 2-aaf, and 3-me - dab . To test the role of the drh1 locus in hepatotoxin susceptibility, donryu, drh, drh.f344-drh1, and f344 rats were administered 200 mg / kg den or 5 ml / kg ccl4 to compare liver injury 24 h after treatment . After den treatment, serum alt and ast levels in drh.f344-drh1 rats were as high as those in donryu and f344 rats, while these levels were significantly lower in drh rats (figure 5(a)). After ccl4 treatment, drh rats exhibited a lower degree of hepatic injury than donryu rats as previously reported, while f344 rats also showed the lower degree of injury than donryu rats (figure 5(b)), presumably because the factor(s) other than the drh1 locus that was involved to the ccl4 susceptibility might be different between donryu and f344 rats . However, drh.f344-drh1 rats exhibited a higher degree of hepatic injury than drh and f344 rats (figure 5(b)). We next addressed whether the drh1 locus plays a role in the proliferative capacity of hepatocytes . For this purpose, hepatocytes were isolated from donryu, drh, drh.f344-drh1, and f344 rats and cultured in the presence of various concentrations of egf (0, 1, 10, or 50 ng / ml) or hgf (0, 5, 20, or 100 ng / ml). Because [h]-thymidine uptake increased beginning on day 1 after growth factor treatment and peaked on day 3 in the hepatocytes of all rats (data not shown), the cells were then incubated with [h]-thymidine between days 1 and 3 . Uptake of [h]-thymidine into hepatocyte dna increased after egf or hgf treatment in all rats, but drh.f344-drh1 and drh hepatocytes exhibited approximately 5070% of the uptake observed in donryu and f344 hepatocytes (figures 6(a) and 6(b)). We therefore compared the nuclear sizes of donryu, drh, drh.f344-drh1, and f344 hepatocytes collected from 8-week - old rats . When the dapi - stained nuclear images of hepatocytes cultured for 2 h on cover glass were analyzed by image j software, the nuclear size could be categorized into small, medium, and large classes (data not shown). Approximately 60% of drh.f344-drh1 and drh hepatocytes had single- or double - small - sized nuclei, while the remainder had single - medium - sized nuclei (figures 7(a) and 7(b)). By contrast, 1015% of donryu and f344 hepatocytes had double - small - sized nuclei, while the remaining 8590% had single- or double - medium - sized nuclei (figures 7(a) and 7(b)). In drh rats, two major genetic loci, drh1 on chromosome 1 and drh2 on chromosome 4, have been demonstrated to have strong effects on the number and size of preneoplastic hepatocytic lesions during hepatocarcinogenesis [5, 6]. Drh hepatocytes, on the other hand, have unique properties, including low levels of proliferation after ln treatment [4, 9], resistance to hepatotoxic chemicals, small cell size and low levels of p38 and jnk activation in vitro . If some of these properties are linked to the genetic loci, then the properties might be related to the mechanism(s) of hepatocarcinogenesis resistance in drh rats . By comparing drh.f344-drh1 hepatocytes with donryu, drh, and f344 hepatocytes, we demonstrated that low levels of proliferation and low levels of p38 dephosphorylation after ln treatment and resistance to hepatotoxic chemicals in drh hepatocytes are linked to the drh1 locus, while low levels of proliferation in vitro and small nuclear size are not linked to this locus (table 1). It has been suggested that the hepatocyte proliferation induced by ln is at least partly due to the soluble factors generated by activated kupffer cells [11, 12]. After ln treatment, the mrna levels of tnf-, il-6, and cox2, which are expressed in activated kupffer cells [23, 24], were increased, and nfb and stat3, which are transcription factors that can be activated, respectively, by tnf- and il-6 to play important roles in hepatocyte proliferation, were activated . However, there was no remarkable difference between drh, donryu, and drh.f344-drh1 livers regarding cytokine and cox2 mrna expression and nfb or stat3 activation after ln treatment, suggesting that the low level of proliferation of drh hepatocytes induced by ln might not be due to the lack of factors that mediate hepatocyte proliferation; instead, they might be dependent on the inherent properties of drh hepatocytes . It has been reported that, although p38 is hyperphosphorylated in normal livers, it becomes hypophosphorylated after treatment with chemicals such as ccl4, d - galactosamine, and thioacetamide [2022] and that p38 hypophosphorylation is mediated by activation of the phosphatase mkp-1 . In the present study, we demonstrated that p38 dephosphorylation also occurred after ln treatment, an effect that was dependent on the drh1 locus . Although the mechanism of p38 dephosphorylation induced by ln is unknown, this phenomenon may be related to the low level of proliferation of drh hepatocytes induced by ln and to hepatocarcinogenesis resistance in drh rats . The resistance of drh hepatocytes to den and ccl4 was also linked to the drh1 locus . Liu et al . Reported that when a 3-me - dab diet was continuously provided, 17.9%, 11.3%, and 5.7% of the total liver tissues were made up of fibrotic areas in f344, drh.f344-drh1, and drh rats, respectively, suggesting that the drh1 locus may also be related to resistance to chronic hepatic injury induced by 3-me - dab . Hepatic injury of the liver tissue is considered important for the selective growth of preneoplastic hepatocytes during hepatocarcinogenesis . In fact, transplanted drh preneoplastic hepatocytes proliferate at high rates in donryu livers treated with 2-aaf / ph, while transplanted donryu hepatocytes proliferate at low rates in drh livers treated with the same regimen . Therefore, the tissue environment of the drh liver may be less effective for the selective growth of preneoplastic hepatocytes during hepatocarcinogen treatment, which may be one of the mechanisms of hepatocarcinogenesis resistance in drh rats . The low level of proliferation of drh hepatocytes in vitro is not linked to the drh1 locus . Hepatocyte proliferation in vitro is associated with complex changes, including the loss of hepatocyte - specific gene expression (e.g., albumin and cytochrome p4502a1), the gain of bile duct epithelium - specific gene expression (e.g., cytokeratin 19), and the activation of specific transcription factors (e.g., ap1 and nfb). In addition, the signals mediated by the extracellular matrix influence proliferation and gene expression in hepatocytes in vitro [17, 2628]. Although the basis for the low level of proliferation of drh hepatocytes in vitro remains to be investigated, drh hepatocytes may have inherently low responsiveness to hepatocyte growth factors . Because the proliferation of preneoplastic hepatocytes is considered to be dependent on extracellular signals rather than on autonomous growth, the low level of proliferation of drh hepatocytes in vitro may be related to the small preneoplastic hepatocytic lesions present in drh livers during hepatocarcinogenesis . The small nuclear size of drh hepatocytes was also not linked to the drh1 locus . Although hepatocytes have single - diploid nuclei during development, double - nuclear diploid and single- or double - nuclear tetraploid cells constitute the main populations in the adult liver . Double - nuclear cells may be generated by a lack of cytokinesis after dna synthesis, while tetraploid cells may be generated by a lack of mitosis after dna synthesis in diploid cells . On the other hand, polyploid hepatocytes increase in number under various pathophysiological conditions, including liver regeneration after partial hepatectomy, exposure to hepatotoxic chemicals such as retrorsine [31, 32], and metabolic defects associated with copper or iron deposition [33, 34]. Moreover, polyploid hepatocytes have been shown to express senescence phenotypes, including increased expression of p21 and -galactosidase, which has been demonstrated in senescent cells [36, 37], low replication capacity, and high levels of apoptosis . It is possible that suppressed or delayed tetraploidization in drh hepatocytes reflects a diminished senescence phenotype, which may be linked to hepatocarcinogenesis resistance in drh rats . In summary, drh hepatocytes possess unique properties, some of which are linked to the drh1 locus however, it remains to be investigated whether other properties are linked to the drh2 locus . Identification of the genes present in these loci may contribute to the clarification of the mechanism(s) and prevention of hepatocarcinogenesis.
Patients presenting a carotid stenosis and contralateral occlusion (co) have been historically considered at high risk for carotid endarterectomy (cea), since results from randomized controlled trials (rcts) on carotid surgery have reported morbidity and mortality rates significantly higher than in the general population affected by carotid stenosis [15]. Hence, that group of patients has been frequently excluded from surgical treatment in some prospective rcts comparing results in cea and carotid artery stenting (cas) in the following years, as in the sapphire trial . On the other hand, some single - center reports have highlighted a nondissimilar rate of complications in patients presenting with a carotid stenosis with or without contralateral occlusion [610]. In those studies perioperative complications are reported in 0.7 to 6.9% of patients presenting with carotid stenosis and contralateral occlusion, thus reaching percentages consistent with international recommendations for cea postoperative complications in symptomatic patients, but surely exceeding those requested for cea in asymptomatic ones [11, 12]. However, within the population of patients affected by carotid stenosis and contralateral occlusion, different risk categories could be identified with respect to general medical conditions and involvement of other vascular districts in the atherosclerotic process, as well as with respect to presence of previous brain infarct on neuroimaging . It has to be noted that frequently patients affected by co have been included in the high - surgical risk category mixed with patients presenting general medical conditions at high risk for surgery, thus generating confusion with regard to the real risk addressed by those patients [5, 7, 8, 10]. We retrospectively reviewed our database on carotid endarterectomy to analyse the incidence of postoperative neurological complications in patients affected by carotid stenosis and chronic contralateral occlusion, to analyse factors associated with the development of complications in this group of patients, and to compare their results with those obtained in the group of patients without co submitted to cea at our vascular surgery division . From january 1997 to december 2012, 1639 patients underwent primary cea at our vascular surgery division . Data on demographics, risk factors, preoperative neurological evaluation and imaging, intervention, and 30-day outcomes were prospectively collected in our institutional database . From the database 136 out of 1639 (8.3%) patients presenting a stenosis of internal carotid artery with a contralateral internal carotid occlusion (co) submitted to carotid endarterectomy in the stenotic side were identified . A carotid stenosis 70% in asymptomatic patients or 50% (nascet stenosis evaluation criteria) in symptomatic patients was considered indication for intervention . Patients were considered symptomatic if they presented carotid - related neurological symptoms in the previous six months before operation . Patients submitted to carotid endarterectomy in urgency (within 2 weeks from last neurological symptom ipsilateral to the carotid stenosis or occlusion) were not considered for analysis in the present series because they represent a subset of patients at higher surgical risk . All patients preoperatively underwent a complete medical examination, assessment of preoperative neurological examination by the neurologist by use of national institute of health stroke scale (nihss) or rankin scale evaluation, blood test, electrocardiogram, and carotid duplex ultrasound imaging to assess the degree of stenosis . Brain computed tomography (ct) or magnetic resonance imaging (mri) was performed in all co patients and in all control sample patients with symptomatic or ulcerated / irregular carotid plaque detected at duplex ultrasound examination . Before admission, all patients classified as having a carotid occlusion had been previously submitted to at least two different imaging modalities to confirm the obstruction (duplex us, contrast - enhanced supra - aorticvessels ct scans and mri). Patients were operated on on cervical block anaesthesia or, alternatively, general anaesthesia whenever a local anaesthesia was not feasible for patient - related causes . Patients under local cervical anaesthesia were assessed for neurological deficit and pain throughout the entire procedure by hand - grip test . During operations under general anaesthesia neurological monitoring was performed by transcranial doppler (tcd) whenever possible and adjunctive quantitative electroencephalogram (qeeg). Cerebral protection by sundt shunting was selectively used when mean velocity in the middle cerebral artery at tcd monitoring decreased to 15 cm / sec or a significant alteration in qeeg recording was detected . Patients were maintained under their scheduled asa (100 mg) or ticlopidine (250 mg) medication and were postoperatively reevaluated by a neurologist . Follow - up was conducted at our vascular ultrasound laboratory by carotid dus and clinical examination at 1, 6, and 12 months and then annually . When a postoperative complication had occurred a neurological assessment outcome measures for analysis were perioperative (30-day) major stroke (assessed as a neurological deficit lasting more than 24 hours and scored as nihss 4), minor stroke (assessed as a neurological deficit lasting more than 24 hours and scored as nihss 3), and stroke - related or neurological death . Water - shed infarction (cerebral border - zone infarctions) and hyperperfusion syndrome (defined as occurrence of severe unilateral headache, acute changes in mental status, vomiting, seizures, focal neurologic deficits, and, ultimately, intracranial hemorrhage) occurrence were also recorded . Neurological morbidity (major + minor stroke) and mortality were analyzed according to clinical preoperative demographics and presentation and intraoperative details and compared between patients with or without carotid occlusion contralateral to the treated carotid stenosis . Univariate predictors that were significant at p <0.05 were then entered into a multivariate model using logistic regression which was used to generate odds ratios (ors) and 95% confidence intervals (cis) for 30-day neurological outcomes, in order to identify a subset of patients at high risk for neurological complications . The demographic characteristics of the two groups (co and control patients) are shown in table 1 . Between groups analysis disclosed that patients with carotid stenosis and contralateral occlusion (co group) were significantly younger (67.02 7.9 versus 69.72 8.13; p <0.0001), were more frequently males (114 versus 1054; 83.8% versus 68.3%; p = 0.001) and more frequently smokers (103 versus 828; 75.7% versus 53.7%; p <0.0001), and presented a significantly higher incidence of peripheral arterial disease (45 versus 248; 33.1% versus 16.1%; p <0.0001) than patients without contralateral occlusion (control group). Co patients referred previous neurological symptoms ipsilateral to the stenosis in 18 cases out of 86, and contralateral, or ipsilateral to the carotid occlusion, in 68 cases . Analysis of neurological presentation before operation disclosed that co patients were more frequently symptomatic (86 co patients presenting preoperative neurological symptoms versus 665 control patients; 63.2% versus 46.4%; p <0.0001) and presented more frequently cerebral infarcts on preoperative brain imaging (65 co patients versus 379 control patients; 47.8% versus 25.2%; p <0.0001). One vertebral artery was occluded in 9 patients in the co group and in 25 patients in the control group . Co patients were more frequently submitted to cea under local anaesthesia (93 versus 873; 68.4% versus 58.1%; p = 0.02) and had more frequently a shunt implantation after clamping test (40 versus 121; 29.4% versus 8.1%; p <0.0001). Shunt use rate was not statistically different in patients submitted to cea under local or general anaesthesia in both groups . Mean clamping time was 23.3 13.5 versus 22.9 12.5 minutes in co versus control group (p = 0.28). Perioperative (30-day) complications analysis disclosed no significant difference in neurological mortality rates between the two groups (p = 0.28). Incidence of postoperative minor stroke was not significantly different in the two groups (0.7% in co group and 0.5% in control group, resp . ), while major stroke incidence analysis disclosed a statistically significant difference (4.4% in co and 1.2% in control group, p = 0.009), accounting for higher overall neurological morbidity and overall neurological complication rates in co group with respect to control group (5.1% versus 1.7%, p = 0.01, and 6.6% versus 2.1%, p = 0.003, resp . ). In co patients 5 out of 9 perioperative neurological complications were related to technical defects (carotid early thrombosis, 55.5%) while in control cases carotid early thrombosis was responsible for neurological events in 24 out of 26 cases (92.3%). In 4 cases in co group and 2 cases in control group neurological complications were related to hypoperfusion or reperfusion injuries (44.5% versus 7.7%). No complication occurred in co patients presenting one vertebral artery occluded and 1 major stroke (carotid thrombosis in the first hours following intervention) occurred in one patient with an occluded vertebral artery in the control group . Analysis of preoperative clinical factors and neurological outcome in co patients disclosed that neurological complications occurred more frequently in elderly patients (patients with complications versus patients without complications mean age 73.55 4.24 versus 66.55 7.9; 95% ci: 1.7612.27; p <0.01; patients with complications versus patients without complications and age> 74 years p = 0.008) and in patients with cerebral infarct on preoperative neuroimaging (p = 0.036). Use of shunt did not show to be related to an increased risk of neurological complications (4 patients in the shunt group and 5 patients in the nonshunt group presented overall perioperative neurological complications combined endpoint). All other variables analysed did not show any statistical difference between favourable and unfavourable outcome . The abovementioned risk factors (> 74 years derived from the intragroup continuous data analysis of age, and preoperative brain damage) were subsequently analyzed for their role in the development of postoperative neurological complications in the whole sample . Odds ratio analysis disclosed that the association of ipsilateral carotid stenosis and contralateral occlusion with bilateral or contralateral preoperative brain ischemic lesion exposed the cea patient to a higher surgical risk of postoperative neurological complications (or 8.1, 95% ci 1.7238.41, p = 0.001; and or 3.2, 95% ci 0.9510.96, p = 0.04, resp ., table 3). Similarly, in cea patients the association of ipsilateral stenosis and contralateral occlusion with age> 74 years increased significantly this risk (or 11.5, 95% ci 4.0832.62, p <0.0001). Further, in co cea patients the association of age> 74 years with ipsi / bilateral or contralateral preoperative brain damage showed an augmented surgical risk of brain lesions (or 19.9; 95% ci 1.77224.56, p = 0.0006; or 40.9, 95% ci 5.61298.05, p <0.0001, resp ., table 4). Among 11 co patients presenting both age> 74 years and a preoperative brain infarct, 5 (45.5%) presented postoperative neurological complications . Analysis of the causes of postoperative neurological complications in those 5 patients disclosed that one patient suffered from hemorrhagic transformation of a preoperative brain infarct and died; one patient suffered from acute postoperative carotid thrombosis with immediate neurological deterioration and slow recovery in the following months after cea; one patient with a very small internal carotid artery (maximal external diameter 4 mm) who did not tolerate carotid clamping, in which a 3 4 mm sundt shunt was employed, presented an intraoperative cerebral hypoperfusion with prompt recovery of the postoperative neurological deficit; and two patients presented a postoperative neurological deficit related to the contralateral carotid occlusion (watershed infarct) with a residual small deficit in the postoperative period . In the present series cea patients affected by carotid stenosis and contralateral occlusion presented significantly higher perioperative major stroke rate (p = 0.009), overall neurological morbidity (p = 0.01), and overall neurological complication rates (p = 0.003) compared to control group of patients . From our experience the co sample, as a whole, represents a subset of patients at higher surgical risk for cea when compared to the general patients affected by carotid stenosis requiring intervention, in accordance with previous reports [1, 3, 8, 1416]. Nevertheless, when dealing with co patients, some peculiarities must be taken into account and some ensuing considerations could be made . First, in our series co patients demographics analysis showed a higher frequency of smoking history, other vascular district involvements in the atherosclerotic process (i.e., peripheral arterial disease), neurological symptoms history, and, of course, presence of previous brain infarction on neuroimaging . Those data are in accordance with previous studies by julia et al . And rockman this seems to define the picture of a population at high risk for any surgical procedure, given the extensive involvement of the blood vessels in the atherosclerotic process [1823]. This peculiarity is underlined by the presence of a preoperative neurological symptomatology in 64% of patients in our series, since the population analyzed presented more frequently carotid - related symptoms and more frequently a brain infraction on neuroimaging contralateral to the side affected by the stenosis . So the brain in those patients is somewhat more fragile and less prone to tolerate carotid clamping during cea or also eventual small and clinically silent ischemic brain lesions (eventually accompanied by perilesional oedema) caused by microembolic plaque particles dislodged during cea . Ultimately, it could be speculated that the brain in co patients could have less cerebral functional reserve . Have recently reported a significant reduction in cerebrovascular reactivity in anterior circulation of the brain hemisphere ipsilateral to a carotid stenoocclusive disease, thus implying that unilateral carotid stenosis affects the vascular reserve of both sides of the brain, so not only the hemisphere ipsilateral to an occlusion, but also the contralateral one . To further support the theory of a hemodynamic impairment in patients with stenosis and contralateral occlusion oka et al . Demonstrated increased cerebral blood flow and cerebrovascular reactivity in both hemispheres 36 months after carotid stenosis treatment . In our series in co patients immediate neurological complications were related to carotid embolism in 5 cases (55%) and to hypo- or reperfusion in 4 cases (45%), thus accounting for a frailer brain in those patients . These data demonstrate relevant differences concerning causes of complications in comparison with the control sample where hypo- or reperfusion were responsible for postoperative complications in 2/36 cases (5.5%), strengthening the assumption that co patients present with an increased perfusion instability . Such susceptibility of the brain with respect to any type of ischemic insult in those patients is sustained also by the increased need for shunt implantation during cea in co, in accordance with previous reports [6, 9, 26]. Some conflicting data are reported in the literature concerning the need for shunt implantation in co patients: while some authors advocate shunt implantation in all patients with co, others recommend selective shunting or no shunting at all [29, 30]. To add to the matter, a recent study by goodney et al . Showed that shunt use for co patients during cea is associated with fewer complications, but only if the surgeon used a shunt as part of his or her routine practice in cea . A careful intragroup analysis of co population has highlighted that some additional preoperative risk factors can enhance the neurological risk in cea . In our experience presence of brain infarct on preoperative neuroimaging in co patients decreases the capability of tolerating any kind of ischemic insult, as demonstrated by a 4.4-fold neurological risk increase in this subset of patients (table 3). From our analysis patients with bilateral or contralateral brain lesions unfortunately, no information was available in our database concerning the size of brain ischemic lesions but we can speculate that contralateral damage in our series, namely, ipsilateral to a carotid occlusion, might be quite wide and so it can reinforce the persuasion that those patients' brains are less prone to tolerate any kind of hemodynamic or embolic ischemic insult . On the other hand, an incomplete willis circle might justify the higher incidence of old brain infarcts and liability to new ischemic events . Moreover, advanced age, defined in our experience by the presence of more than 74 years, further increases the neurological risk by 11.5-fold (table 4). The elderly seem at higher risk of brain hemodynamic impairment during carotid clamping per se . When advanced age and preoperative brain infarct are combined, the risk of postoperative neurological complications reached prohibitive values in our series (or 28.6 in co population, table 4). Analysis of specific complications developed in 5 out of 11 co patients presenting both age> 74 years and a preoperative brain infarct has confirmed the higher fragility of the brain in those patients . Except for one case of carotid thrombosis, probably related to a technical defect in cea, in two patients the postoperative neurological deficit was related to a global brain hypoperfusion not clinically evident during cea and in another one to a hypoperfusion due to a very small carotid artery in which a small shunt had been implanted, thus causing a damage in the hemisphere ipsilateral to the occluded carotid artery . In one last case a preoperative brain lesion underwent a hemorrhagic transformation thus underlining the weakness produced in the blood - brain barrier by a previous damage . Even if a higher neurological surgical risk in patients affected by carotid stenosis and contralateral occlusion has been reported in numerous series [8, 14, 15], some single - center experiences have derived different conclusions [27, 33]. This is why nowadays no clear indications for carotid treatment are recognized for this group of patients . Historical data on cea patients have shown co to be a significant risk factor for perioperative development of neurological complications [13, 15]. A post hoc analysis of patients enrolled in nascet and in the ontario carotid endarterectomy registry reported a significantly higher incidence of 30-day adverse neurological events in patients with co. more recently two meta - analyses of patients undergoing revascularization when presenting a contralateral carotid occlusion were independently published in 2013 [16, 35]. Antoniou et al . Performed a systematic review of electronic information sources to identify studies comparing perioperative and early outcomes of cea in patients with occluded and patent contralateral carotid arteries . Thirty articles were included for meta - analysis, comprising 27265 patients having undergone 28846 ceas between 1961 and 2009 . Among them the incidence of stroke was 3.3% in the occluded contralateral carotid group and 1.9% in the patent contralateral carotid group (or 1.65; 95% ci 1.302.09; p <0.001). No significant heterogeneity among the studies was identified and no statistically significant association between the time of publication of studies and the likelihood of developing perioperative neurological complications was found . Analysis of complications related to routine or selective shunting was not performed given the lack of significant heterogeneity of outcome in the selected studies . Patients undergoing cea in the presence of an occluded contralateral carotid artery had increased perioperative and early postoperative risk, but they also pointed out a wide variety among studies of degree of stenosis, indication for treatment, exclusion of recurrent stenosis, carotid disease diagnostic methods, selection criteria for patients enrolment, examination of the status of collateral vertebrobasilar circulation, combination of results in symptomatic and asymptomatic patients, anesthetic methods, surgical techniques, and shunt use . Performed a systematic review of papers published up to march 2012 reporting results on patients with carotid stenosis and contralateral occlusion submitted to either cea or cas . The authors reported co to be considered a significant risk factor for stroke and death in cea (or 1.82; 95% ci 1.572.11; p <0.00001) but not in cas (or 1.22; 95% ci 0.602.49; p <0.58). In cea studies the authors performed a subgroup analysis reporting separate results for symptomatic patients (or 2.43; 95% ci 1.075.50; p = 0.03), asymptomatic patients (or 1.83; 95% ci 1.252.68; p = 0.002), and patients included in studies with higher statistical power (or 1.75; 95% ci 1.442.12; p <0.00001), thus concluding that in all analysis co represents a significant risk factor for adverse neurological events . Furthermore, the analysis performed with respect to shunt use revealed that in studies reporting both selective and routine shunting an increased risk of stroke and death can be encountered in co patients (resp ., or 1.83; 95% ci 1.342.52; p <0.0002; or 2.14; 95% ci 1.283.32; p <0.0007), while in studies reporting no shunt use that risk was increased but not significantly (or 2.61; 95% ci 0.917.46; p <0.07). No cas studies were deemed appropriate for the subgroup meta - analysis . Nevertheless, based on historical data derived from rcts, showing an increased risk of neurological complications in co patients, a cas treatment has been proposed and performed in the majority of those patients for many years . Retrospectively reviewed 417 cas procedures performed between may 2001 and july 2010 and concluded that a preexisting co does not seem to adversely impact cas outcomes . On the contrary, a report by brewster et al . Has pointed out that although cea and cas can both be performed with satisfactory perioperative results, the observed outcomes do not support the presence of contralateral carotid occlusion as a selection criteria for cas over cea in the absence of other indications . More recently, more papers have fuelled the debate on co patients [3840]. Yang et al . Have found that co has no adverse impact on the development of postprocedural stroke after either cea or cas, reporting a stroke rate of 2.3% in 698 cea patients routinely submitted to shunt implantation and 4% in 455 cas patients . Have derived quite different conclusions, showing that in cea patients co significantly affects perioperative stroke rates (3.1% versus 1.1% in patients without co, p <0.0001), while in cas patients co does not affect periprocedural stroke rates (2.1% versus 2.3% in patients without co, p = 0.82). Kang et al . Reported again co to be among predictors of any stroke at 30 days after cea and also of long - term ipsilateral stroke (hr 2.06; p = 0.025). On the other hand, alternative strategies, such as medical therapy alone, appeared to be of low efficacy in patients affected by co in historical studies [15, 41]. In those papers reporting on patients suffering from internal carotid artery stenosis and contralateral occlusion alarmingly high rates of recurrent stroke, ranging from 20% within 3 years to 34% within 51 months of follow - up in medically - treated patients, data from nascet reported a 14.3% risk of stroke in co patients submitted to cea, but a 69.4% 2-year risk of stroke in those patients treated by medical therapy alone, thus leaving very short room to the latter, when considering the whole population of patients affected by co. it must be acknowledged that optimal medical treatment has significantly improved since those cited studies were conducted, making it possible that medical therapy alone in asymptomatic patients with co, as well as the so - called high - risk categories, should be considered the best treatment in the near future . Hence, it is of outmost importance to identify those co patients with additional risk factors for carotid surgery who can better be treated by optimized medical therapy and strict surveillance in absence of recent neurological symptoms . In our experience cea patients with co present with a heavier burden of diffuse atherosclerotic disease when compared to control sample of cea patients . Moreover, they are at higher risk for postoperative neurological complications because of a higher brain susceptibility mainly encountered in a small subset of patients presenting association of two most significant risk factors for developing complications (advanced age, i.e.,> 74 years, and preoperative brain infarction on preoperative ct scans). In those cases the risk of surgery seems to be excessive, and exclusion from surgery with an alternative interventional or conservative approach could be worthwhile . Future studies evaluating the role of medical therapy over carotid intervention in this subgroup of patients are mandatory.
Malaria is a parasitic disease of global importance, with more than 3000 million people in over 100 malaria endemic countries being at risk, and is responsible for the death of approximately a million people annually . Over 90% of yearly deaths resulting from malaria drugs such as chloroquine and sulphadoxine - pyrimethamine which had been useful against the lethal falciparum malaria infection for over fifty years, are no longer effective due to wide spread resistance in all malaria endemic regions . In addressing the problem of drug resistance, the world health organization (who) has advocated the use of artemisinin and its derivatives in combination for the treatment of malaria . Artesunate is one of the most extensively used artemisinin derivatives and is employed both as monotherapy and in combination with many of the older anti malarial medications . The who has advised the use of presumptive diagnosis as basis for treatment of uncomplicated malaria where microscopy based diagnosis is unavailable or cannot be conducted . In many of such instances, artesunate and other artemisinin derivatives are used in combination or as monotherapy . As a result of the geographic overlap in malaria and hiv / aids especially in africa, the co morbid state of malaria and hiv result in serious concerns all around the globe and both diseases are known to affect each other negatively . Malaria appears to reduce cd4, while an increase in the incidence and severity of malaria in the hiv population may also result in worsening of the hiv burden . Hiv / malaria co - infection thus presents the challenges of not only multiple drug therapy, but also possible adverse events that can alter haematological recovery following co infection . The integrity and function of other important organs such as the liver may also be affected . The liver is the second largest organ in the body involved in a host of functions including synthesis of clotting factors, detoxification and metabolism of lipids and carbohydrates . Substantial disruption in its anatomy or function may result in severe alteration in its metabolic roles, and this may adversely affect physiological functions . The co existence of malaria and other conditions that necessitate the concurrent administration of lamivudine and artesunate may result in adverse effects, leading to possible structural and functional alterations of the liver . The objective of this study was to investigate the possible effects of lamivudine - artesunate co administration on the histopathology of the liver of rats in the diseased state of rodent malaria with concurrent immunosuppression . Drugs used for this study were lamivudine (evans), artesunate (tuyil pharmaceuticals) cyclophosphamide (klab), sodium bicarbonate (martindale). The animals were obtained from the animal facility of the department of pharmacology and therapeutics, ahmadu bello university zaria and were used according to the nih animal care guidelines with approval of the departmental animal committee (dac / iw - ot/1 - 07). Animals were placed on food and public water supply ad libitum for the entire duration of the experiment . The animals were allowed to acclimatize with the experimental room for a period of two weeks prior to the commencement of the study . Both cyclophosphamide and lamivudine are very soluble in distilled water and were prepared using distilled water as vehicle . Artesunate was prepared by dissolving 60 mg of the powder in 1 ml of 5% sodium bicarbonate, and this was made up to 6 ml with distilled water . Required concentrations for administration animals were grouped into five groups of six animals (equal distribution of male and females). Animals in the first group served as vehicle control . The animals in the second group served as diseased control . Animals in the third group received lamivudine (20 mg / kg) alone while the animals in the fourth group received a combination of lamivudine (20 mg / kg) and artesunate (10 mg / kg). All animals belonging to groups 2 - 5 were immunosuppresed with an intraperitoneal stat dose of 100 mg / kg cyclophosphamide, followed by a booster dose of 50 mg / kg on day 8 . Malaria parasite was inoculated into the rats by intraperitoneal injection of approximately 110 parasites on day 12 . Animals that were treated with lamivudine received 20 mg / kg daily for 21 days while those that received artesunate were treated with 10 mg / kg of artesunate starting from day 15 of the study . The total length of the study was 21 days . At the end of the 21 days the rats were dissected and their livers were then removed after which they were fixed in 10% formalin solution . Thereafter, heamatoxylin and eosin stained 6 micrones sections were prepared as previously described . Drugs used for this study were lamivudine (evans), artesunate (tuyil pharmaceuticals) cyclophosphamide (klab), sodium bicarbonate (martindale). The animals were obtained from the animal facility of the department of pharmacology and therapeutics, ahmadu bello university zaria and were used according to the nih animal care guidelines with approval of the departmental animal committee (dac / iw - ot/1 - 07). Animals were placed on food and public water supply ad libitum for the entire duration of the experiment . The animals were allowed to acclimatize with the experimental room for a period of two weeks prior to the commencement of the study . Both cyclophosphamide and lamivudine are very soluble in distilled water and were prepared using distilled water as vehicle . Artesunate was prepared by dissolving 60 mg of the powder in 1 ml of 5% sodium bicarbonate, and this was made up to 6 ml with distilled water . Required concentrations for administration animals were grouped into five groups of six animals (equal distribution of male and females). Animals in the first group served as vehicle control . The animals in the second group served as diseased control . Animals in the third group received lamivudine (20 mg / kg) alone while the animals in the fourth group received a combination of lamivudine (20 mg / kg) and artesunate (10 mg / kg). All animals belonging to groups 2 - 5 were immunosuppresed with an intraperitoneal stat dose of 100 mg / kg cyclophosphamide, followed by a booster dose of 50 mg / kg on day 8 . Malaria parasite was inoculated into the rats by intraperitoneal injection of approximately 110 parasites on day 12 . Animals that were treated with lamivudine received 20 mg / kg daily for 21 days while those that received artesunate were treated with 10 mg / kg of artesunate starting from day 15 of the study . The total length of the study was 21 days . At the end of the 21 days the rats were dissected and their livers were then removed after which they were fixed in 10% formalin solution . Thereafter, heamatoxylin and eosin stained 6 micrones sections were prepared as previously described . Drugs used for this study were lamivudine (evans), artesunate (tuyil pharmaceuticals) cyclophosphamide (klab), sodium bicarbonate (martindale). The animals were obtained from the animal facility of the department of pharmacology and therapeutics, ahmadu bello university zaria and were used according to the nih animal care guidelines with approval of the departmental animal committee (dac / iw - ot/1 - 07). Animals were placed on food and public water supply ad libitum for the entire duration of the experiment . The animals were allowed to acclimatize with the experimental room for a period of two weeks prior to the commencement of the study . Both cyclophosphamide and lamivudine are very soluble in distilled water and were prepared using distilled water as vehicle . Artesunate was prepared by dissolving 60 mg of the powder in 1 ml of 5% sodium bicarbonate, and this was made up to 6 ml with distilled water . Required concentrations for administration animals were grouped into five groups of six animals (equal distribution of male and females). Animals in the first group served as vehicle control . The animals in the second group served as diseased control . Animals in the third group received lamivudine (20 mg / kg) alone while the animals in the fourth group received a combination of lamivudine (20 mg / kg) and artesunate (10 mg / kg). All animals belonging to groups 2 - 5 were immunosuppresed with an intraperitoneal stat dose of 100 mg / kg cyclophosphamide, followed by a booster dose of 50 mg / kg on day 8 . Malaria parasite was inoculated into the rats by intraperitoneal injection of approximately 110 parasites on day 12 . Animals that were treated with lamivudine received 20 mg / kg daily for 21 days while those that received artesunate were treated with 10 mg / kg of artesunate starting from day 15 of the study . The total length of the study was 21 days . At the end of the 21 days the rats were dissected and their livers were then removed after which they were fixed in 10% formalin solution . Thereafter, heamatoxylin and eosin stained 6 micrones sections were prepared as previously described . The liver from the healthy controls that were neither immunosuppressed nor infected with p berghei showed no pathology and had distinct hepatic cords and sinusoids . Animals that received immunosuppressive therapy and were simultaneously infected with p berghei showed different degrees of haemosiderosis and pathologic involvement ranging from focal necrosis to some congestion in sinusoidal spaces, while some perivascular cuffing was also observed in the group that received artesunate alone (figures 15). 400) section of liver from a rat immunosuppressed with cyclophosphamide and infected with p. berghei . Section of liver from a rat that received lamivudine and was immunosuppressed with cylcophosphamide, and infected with p. berghei . Section of the liver of a rat that received lamivudine and artesunate treatment, and was immunosuppressed with cylcophosphamide, and infected with p. berghei . The section shows congestion in the liver, intact hepatocytes with few necrotic hepatocytes and well defined hepatic cords and sinosoidal spaces . Section of liver of a rat that received artesunate alone, and was immunosuppressed with cylcophosphamide, and infected with p. berghei . Many xenobiotics, drugs and chemicals are able to result in diverse forms of liver injury, and this may result in distortion in liver histology . The histological picture of the liver in the diseased control animals and animals that received only lamivudine appeared largely similar, showing mild haemosiderosis and some focal areas of necrosis and congestion . Studies reported earlier have also shown artesunate treatment resulting in congestion of hepatic sinusoids in healthy rats . However, in the current study, the liver of animals that received artesunate alone also showed haemosiderosis, perivascular cuffing and some fatty degeneration . Fatty degeneration is often seen with severe weight loss as was the case in this study with most of the groups . Artesunate has shown diuretic effect and may account for the loss in weight with reduction in food intake as a result of the signs of malaria which includes anorexia . The majority of the altered hepatic architecture associated with the diseased animals in all of the groups derive from the pathophysiology of malaria infection, which affects several major organs in the body . The increase level of circulating iron due to haemolysis accounts for the associated haemosiderin . Previous work in healthy animals have shown no toxicity with low oral doses of artesunate in rats, while 15 mg / kg doses have been reported to result in focal necrosis in rats . The nature of hepatic involvement in severe malaria has long been described with necrosis of the liver which is particularly a more pronounced problem in children or non immune individuals . This is the case with the animals in this study which were not previously exposed to the parasite thus making them exhibit atypical response to malaria parasite as against what obtains in semi immune individuals . The histological observations from this study has not shown any significant histological effects that could be directly attributed to the combination of lamivudine and artesunate as most of the observed effects appear to be consequence of the disease condition . However, there is consistent data to support the histopathological changes that are a result of artesunate administration in other studies, which appear not to differ in the disease model used . The outcome of lamivudine and artesunate co administration in the presence of immunosuppression and plasmodiasis does not appear to show clearly distinct differences in hepatic histology in comparison with the immunosuppressed and parasitized controls . However there remains need for caution and urgency of treatment of malaria in the presence of immunosuppression due to the possible consequences of recurrent malaria infection and its possible attendant damage on the liver . The histopathological consequences of artesunate which have been previously reported in healthy animals and confirmed in this disease model, may suggest the need for caution and monitoring of hepatic parameters that may be possible markers of histopathological consequences of the drug treatment.
Protection of women from domestic violence act, 2005, which states that any act, conduct, omission and commission that harms or injures or has a potential to harm or injure will be considered as domestic violence by the law. (2) data from national health family survey suggest that approximately 37% of the married women experience physical or sexual violence in their lifetime. (3) health issues related to ipv include injuries, chronic pain syndrome, substance abuse, depression, suicidal attempts, sexually transmitted infections, adverse pregnancy outcomes, and less frequent contraception adoption . Studies have highlighted the association of ipv with reproductive health problems such as unwanted pregnancies, lesser adoption of contraception, fetal loss, abortions, and higher incidence of infertility . Existing data reveal that these associations co - occur with reproductive control, i.e., male partners' attempts to control a woman's reproductive choices . Women in abused relationships have limited decision - making regarding contraceptive use and family planning . Women's lack of control over her reproductive health is increasingly recognized as a critical mechanism underlying abused women's elevated risk for unintended pregnancy. (45) much of the evidence for ipv and contraception use has been from studies conducted in developed countries; very few studies have examined this relationship in the indian scenario . Stephenson et al . Found lower contraceptive adoption among indian women experiencing physical domestic violence from their husbands. (6) in this study, we tried to evaluate the prevalence of ipv, response to it by the women, and the impact of partner violence on contraceptive adoption . Partner violence and population control are two big social problems the indian society is facing . Existing data suggest that women in abusive relationships exhibit fear and less decisive powers; hence, we hypothesized that contraception adoption would be lesser among women with ipv compared to those without ipv . Thus, the present study was designed with the following objectives: to find out the prevalence of various types of domestic violence.to identify associated risk factors for ipv.to find out the relation between ipv and contraceptive adoption among women who are victim of ipv . To find out the prevalence of various types of domestic violence . To identify associated risk factors for ipv . To find out the relation between ipv and contraceptive adoption among women who are victim of ipv . This questionnaire - based, cross - sectional study was conducted in the department of obstetrics and gynecology at a tertiary care hospital in delhi . Four hundred and one postpartum females were randomly selected over a period of 5 months from march 2014 to july, 2014 . The women were questioned about their age, parity, educational status, occupation, husband's education, and monthly family income . Questions pertaining to ipv and contraceptive adoption are mentioned in the questionnaire as in table 1 . Questions pertaining to ipv and contraceptive adoption (self - made) data were analyzed using statistical package for the social sciences (spss) software 22.0.0 (spss - inc ., chi - square and fisher exact tests were used to assess differences in patient characteristics between women who experienced ipv and those who did not . Four hundred and one women were interviewed . Out of them, 195 (48.63%) women were victim of at least one form of violence and the remaining 206 (51.37%) women denied any kind of violence . Among 195 cases of ipv, 12.8% were in the age group of 15 - 20 years, 52.3% were in the age group of 21 - 25 years, 27.7% were in the age group of 26 - 30 years, and 7.2% were in the age group of 31 - 45 years . Around 28.2% such women were illiterate, but women with higher educational status (graduate and above-7.2%) also victim of ipv . Most of the cases (65%) of ipv happened between 2 - 10 years of marriage . Among such women, statistically significant association of ipv was seen with women's and her partner's education status and the family income [table 2]. Sexual violence was seen in 38.4% of the cases, physical violence in 22.4% of the cases, and verbal abuse was seen in nearly 32.7% of the cases . Physical violence mainly consisted of pushing (9.4%), slapping (19.45%), being punched (9.4%), kicked (4.98%), beat with weapon (2.49%), and inflict burns (0.99%) [table 3]. In response to any of the three violence perpetrated, only 23 cases (11.79%) reacted . In 4.61% cases, approximately, 88.2% of the women never complained and silently tolerated the torture thinking it to be her destiny [table 4]. Demographic characteristics with respect to partner violence type of violence faced by women response to any of the violence approximately, 45.14% of the women could not use condoms just because it was their husbands' decision . Among the remaining women, 15.71% were not aware of the use of condoms and 8.73% felt shy to ask their husbands to use it . In nearly 25.68% of the cases, approximately, 81.5% women who are victim of ipv were aware of contraception as compared to 86.89% who are not victim of ipv, and around 49.2% women with ipv had used contraception as compared to 47.1% cases without ipv . Around 25% of women with history of ipv agreed to use postpartum cu - t / progestin only pill in immediate postpartum period, whereas 35.9% of those without history of ipv accepted them (p value = 0.023). It is also noted that 7.69% of the husbands got violent in cases with ipv as compared to only 0.48% cases without ipv . On the other hand, 17.39% of the husbands got angry in cases with ipv as compared to 3.39% cases without ipv . Only 2.05% husbands accepted wife's request of contraception in ipv cases in contrast to 14% husbands of women without ipv [table 6]. Reasons for not using condoms among the subjects contraception characteristics of women with or without ipv partner violence in any household is a common problem . In spite of many initiatives set up by the government of india to decrease its occurrence, it is a universal phenomenon in some or other form in every household . In this study, at least 48.63% of the cases were victim of at least one form of the violence . Forced sex was seen in 38.4%, physical abuse in 22.4%, and verbal abuse in 32.7% of the cases . Jeyaseelan et al. (7) found the prevalence of physical violence as 26%, whereas shrivastava et al. (8) found it as 63.4% . In the present study, this could be due to the fact that these women were mainly dependent on their husband for basic needs, thereby kept on tolerating the violence . Approximately, 93% of the abused women were housewives . A statistically significant odds ratio of 2.24 [95% confidence interval (ci): (1.14 - 4 - 37)] was noted between the literacy status of less than 10th standard education and being abused by mishra et al. (9) kimuna et al . Also noted it to be one of the key determinants for domestic violence in india. (10) husband's literacy also carried significant impact on ipv in our study . Moreover, families with lower monthly income had higher ipv incidence (85% cases of ipv occurred in families with income <10,000). This study showed that only a small proportion of suffering women reacted to the violence (11.79%). Majority of the women silently tolerated the violence . Reason behind this could be her concern for her children or lack of an alternative economic support . Majority of the women in this study were housewives and economically dependent only on their husbands . Moreover, she felt that her love and care for her husband would change him and as such she never reacts aggressively . Her concern for her parents also prevented her from taking any drastic step against her husband . Ipv is one of the factors associated with women not being able to use or access contraceptives(1112) (silverman et al ., 2007; williams et al ., 2008). In our male - dominated society, there is no reproductive autonomy that means, a woman is never allowed to make independent choices regarding family planning . Decisions regarding getting pregnant or to avoid pregnancy are mostly taken either by husband or mother - in - law . This reproductive control by a man can exist in several forms economic or monetary control (depriving a women by not giving her money to buy contraceptives), emotional (accusing her of not trusting him, telling her that due to work stress he forgot to buy contraception), and physical (getting angry and violent on her contraceptive requests). All these behaviors expose her to the risk of recurrent pregnancy and at times, recurrent induced abortion . Previous authors have postulated that contraceptive use may be more difficult for women experiencing violence, leading to a higher incidence of unintended pregnancy. (13) ipv is associated with a reduced likelihood of modern method adoption in india. (6) however, these associations are not always true as many other studies have found that partner violence has increased likelihood of ever using contraceptives and even subsequent contraceptive use. (141516) the present study revealed that women exposed to ipv were more likely to use contraception (49.2%) as compared to those who did not report any violence (47.1%). The reason behind increased contraception among ipv suffering women could be comparatively increased desire to avoid unwanted pregnancies in these women . Many studies have found that women who suffer from domestic violence are more likely to seek induced abortions . Moreover, the government's initiatives to make contraceptive services freely available to all could be another reason for its increased use, despite the violent behavior of the husband . Though this study showed that higher percentage of women without ipv accepted immediate postpartum contraception methods as compared to those with ipv (35.9% vs. 25%), still women with ipv had higher overall frequency of using contraceptive methods . The present study revealed that women exposed to ipv were more likely to use contraception (49.2%) as compared to those who did not report any violence . However, higher percentage of women without ipv accepted immediate postpartum contraception methods as compared to those with ipv . Health - care providers need to be more sensitive to the issue of ipv while offering and prescribing contraceptive advice as the acceptance and persual may be affected.
Spasticity is defined as a velocity - dependent increase in muscle tone that is usually caused by damage to the central nervous system, which controls voluntary movement . Disease conditions that cause spasticity include cerebral palsy, stroke, multiple sclerosis, traumatic brain injury, and so on . Continuous spasticity causes various signs and symptoms such as muscle stiffness and shortening, muscle fatigue, pain or tightness around the joints, abnormal posture, and joint contractures . Relief of spasticity is a major task of rehabilitation to improve patients activities of daily living (adl). Although botulinum - toxin injections and neurosurgical intrathecal baclofen pump implantation have been recently developed as effective approaches, both have some problems: the former is expensive and limited by the dose and number of injections that can be administered, and also the short shelf life, whereas the latter requires a specialized technique and is invasive . Fetal - type minamata disease (fmd) is caused by methylmercury (mehg) intoxication in fetal period in utero because mehg accumulates in the brain of the fetus by crossing the placenta and the blood fmd shows cerebral palsy - like clinical features (delay of psychomotor development, motor dysfunction, abnormal muscle tone, and involuntary movement) with mental disturbance . Recently, we have reported that vibratory stimulation of the plantar fascia using a hand - held vibration massager attenuated severe planter pain and lower - limb spasticity in 1 patient with fmd . The spherical - shaped head of the frequency generator used in that study fitted the plantar curvature . The vibratory stimulus has a history of influencing not only pain but also muscle tone ., we present 3 cases of fmd who received vibratory stimulation generated by a hand - held vibration massager to plantar fascia to draw lessons from the treatment experience . We demonstrated that long - term vibration therapy of the plantar fascia effectively provides a relief of lower - limb spasticity and improves motor function of patients with fmd . Three patients with fmd first received vibration therapy at 48, 53, or 54 years of age . Case 1 was diagnosed at 7 years of age by high mehg concentration of his umbilical cord . Cases 2 and 3 were diagnosed as probably having fmd by clinical findings and epidemiological data, though mehg concentration of their umbilical cords could not be estimated . All of them showed delay of psychomotor development and cerebral palsy - like neurological signs with bilateral lower - limb spasticity, although their severity and clinical features were different . He had difficulty in flexing his knee in walking and often fell down when walking . Although past interventions for spasticity included oral medications and conventional rehabilitation such as stretching and limb - positioning in case 1, spasticity and adl progressively got worse . Although case 3 showed spasticity, his modified ashworth scale (mas) score was undeterminable because the muscle tension was often induced by psychological reactions to external stimuli . Changes in mas score, rom, and motor function after vibration therapy patients were placed in a supine position to relax their muscle tone . The head of a hand - held vibration massager (thrive md-01; thrive co., ltd, osaka, japan) was fixed with a velcro tape on the planter fascia as shown in figure 1 . The frequency of the vibratory stimulus was 90 hz and the amplitude was 1.0 mm peak to peak . The vibratory stimulus was performed for 15 minutes twice a week for case 1 and once a week for cases 2 and 3 depending on their rate of clinic visits . The repetitive facilitation exercise (kawahira method) of the lower limbs followed after vibration therapy for cases 1 and 2 . For case 3, kawahira method was not suitable for case 3 because of his psychological reactions to external stimuli . We assessed the effects of vibration stimuli on lower - limb spasticity by the changes in the mas score of the ankle flexor muscle, passive range of motion (p - rom) and active range of motion (a - rom) of ankle dorsiflexion, and functional activity . Clinical assessments were performed every 2 to 3 months . For case 3, 10-m straight forward free walking time a patient was placed in the decubitus position and the head of a hand - held vibration massager was fixed to the planter fascia using velcro tape . The effect of vibratory stimulation on spasticity was observed soon after the first 15-minute treatment and resulted in better performance of the repetitive facilitation . Although spasticity rebound was observed, the duration of attenuation of spasticity became progressively longer . Table 1 shows the changes in the mas scores, rom, and motor function 1 or 2 years after vibration therapy . The labored gait improved and gait speed increased in case 3 (figure 2). After the intervention for 8 weeks, his free walking velocity became 12 s/10 m, which neared the velocity 10 s/10 m that most people can walk on a crosswalk before the light to change . He became able to flex his knee in walking, resulting in the improvement of gait unsteadiness and a decrease in falling down when walking . Change in 10-m straight forward free walking time after the intervention of vibration therapy in case 3 . Case 1 continued vibration therapy for more than 2 years and showed further improved mas score and rom . His motion behavior of standing and transferring became better balanced due to the improvement of lower - limb spasticity and ankle movement . None of them showed adverse and unanticipated effects caused by vibratory stimulation of the plantar fascia . The right soleus h - reflex was evoked by a stimulus with a 1-millisecond duration and the intensity was set to elicit the maximum h - reflex using a neuropack s1 electromyography unit (meb-9402; nihon kohden company, tokyo, japan). The amplitude of soleus h - reflex decreased by 50% after the 15-minute vibration therapy (figure 3), indicating that -motor neuron excitability was suppressed . Attenuated amplitude of soleus h - reflex after vibration therapy of the plantar fascia at 90 hz for 15 minutes in case 1 . The averaged wave was demonstrated . Informed consent for this study was obtained from the subjects according to the ethical guidelines of the national institute for minamata disease . In this study, we demonstrated that long - term vibration therapy of the plantar fascia was effective for improving lower - limb spasticity and motor function of 3 patients with chronic fmd . The different improvements in 3 patients after vibration therapy observed in this study may be due to their different severity and clinical features . The device was a commercial hand - held vibration massager that could generate an effective frequency of 90 hz . Since the method is inexpensive, noninvasive, and convenient, we believe that this approach could be widely used for lower - limb spasticity in patients with chronic neurological disorders . The effects of vibration therapy of the plantar fascia were assessed using mas scores of the ankle flexor muscle, and also p - rom and a - rom of ankle dorsiflexion . Mas score improved 1 year postintervention in 2 cases, and p - rom and a - rom were improved 2 years postintervention in 1 case who continued vibration therapy . We applied the repetitive facilitation exercise (kawahira method) subsequently after vibration therapy to enhance voluntary ankle dorsiflexion . The improved a - rom in case 1 suggests that vibration therapy enhanced the effect of the kawahira method by suppressing spinal -motor neuron excitability . Further, vibration therapy improved gait unsteadiness in case 3; labored gait improved and gait speed increased . The previous study demonstrated the importance of vibration frequency of 80 hz to activate the cerebral cortex motor area . As such, vibration stimulation of the plantar fascia with a frequency of 90 hz may also work to coordinate motor function by stimulating the cerebral cortex and cerebellum . Further studies of the effect of vibratory stimulation on the central nervous system will uncover the mechanism . Since the tonic vibration reflex was described, vibratory stimulation has been traditionally applied to the antagonist of the spastic muscle . But the direct application to the spastic muscle using the same device as ours has been recently reported to be effective for short - term attenuation of spasticity in patients with stroke . Here we applied vibratory stimulation to the plantar fascia because our previous fmd study demonstrated that the application of vibratory stimulation to the plantar fascia was more effective than that to the spastic muscle directly . The mechanoreceptor reactive to vibration stimuli with a frequency> 80 hz has been known to be the pacinian corpuscles . The pacinian corpuscles, which are widely distributed in the mammalian body, were found in the dermis and subcutaneous tissues of the fingers, palm, and sole in addition to the muscles and tendons . The effectiveness of vibration therapy may be dependent on the individual difference in the distribution of the pacinian corpuscles . In this study the duration of the vibration therapy of the plantar fascia for 10 minutes in case 1 induced an adverse increase in the amplitude of the h - reflex, an index of spinal motor neuron excitability (data not shown). Motor unit activity is determined by the integration of excitatory and inhibitory neural inputs to the -motor neuron; activation of the cerebral cortex, ib afferent from the golgi tendon organ via the interneurons, and ia afferent from the intrafusal muscle spindle that receives presynaptic inhibition . Ia fibers have been found to be sensitive to vibratory stimulation, and excitatory input to the -motor neurons from ia afferents is enhanced by brief vibration but suppressed by prolonged vibratory stimulation . The decreased activities of the reflection circuit and motor neuron by prolonged vibratory stimulation are thought to be due to increased discharge thresholds of the ia fibers, presynaptic inhibition of the ia terminals, and transmitter depletion at the ia terminals . The previous autopsy study demonstrated a decreased number and morphological changes of the mechanoreceptors in the myotendinous junction of the spastic paralyzed human muscle . Further, experimental study demonstrated that unloading hindlimb caused a decrease in calbindin - d28, a marker of mechanosensory afferent nerve terminals, in intrafusal fibers, and the impairment of muscle spindle . The long duration of the vibratory stimulation for the suppression of h - reflex amplitude may be needed due to a decreased mechanoreceptor number and its impaired function in patients with chronic spasticity . In conclusion, we demonstrated that long - term vibration therapy of the plantar fascia using a hand - held vibration massager at a frequency of 90 hz relieved lower - limb spasticity and improved motor function in 3 patients with cerebral palsy - like syndrome, fmd . It could be widely used for lower - limb spasticity in patients with chronic neurological disorders.
Currently, there are no recommendations for the management of a second local recurrence of prostate adenocarcinoma except for the introduction of palliative androgen deprivation therapy (adt). Improved diagnostic techniques including functional magnetic resonance imaging (mri) and positron emission tomography imaging (pet) with choline derived tracers enables to more accurately detect the site of recurrence in case of subsequent biochemical relapse . The development of new therapeutic approaches, including local treatment, allows to consider other strategies . Androgen deprivation therapy can often be poorly tolerated (psychologic, metabolic, cardio - vascular and bone complications), with an unclear impact on specific survival; it could be delayed for a selected group of patients . Two radiotherapy techniques have already been investigated in the context of first salvage treatment for prostate cancer local recurrence: brachytherapy (low- or high - dose - rate) and stereoatactic radiosurgery (srs). We report the case of a 60-year - old man who underwent high - dose - rate brachytherapy (hdrb) as a salvage treatment for second biochemical relapse due to second local recurrence . In november 1997, he presented an intermediate - risk prostate cancer (histological details not available). He underwent an external beam radiation therapy delivering a total dose of 70 gy in 35 fractions combined with a 6 months adt . In 2006, prostate specific antigen (psa) level increased to 3.87 ng / ml . While computed tomography (ct) scan and bone scan were considered as normal, transperineal ultrasound - guided prostatic biopsies confirmed a gleason score 9 (4 + 5) local recurrence (all the left lobe and the right median apex). A local treatment by high - intensity focused ultrasound (hifu) was performed, which led to a psa decrease to 0.04 ng / ml . In 2011, due to rising psa (3.84 ng / ml), extension work - up based on ct - scan, mri, and bone scan was performed to exclude the possibility of distant metastases . New biopsies were carried out confirming second local recurrence, gleason score 10 (5 + 5). A salvage hdrb was proposed as a treatment and patient was informed about the risks of urinary and rectal side effects . An institutional multidisciplinary expert committee confirmed this therapeutic option, and a signed - consent form was obtained . Brachytherapy was performed under general anaesthesia after urinary catheterization . Using trans - rectal ultrasound (trus) guidance, 10 catheters (sharp needles, elekta ab, stockholm, sweden) were implanted trans - perineally and peri - urethrally through a dedicated template (fig . Dosimetric ct - scans were performed daily, before each fraction, followed by re - optimization in regards to the risk of catheter migration . Clinical target volume (ctv) consisted in the whole prostate (prostatic volume 5 cc, due to a previous hifu treatment) extended to the proximal part of left seminal vesicle . Urethra and rectum were considered as organs at risk (oars) and were delineated . Brachytherapy delivered a total dose of 35 gy in 5 fractions (7 gy / fraction) over 5 consecutive days . The equivalent dose / = 3 at 2 gy (eqd23) was 70 gy for prostate cancer . Dose constraints for oars were: vu115 (percentage of the urethra volume receiving 115% of the prescribed dose) and vr90 (percentage of the rectum volume receiving 90% of the prescribed dose). Both of them were to be less than 1% of the prescribed dose . Patient was hospitalized 6 days, from the day before the implantation to the day after the last fraction . It led to a common macroscopic haematuria managed with bladder irrigation until the bleeding stopped (24 hours). After removal of the urinary catheter and normalization of urinary function, patient was discharged . Brown: rectum due to the multiplications and accessibility of new technologies, we decided to compare hdrb to srs based on the same dosimetric ct scan, since the algorithm for calculating the srs does not take into account the heterogeneity of volumes . We report the case of a 60-year - old man who underwent high - dose - rate brachytherapy (hdrb) as a salvage treatment for second biochemical relapse due to second local recurrence . In november 1997, he presented an intermediate - risk prostate cancer (histological details not available). He underwent an external beam radiation therapy delivering a total dose of 70 gy in 35 fractions combined with a 6 months adt . In 2006, prostate specific antigen (psa) level increased to 3.87 ng / ml . While computed tomography (ct) scan and bone scan were considered as normal, transperineal ultrasound - guided prostatic biopsies confirmed a gleason score 9 (4 + 5) local recurrence (all the left lobe and the right median apex). A local treatment by high - intensity focused ultrasound (hifu) was performed, which led to a psa decrease to 0.04 ng / ml . In 2011, due to rising psa (3.84 ng / ml), extension work - up based on ct - scan, mri, and bone scan was performed to exclude the possibility of distant metastases . New biopsies were carried out confirming second local recurrence, gleason score 10 (5 + 5). A salvage hdrb was proposed as a treatment and patient was informed about the risks of urinary and rectal side effects . An institutional multidisciplinary expert committee confirmed this therapeutic option, and a signed - consent form was obtained . Brachytherapy was performed under general anaesthesia after urinary catheterization . Using trans - rectal ultrasound (trus) guidance, 10 catheters (sharp needles, elekta ab, stockholm, sweden) were implanted trans - perineally and peri - urethrally through a dedicated template (fig . Dosimetric ct - scans were performed daily, before each fraction, followed by re - optimization in regards to the risk of catheter migration . Clinical target volume (ctv) consisted in the whole prostate (prostatic volume 5 cc, due to a previous hifu treatment) extended to the proximal part of left seminal vesicle . Urethra and rectum brachytherapy delivered a total dose of 35 gy in 5 fractions (7 gy / fraction) over 5 consecutive days . The equivalent dose / = 3 at 2 gy (eqd23) was 70 gy for prostate cancer . Dose constraints for oars were: vu115 (percentage of the urethra volume receiving 115% of the prescribed dose) and vr90 (percentage of the rectum volume receiving 90% of the prescribed dose). Both of them were to be less than 1% of the prescribed dose . Patient was hospitalized 6 days, from the day before the implantation to the day after the last fraction . It led to a common macroscopic haematuria managed with bladder irrigation until the bleeding stopped (24 hours). After removal of the urinary catheter and normalization of urinary function, patient was discharged . Brown: rectum due to the multiplications and accessibility of new technologies, we decided to compare hdrb to srs based on the same dosimetric ct scan, since the algorithm for calculating the srs does not take into account the heterogeneity of volumes . Immediate tolerance was acceptable with the macroscopic hematuria managed within the 24 hours after the folley catheter removal . One month after salvage hdbr, we noticed a nocturnal urinary frequency of 6 per night and dysuria grade 2 according to the common terminology criteria for adverse event (ctcae v4.0) with no grade 2 digestive toxicities . At 24 months, late toxicities were nocturnal urinary frequency from 4 to 5 per night, urinary incontinence grade 2 with no grade 2 digestive complication . Regarding sexual abilities, we observed an erectile dysfunction grade 3, which was already present before treatment (the impact of salvage hdrb on sexual function has been difficult to evaluate due to a pre - therapeutic grade 3 erectile dysfunction). Efficacy at 24 months was achieved with a psa nadir at 0.03 ng / ml without any adt (fig . 2). Prostate specific antigen (psa) evolution (ng / ml) dosimetric results related to hdrb are detailed in table 1 . The comparison of the two irradiation techniques, on the same ct - scan, was based on: percentage of the ctv receiving 100% of the dose (ctv v100%); dose delivered to 90% of the ctv (ctv d90); percentage of the urethra volume receiving 115% (v115%) of the prescribed dose; dose delivered to 0.1 cc, 1 cc and 2 cc of the urethra; percentage of the rectal volume receiving 90% (v90%) of the prescribed dose; dose delivered to 0.1 cc, 1 cc and 2 cc the rectum; median dose delivered to right and left femoral heads and volume of the 0.5 gy isodose (eqd2/3 = 1 gy), intimately linked to the integral dose ([total volume receiving 0.5 gy] [ctv + oars]) (table 2 and fig . 3). Prostatic dose - distribution analysis on ct - scan axial slices for each treatment type . Prescribed dose: 35 gy in 5 fractions . B) stereotactic radiosurgery high - dose - rate brachytherapy dosimetry ctv clinical target volume; v100%, v150%, v200%, v115%, v97% volume of the anatomic volume receiving 100%, 150%, 200%, 115%, 97% of the prescribed dose; d100, d90 the minimum dose to 100%, 90% of the ctv dosimetric comparison of high - dose - rate brachytherapy (hdrb) and stereoatactic radiosurgery (srs) ctv clinical target volume, oar organs at risk, d0.1cc, d1cc, d2cc minimum dose to the most exposed 0.1 cm, 1 cm, 2 cm coverage of the target volume (v100% and d90) appears better with srs (v100%: 99.2% vs. 90.3%, and d90: 37.5 gy vs. 35.09 gy). Regarding the dosimetric parameters related to the oars (urethra and rectum), the results appeared quite similar except for the rectum with higher dose with hdrb (d0.1cc rectum: 32.4 vs. 29.6 gy; d1cc rectum: 28.1 vs. 25.3 gy, and d2cc rectum: 25.5 vs. 18.5 gy). Median doses delivered to right and left femoral heads were lower with hdrb (median dose: 0.15 vs. 6 gy, 0.17 vs. 3.7 gy for right and left femoral heads, respectively). In terms of integral dose compared to the results obtained with srs, hdrb reduced divided the 0.5 gy isodose volume by 2 (13 021 vs. 7200 cc). Immediate tolerance was acceptable with the macroscopic hematuria managed within the 24 hours after the folley catheter removal . One month after salvage hdbr, we noticed a nocturnal urinary frequency of 6 per night and dysuria grade 2 according to the common terminology criteria for adverse event (ctcae v4.0) with no grade 2 digestive toxicities . At 24 months, late toxicities were nocturnal urinary frequency from 4 to 5 per night, urinary incontinence grade 2 with no grade 2 digestive complication . Regarding sexual abilities, we observed an erectile dysfunction grade 3, which was already present before treatment (the impact of salvage hdrb on sexual function has been difficult to evaluate due to a pre - therapeutic grade 3 erectile dysfunction). Efficacy at 24 months was achieved with a psa nadir at 0.03 ng / ml without any adt (fig . The comparison of the two irradiation techniques, on the same ct - scan, was based on: percentage of the ctv receiving 100% of the dose (ctv v100%); dose delivered to 90% of the ctv (ctv d90); percentage of the urethra volume receiving 115% (v115%) of the prescribed dose; dose delivered to 0.1 cc, 1 cc and 2 cc of the urethra; percentage of the rectal volume receiving 90% (v90%) of the prescribed dose; dose delivered to 0.1 cc, 1 cc and 2 cc the rectum; median dose delivered to right and left femoral heads and volume of the 0.5 gy isodose (eqd2/3 = 1 gy), intimately linked to the integral dose ([total volume receiving 0.5 gy] [ctv + oars]) (table 2 and fig . 3). Prostatic dose - distribution analysis on ct - scan axial slices for each treatment type . Prescribed dose: 35 gy in 5 fractions . B) stereotactic radiosurgery high - dose - rate brachytherapy dosimetry ctv clinical target volume; v100%, v150%, v200%, v115%, v97% volume of the anatomic volume receiving 100%, 150%, 200%, 115%, 97% of the prescribed dose; d100, d90 the minimum dose to 100%, 90% of the ctv dosimetric comparison of high - dose - rate brachytherapy (hdrb) and stereoatactic radiosurgery (srs) ctv clinical target volume, oar organs at risk, d0.1cc, d1cc, d2cc minimum dose to the most exposed 0.1 cm, 1 cm, 2 cm coverage of the target volume (v100% and d90) appears better with srs (v100%: 99.2% vs. 90.3%, and d90: 37.5 gy vs. 35.09 gy). Regarding the dosimetric parameters related to the oars (urethra and rectum), the results appeared quite similar except for the rectum with higher dose with hdrb (d0.1cc rectum: 32.4 vs. 29.6 gy; d1cc rectum: 28.1 vs. 25.3 gy, and d2cc rectum: 25.5 vs. 18.5 gy). Median doses delivered to right and left femoral heads were lower with hdrb (median dose: 0.15 vs. 6 gy, 0.17 vs. 3.7 gy for right and left femoral heads, respectively). In terms of integral dose compared to the results obtained with srs, hdrb reduced divided the 0.5 gy isodose volume by 2 (13 021 vs. 7200 cc). This case report highlights the possibility to perform a second salvage treatment for local recurrent prostate cancer using hdrb . The psa nadir was reached within 24 months . As expected, the main side effect was a urinary toxicity . Fortunately, for this specific patient, no digestive toxicity was observed, however, rectal toxicity remains an important issue and could be reduced by using transperineal injection of hyaluronic acid in the anterior perirectal fat . The benefit of this second salvage local treatment is to delay the beginning of adt and its side effects (cardiovascular, osteoporosis, sexual, and psychological) [57]. After two salvage treatments, we noticed a rising gleason score that could be due to either more aggressive selected cancer cells or an overestimation in correlation with the difficulty of histopathological interpretation . The comparison between the two techniques does not allow to conclude on the best method . The percentage of ctv receiving 100% of the prescribed dose is greater than 90% in the two techniques . Protection of oars is respected with both techniques . However, median dose to the femoral heads is higher with srs, which has to be considered with the dose already delivered during the initial external beam radiation therapy . The most important difference highlighted related to the volume receiving 0.5 gy (eqd23 = 1 gy), which was reduced with hdrb and seems to be correlated to the risk of radiation - induced cancers . Regarding these results and imaging improvement (mri and pet with choline derived tracers), both hdrb and srs appear to be an attractive treatment option of localized prostate cancer recurrence . The type of treatment should be made based on the availability and experience of the techniques . The accuracy of hdrb and srs, and the ability to precisely define the site of prostate cancer could allow considering focal therapy for intra - prostatic small local recurrence in order to significantly reduce toxicity [11, 12].
At present, low back pain (lbp) is a great social and economic problem because the ongoing prevalence of this condition is between 6085%, and its incidence has been increasing in developed countries since the second half of the last century1 . The highest incidence of lbp is observed in patients between 3035 years of age2 . From many randomized studies, it is clear that not only prevention, but in particular follow - up care of lbp patients must include regular physical activity together with appropriately indicated rehabilitation, which does not strain the musculoskeletal system3 . If a patient is susceptible to dysfunction of the neurological system, additional ways of treatment should include other appropriate therapy methods, including surgery . Early and correct diagnosis is essential to establish the severity of patients conditions4, 5 . Currently, there is no precise definition of chronic low back pain . In some cases, generally, it can be classified as frequently recurring back pain, which intermittently affects individuals over an extended period of time6 . In many lbp patients, it is often difficult to properly diagnose and identify the cause, despite the significant advances in currently available diagnostic methods . In some cases, it is difficult to clearly connect the results of imaging methods, the subjective symptoms described by the patient, and changes in the pathology of the musculoskeletal system7 . Another complication for making a correct diagnosis is the fact that lbp can have a variety of different etiologies . The most significant ethiopathogenetic factors of vertebrogenic dysfunctions cited by richardson et al.7 include disorders of the deep stabilization systems of the spine (dss). The deep stabilization system of the spine is responsible for stabilization of the spine as well as the entire body during movement and under static pressure7 . When it is compromised or weakened, the entire body is destabilized, while some muscle structures can be overload and others can be weakened including the deep stabilization system . Even at present, when a great variety of different diagnostic tools are available, we cannot accurately elucidate the connection between objective findings during physical examination, subjective complaints of the patient and discrepancies between them . According to ricci et al ., approximately 39% of patients suffer from herniated disc and do not describe any subjective complaints, and during radiculography protrusion of the intervertebral disc was found in 50% of cases and herniation of the disc in 24% of cases, data in a study on workers in the usa8 . To our knowledge, similar research evaluating the effects of rehabilitation using plantography and posturography has not been performed in the past . We hypothesized that there would be certain differences in all measured values of patients before and after treatment with the infinity method . The purpose of this study was to verify the presence of differences between the measured values of the center of force (cof) and subjective pain described by a visual analogue scale (vas) in patients with lbp before and after rehabilitation therapy, and used the results to evaluate the efficacy of our special rehabilitation method for the treatment of patients with lbp . All participants read and signed an informed consent form, and the study was approved by the ethics committee of the rehabilitation clinic brandys nad orlici . Patients with the diagnosis of lbp who were treated at the rehabilitation clinic brandys nad orlici from february to november 2013 were evaluated . The goal was to confirm the efficacy of our rehabilitation therapy, the infinity method . During the study period, a total of 198 patients were treated and examined using a matscan device (tekscan inc ., south boston, massachusetts, usa). All these patients suffered from lbp and the most frequent causes of the pain were: osteochondrosis, spondylarthrosis, and spondylosis . The ages of the females and males in this group were 64.42 11.52 and 58.33 12.11 years, respectively . All the patients received at least five individual 30-minute therapy sessions per week using the infinity method, and six group therapy sessions per week in the gymnasium and swimming pool, with each session lasting 30 minutes and including the infinity method . The infinity method is a special rehabilitation method developed at the rehabilitation clinic brandys nad orlici . Its name comes from the english word infinity because it utilizes movement in the shape of the infinity sign in part of the exercises . The method focuses on stabilization and strengthening of trunk muscles, dorsal and abdominal muscles, including the deep stabilization system closely linked with diaphragmatic breathing . It contributes to increasing body mobility and flexibility based on relaxation, extension, and mobilization of the soft tissues of the motor system . It activates subconscious and conscious setting of the postural system of the body, efficiently involves the stabilization system of the spine, and does not overload musculo - fibrous tissues . The first is called macro - movement (in a range of centimeters) and it resembles tai - chi . The second is called micro - movement (in a range of millimeters) and it is especially designed for patients with significant pain and patients with limited movement range (either because of inability or restriction due to medical indication). Micro - movement is a fine movement that minimally loads the motor system, improves muscle activity and trains the higher motor centers in the central nervous system . The third type of movement is movement with visualization during which the patient only imagines the movement . One of the advantages of the infinity method is that the treatment can be applied even in the acute phase when a patient may be suffering from intense pain, and that it offers extended variability of auto - therapeutic exercises . The therapy includes special exercises and training of breathing, which allow both muscle relaxation and activation of several muscle groups, including the deep stabilization system, as well as improving psychological factors . We evaluated the efficacy of the rehabilitation therapy using the matscan pressure mat system . At the beginning and at the end of the intervention the measurements were carried out with the patients standing upright with their eyes open . We compared the values measured before and after the intervention with the infinity method . Measurements were taken for 30 seconds with a scan frequency of 30 hz . Balance of the body is quantified by monitoring fluctuations of the coordinate center of supporting forces . For example, brumagne et al.9 use the notation center of foot pressure (cop). In our case, we use the notation center of force (cof). The patients plantar function was evaluated using a graphic method (computer plantography) which graphically quantifies postural control during quiet upright standing (posturography). The variables measured were: bilateral pressure on the right and left soles of the feet, gravitational forces between both soles, anteroposterior (a - p) and mediolateral (m - l) displacement of cof, the center of gravity of the body between both soles of the feet . Using the sway analysis module (sam), we measured the area within which the values of cof, a - p and m - l excursion of cof moved in a defined time interval (30 seconds). The shift in cof and difference in the cof area between pre- and post - intervention for measurement of subjective pain of patients, a visual analogue scale (vas) was used . Vas has been used by many authors and is considered a reliable assessment of pain . A scale of 10 cm in length was divided into ten equally long sections and numbered from 0 on the left side for no pain to 10 on the right side for very severe pain . Data were analyzed using the paired t - test for the parametric tests and wilcoxon signed - rank test was used to analyze the vas data . Descriptive statistics for the outcome measures are presented as mean standard deviation (sd). The statistical power of the tests was 0.8 for the differences in the variables noted in this study . Statistical significance was based on an alpha level set at 0.05, furthermore the median difference and the 95% confidence intervals were calculated . The statistical analyses were performed using the statistical package spss for windows, version 22.0 (ibm, armonk, ny, usa). The demographic characteristics of the individuals are shown in table 1table 1.characteristics of patients with low back paingenderfemales24 (72.7%) males9 (27.3%) mean age (sd)females64.42 11.52males58.33 12.11number of improved items5 (all)20 (60.6%) 45 (15.2%) 36 (18.2%) 22 (6%) vas (visual analog scale)better30 (90.9%) same3 (9.1%) sd: standard deviation . All the measured values of the test group of the lbp patients showed statistically significant differences (p = 0.001) after treatment . Five of these parameters were related to plantographic and posturographic measurements, which showed decreases in measured values (table 2table 2.plantographic and posturographic parameters results of the paired samples testdependent variablespaired differencesmeanstandard deviationstandard error mean95% confidence interval of the differencelowerupperap before - after0.942550.951160.165580.605291.27982ml before - after1.176181.233130.214660.738941.61343area before - after2.304682.999120.522081.241233.36812distance before - after7.6797011.046211.922903.7628811.59651variation before - after0.00916450.01214160.00211360.00485930.0134698ap: anteroposterior directions; ml: mediolateral directions . There were significant differences between pre- and post - rehabilitation treatment in the antero - posterior direction of movement of the cof (p <0.001), medial - lateral movement of the cof (p <0.001), the area covered (p <0.001), the cof distance (p <0.001), and the variation of cof (p <0.001). The sixth assessed dependent variable was pain which also showed a statistically significant reduction (p <0.001). statistically significant (p <0.001) the results of this study show that the rehabilitation treatment of patients using the infinity method resulted in statistically significant improvements in the observed plantographic and posturographic parameters of stance stability and reduced subjectively reported pain as measured by vas, which can also be considered to be a significant improvement in the patients overall health . The patient group was further divided into two groups: patients with radiculopathy (n=8), and patients without radiculopathy (n=25). Using the t - test, we compared the results of both groups . In both groups we found statistically significant changes in vas pain . Although most of the patients with radiculopathy showed improved values of measured parameters after treatment, we did not find statistically significant changes for most of the parameters of posturography measurement . In patients with radiculopathy, we succeeded in achieving stabilization of the lower back region, which was shown by the statistically significant decrease in vas pain . However, several patients retained trigger points in the area of the hamstrings, gluteus medius and minimus, and musculus quadriceps femoris, which could have resulted in imbalance in the measured posturographic parameters . We also divided the subjects according to age into two groups: patients younger than sixty years (n=13) and patients over sixty years (n=20). In both groups however, we did not find statistically significant changes in most of the parameters measured in posturography in the younger group, in contrast to the older group of patients . This may be because the younger patients were treated for a shorter time (due to work reasons) than the older patients . Thus, the younger patients did not achieve the maximum possible improvement in their health state . Various randomized studies have demonstrated the efficacy of maintaining the physical activity of patients with low back pain . Rehabilitation therapy is recommended as back pain onset prevention as well as treatment of patients who already suffer from pain . For acute pain, it is also recommended to initiate treatment with non - opioid analgesics or nonsteroidal anti - inflammatory drugs1 . For restoration of locomotion, it is advisable to choose an individual rehabilitation program which the patient can continue at home after proper training with a physiotherapist . The rehabilitation program should focus on strengthening the deep stabilizer muscles of the spine and stretching and relaxing muscles and connective tissues7 . However, if the patient suffers from acute pain, severe chronic pain, or already has some other physical limitations, the usual rehabilitation treatment cannot always be started immediately . The results of clinical studies confirm the statistically significant efficacy of our rehabilitation therapy infinity method for patients with lbp . The present study demonstrated that the therapy not only effectively reduces pain, but also, the measured cof parameters show, that this therapy can effectively center and stabilize the entire posture . Changes in vas pain were statistically significant in both cases and the improvements ranged from 20 to 30% . With the infinity method, we achieved statistically significant improvements of 46.6% on average, and compared to conventional methods, the infinity method achieved 16.6 to 26.6% better improvement in the treatment of patients with lbp . Yoo et al.12 compared the effect of core stabilization exercises, which are similar to the body stabilization used by the infinity method using vas for comparison . They found there were statistically significant differences between values of vas before and after exercise in similar patient sets . Their results are in agreement with our results of a decrease in the vas value of patients after rehabilitation . Han et al.13 reported a statistically significant decrease of vas of 52.1% in patients with lbp after aquatic therapy . In the present study lee et al.14 utilized posturography for the comparison of patients with lbp with a control group of healthy individuals . In their study, they mainly found significant differences in a - p excursion of cof between patients and the control group . Hsieh et al.15 verified the effectiveness of four different standard methods used for the treatment of patients with lbp using vas as the evaluation measure . After three weeks of rehabilitation treatment, all four groups showed significant improvements of 1.08 to 2.01 cm on average on the vas . In the present study, we achieved statistically significant improvements of 2.5 cm on the vas on average . We think that the difference between the results of previously conducted studies and our study lies mainly in the movement therapy, especially the micro - movements, both passive and/or active movements in the range of millimeters . Therapy and exercises place load on the musculoskeletal apparatus without overloading it and the activation occurs simultaneously often with relaxation of these structures . Especially during the 1980s and 1990s, many studies on the effectiveness of rehabilitation for patients with lbp were conducted as this condition was (and still is) associated with great social and economic problems . Popa et al.16, 17 reviewed 69 case reports and their various treatments as well as the quality of their implementation and evaluation . These studies provide an overview of basic methods that are used in the treatment of patients with lbp and their potential positive effects . For comparison with the results of our study all these studies investigated the effectiveness of certain rehabilitation methods that were more or less proven to be successful . This study demonstrated the superior efficacy of our special rehabilitation method compared to the results of previously conducted studies assessing the effects of commonly used methods of rehabilitation treatment for patients with lbp . This study was limited by the fact that the results were partially influenced by co - therapies such as electrotherapy, hydrotherapy, or massages, which the patients received in addition to the rehabilitation therapy . Certain improvements may occur even when using conventional methods of treatment, as evidenced by the results of other studies carried out on groups of patients with lbp . However, our results showed greater improvements in the monitored parameters including pain . Therefore, we think that the use of the infinity method achieves better results in the treatment of patients with lbp . Given the large number of options in active and passive exercises and therapies, patients confirmed to us that the method was fun and body reshaping . They reported that they would continue to exercise after the end of treatment thanks to the large number of exercises which they can perform at home . In conclusion, after the rehabilitation therapy, the evaluated patients demonstrated significant improvements in objectively monitored posturography and plantography measurement parameters . In addition, the patients reported subjective pain reduction as measured by the visual analogue scale . Our goal was to achieve optimal balance of structure and function of the patients musculoskeletal systems . This study proved that application of the infinity method improves stabilization, centralization, postural correction of the body, and distribution of weight on the foot soles, and it fully improves the position of the center of force . In the future, we anticipate further objective evaluation of the effectiveness of therapeutic methods using advanced sensor device(s) for measurement.
The registration system and the active vaccination since 1962 have continuously declined the incidence of tuberculosis (tb) in korea . Despite of these efforts, the incidence of tb in korea is high among the organization for economic cooperation and development countries . In 2009, in korea, the reported tb cases per 100,000 were 74.3, and the actual incidence is presumed as 90 cases per 100,000 . According to data from the registration center, the proportion of extra pulmonary tb shows increased tendency since 2003 . Of extra pulmonary cases, the reported incidence of primary tb in performed appendectomies varies from 0.1 to 3.0%, and it is 1.5 to 30% of all patients who were diagnosed with tb . Herein, we report the case of a child with primary tuberculous appendicitis accompanied with mesenteric mass . A 14 year - old boy complained of intermittent right lower quadrant abdominal pain for one day . He had no medical history and received all vaccinations, including bcg (bacille de calmette - guerin) vaccine, as scheduled . One month prior to admission, he started a baseball camp training in his school . He did not experience febrile sensation, weight loss or cough . On physical examination, laboratory findings showed that the white blood cell count and the c - reactive protein were within normal range . Abdominal computed tomography revealed diffuse enlargement of the entire appendix (about 12 mm), with cystic change at the tip of the appendix (fig . 1). Also, the abnormal calcified mass was detected on abdominal computed tomography . The 3.3 27 cm mass was located at the right side of the right iliac artery, inferior to the second portion of the duodenum and anterior to the right psoas muscle (fig . It was accompanied by internal necrosis and was assumed to be the lymph - node from the mesentery . We decided to perform laparoscopic appendectomy and excision of the mesenteric mass . Under general anesthesia, we used three trocars, as follows: a 12-mm trocar at the infra umbilical portion, a 5-mm trocar at the left lower abdominal quadrant, and a 5-mm trocar at the suprapubic portion . We found no ascites . The appendix was diffusely enlarged and distended, and it showed suppurative change (fig . The intra - abdominal mass was protruded from the mesentery, was adjacent to the second portion of the duodenum and showed as a yellowish, calcified mass . It was dissected from the mesentery by hook and harmonic scalpel . After specimen retrieval, the necrotic material from the mesenteric lymph - node showed cheese - like changes . We obtained the acid - fast bacteria (afb) stain and culture from the specimen . The afb stain and culture revealed growth of the mycobacterium tuberculosis complex . From the histologic diagnosis, both the appendix and the mesenteric lymph - node showed chronic granulomatous inflammation, with caseous necrosis (fig . Tb can affect all organ systems, and the most commonly affected region is the lung . Of extra - pulmonary locations of tb, bones and the urinary system are the most frequently affected (30% and 24%, respectively), followed by lymph nodes (13%). Abdominal tb may involve the gastrointestinal tract, peritoneum, lymph nodes, or solid organ; however, peritoneum and abdominal lymph nodes are the most common sites . In the pediatric literature, abdominal tb has been described infrequently, in 2 of 1,000 and 5 of 1,700 patients in industrialized countries ., 0.6% by singh et al ., 2.3% by gupta in india and 0.13% by sohn et al . In korea . In japan, until 2010, primary tb of the appendix was observed in only 11 out of 29 cases of all tb of appendix . The cause of this rarity may be explained by the fact that the intestinal contents do not make direct contact with the luminal mucosa of appendix . As early as 1914, william j mayo said that the appendix is an interesting organ because, although it has abundant lymphoid tissue as adenoid or peyer's patch, was little involved in tb . It has been suggested that the appendix was primarily infected either hematogenously, from a distant focus which was not clinically detectable, or by direct contact with contaminated sputum or food . Also, the appendix was secondarily infected from: 1) direct invasion of adjacent organs, such as the ileocecal region or the urinary system, 2) retrograde lymphatic spread from distant lesions in either the ileum or the ascending colon, and 3) via appendicular serositis or periappendicitis, in peritoneal tb . This makes pre - operative diagnosis difficult and delayed, resulting in a high percentage of complicated appendicitis . Borow and friedman suggested three clinical features: 1) the chronic form - mild to moderate right quadrant abdominal pain, with nausea and vomiting, 2) the acute form - sudden onset of right quadrant abdominal pain, 3) the latent form . It showed typical granuloma, with caseous necrosis surrounding the epithelioid cells and the langhans giant cells . In this case, the appendix and the mesenteric mass also showed the tb infection . We considered that the mesenteric lymphnode involvement did not require further evaluation of intestinal tb, and we administered anti - tb medication . The regimen for anti - tb medication was the standard four drug chemotherapy, including isoniazid, rifampin, pyrazinamide and ethambutol . The anti - tb medication after appendectomy is known to decrease the incidence of the usual complications, such as stump blowout or fecal fistula . In conclusion the involvement of the mesenteric lymph node presenting with abdominal mass was possible with primary tuberculous appendicitis.
Population growth has increased the demand for food; rising prosperity has increased the demand for quality food . At the same time, consumers demand convenience foods, since they are becoming increasingly health conscious; therefore, there is a need to diversify food products . Wheat is being used as a staple food for most part of the world, because of its special dough characteristic like cohesiveness and thus being used in the preparation of bread and other wide ranges of products like noodles, soups, pasta, and other foods like biscuits, cookies, cakes, and breakfast cereal . Pastas include noodles in various lengths, widths and shapes and varieties that are filled with other ingredients like ravioli and tortellini . Pasta is an excellent source of complex carbohydrates, which provide a slow release of energy . Unlike simple sugars that offer a quick, yet fleeting boost of energy enriched varieties provide a good source of several essential nutrients, including iron and several b - vitamins . In recent years increase in popularity of pasta products and their increased consumption make it very important for increase in availability of raw materials . Durum wheat, being the hardest of wheat's is used at large scale for pasta production . Being commercially expensive to produce due to the limited availability makes it a case of study . The endosperm made up of durum is completely different from other wheat species because the mineral was distributed throughout endosperm and has more carotenoids contents, low protein efficiency ratio, and excellent rheological characteristics which are desirable for making pasta products . However, over the years, numerous studies on alternate methods for production of pasta from different raw materials have been conducted, where alternates for raw materials were used for production focusing on reduced cost and to match similar parameters and to improve nutritional value . Limited availability of durum wheat is noticed due to the increase in consumption of pasta products, making it commercially expensive for procurement of raw material . On the other hand however, the availability of durum wheat for production of pasta products is very limited . Alternates are thus researched upon, where it is found that the common wheat widely available throughout the world is similar in comparison with durum wheat . The percentages of starch, protein, minerals, lipids, and amino acids are roughly equivalent . Common bread wheat occupying 90% of the world total production makes it a cheap and readily available alternate for pasta production, thus saving money and creating ample opportunities to improve nutritional quality . The objective of the present study is to find suitability of t. aestivum milled products (refined wheat flour, semolina, and whole wheat flour) in the place of durum semolina for preparation of pasta, thereby reducing the cost of production, maintaining or improving the quality of the product, and then study in detail the rheological properties of the pasta dough, chemical composition, nutritional profile, and quality of developed pasta products . Freshly prepared refined wheat flour, whole wheat flour, and semolina were obtained from narasu's roller flour mills, salem, tamil nadu, india . All reagents and chemicals used are of analytical grade (ar) unless otherwise specified . Raw materials were analyzed for particle size (aacc 55 - 30.01), moisture content (aacc-44 - 15a), ash content (aacc-80 - 01), gluten content (aacc-38 - 10), and micro - kjeldahl method was used to determine nitrogen contents of pasta samples (aacc, 2000), sedimentation value (aacc-56 - 70), farinograph (aacc-54 - 21), mixograph (aacc 54 - 40.02), and alveograph characteristics (aacc-54 - 30a) using the mentioned standard methodologies . Raw materials and water were premixed in a spar mixer at speed 1 (60 rpm) for 10 min to facilitate uniform distribution of water . The premixed dough (500 g) was transferred to a laboratory pasta machine (la monferrina, model dolly, asti, italy). The dough was then extruded through the brass die for pasta type shells in the required size and was dried in sakar drier (shirsat, mumbai) at 75c for 4 h. the pasta samples were then allowed to cool at room temperature and then packed in polyethylene covers for storage . Cooking loss was determined according to the bureau of indian standards (bis 1976). Twenty - five grams of pasta sample was weighed and put in the 250 ml of boiling water . Start timer count is and stirs well to make sure that the pieces are separated . Check the piece of pasta after every 30 sec intervals for its hydration and cooking by squeezing the sample . Firmness of cooked pasta was measured according to method adopted by krishnan and prabhasankar using a universal texture measuring system (lloyds instruments, lr-5 k, hampshire, uk). A panel consisting of 25 panelists (n = 25), who were regular eaters of pasta, was employed for the sensory evaluation of pasta samples . Briefly, panelists evaluated the randomly coded pasta samples for their colour, appearance, aroma, texture, taste, and overall acceptability . Assessors were instructed to cleanse their palate with cold, filtered tap water before tasting each sample . The overall sensory attributes were measured using hedonic scale of 19 where 9 = like extremely, 8 = like very much, 7 = like moderately, 6 = like slightly, 5 = neither like nor dislike, 4 = dislike slightly, 3 = dislike moderately, 2 = dislike very much, and 1 = dislike extremely . All the parameters were carried out in quadruplicates and the means values were reported . The values of surface colour (l, a and b) of raw pasta in terms of lightness (l) and colour (+ a: red a: green; + b: yellow; b: blue) and e were measured using hunter lab colour measuring system (colour measuring labscan xe system, usa). The cooked pasta samples were freeze - dried using heto freeze dryer (dw3, allerod, denmark). Surface and cross section of freeze - dried samples were mounted on the specimen holder and sputter - coated with gold (2 min, 2 mbar). Finally, each sample was transferred to the microscope where it was observed at 15 kv and a vacuum of 9.75 10 torr . A scanning electron microscope (leo 435 vp, leo electronic systems, cambridge, uk) was used to scan the images . The method was followed from aoac method 32.1.17 . In vitro digestibility of starch freeze dried and ground sample (50 mg) was dispersed in 4 ml of sodium acetate buffer (ph 4.6, 0.4 m) containing amyloglucosidase and was incubated in water bath for 30 min at 60c . Then, the enzyme was inactivated by placing the tubes in boiling water bath (100c) for 15 min . The tubes were cooled to room temperature and then centrifuged at 5000 rpm for 10 min . Supernatant was measured for its glucose content using a glucose oxidase - peroxidase (god - pod) kit (autospan, span diagnostics limited, india). Absorption was measured at 505 nm, and the glucose concentration was converted into starch content using a 0.9 factor . The particle size distributions of the flour samples were determined with a series of standard sieves, and the results were expressed as a percentage of the sample weight (table 2). Ideally, the majority of semolina particles should fall within a narrow range of particle size range so that pasta dough water uptake will be homogenous . It was observed that the refined wheat flour (refined wheat flour) is much finer than the control (semolina). The nonuniformity in the particle size can be attributed to the grinding of the semolina particles, which was carried out by an external minimill grinder . However, the results obtained were good within the requirements for good pasta making quality . Whole wheat was ground to a granulation similar to that of the semolina (table 2). Incomplete hydration of semolina or ground whole wheat would result in white specks in the spaghetti . Thus, white specks would affect the appearance, mechanical strength, and cooking quality of the spaghetti . Proximate analysis of all the raw materials (refined wheat flour, whole wheat flour, semolina) used were shown in the table 3 . It is noticed that comb1 (t. aestivum wheat flour & semolina with additives) with the presence of additives has moisture of 10.87% is very much higher compared to the control (9.8%). The other two blends (comb2 & comb3) had acceptable levels of moisture compared to the control as they have the presence of semolina . The proximate composition of all the samples was found to be within limits of pfa and isi standards . Similar results of semolina were also reported by where moisture of indian durum varieties varied from 9.0 to 11.5%, ash content varied from 0.79 to 0.86%, and the protein content varied between 12.1 and 15.9% . Ash, an index of the mineral content of the flour, is of much relevance, and in that it gives the indication of the grade or the extraction of the flour . This is because of the low level of mineral content present in the endosperm when compared to the outer bran content . The bran layer is rich in ash and protein, so removing bran during milling would lower the protein and ash content . It is noticed that all the comb samples have lower ash content when compared to the control, where comb3 (semolina (t. aestivum) and semolina (t. durum)) (table 3) has 0.596% . Comb2 & comb1 samples also have lower ash values, thus improving the quality of the flour . A significant increase in protein content is noticed with the comb samples (table 2), which could be due to the addition of additives . This is similar to the results reported by prabhasankar et al . For increase in nutritional attributes of pasta samples with the addition of additives . The maximum increase is noticed in comb2 (refined wheat flour and semolina (t. durum)) which is 15.40% (table 3) when compared to the control which has 12.8% (table 3) of protein . Increase is also noted in comb1 at a level of 13.70%, thus imparting better nutritional value . The mixograph is a primary physical dough testing procedure in the us durum wheat - breeding program . Figure 1 details the results of the monograms obtained from the pasta samples . Comparing the two raw materials used, dough strength is greatly deferred between the samples where figures 1(b) and 1(c) were comparatively stronger than the others . This is because of the presence of refined wheat flour, which is generally high in strength compared to semolina . Regardless of samples, refined wheat flour had higher peak heights as shown in figures 1(b) and 1(c), but semolina had higher peak width as shown in figures 1(a), 1(d), and 1(e). Higher peak proves that the flour is very resistant to extensibility and also has greater mixing ability making it more stable as seen in control (figure 1(b)). But semolina generally is seen to have a lower peak height (figure 1(a)), thereby reducing the extensibility of the flour making it better for pasta making . Mixogram dough development time (the time required for the mixogram curve to reach maximum height) was greater for comb1 had the highest (4.26) which was made up of refined wheat flour . Based on the mixograms obtained, refined wheat flour had higher dough stability when compared to semolina as evidenced by the rapid decline of the mixogram curves after 3.5 min . Dough stability indicates the time during which the dough resists mechanical action without undergoing a change in consistency . Lack of dough stability or tolerance to over mixing could be related to the dilution of semolina with bran or germ as fewer storage proteins are available to form a gluten matrix . Other researchers have reported that dough stability decreases with increase in bran concentration . In spite of all these issues, all the parameters were in accordance was the norms and were able to produce good pasta quality . It is seen from (figure 1(d)), that the addition of refined wheat flour along with semolina has given the flour additional properties showing a higher peak height when compared to control (figure 1(a)), thus aiding in better pasta making ability . The results obtained along with the amount of water absorption required to centre the farinogram curve on the 500 bu (brabender units) line varied as shown in table 4 . It is well noticed from the results that the samples with a higher concentration of refined wheat flour exhibit stronger dough characteristics (increased water absorption, dough development time and dough stability), that is, refined wheat flour & comb1 in contrast to weak dough development . The whole wheat flour showed higher water absorption 63.9% due to high damages starch, whereas the dough development time and dough stability were lower than those in the whole - wheat flour . The addition of gluten as indicated in samples combs 1, 2, and 3, increased water absorption, dough development time and dough stability was seen . It was also noticed that stability and dough development time are related with each other, and increase in development time caused increased stability, thus leading to stronger dough . Samples with maximum wheat bran concentration caused increase in water absorption with whole wheat flour (whole wheat flour) having maximum absorption (65.9%) (table 4) which was also noticed by and, lowest for refined wheat flour (comb1). Reported that the differences in water absorption are mainly caused by the greater number of hydroxyl group which exist in the fibre structure and allow more water interaction through hydrogen bonding . Alveograph is used to measure the viscoelastic properties (strength and extensibility) of the gluten protein that correlate's well with the firmness and springiness of cooked pasta . Gluten strength and tenacity / extensibility ratio pl is a good predictor of cooking quality, thus making it relevant for pasta - making ability . Figure 2 results obtained from the alveograph of the various samples of flour . Regarding the bread making and pasta characteristics of the flour under examination, the most dramatic effect of additives addition on semolina was observed when the biaxial properties of the wheat dough were assessed in the alveograph . The addition induced a significant modification of the alveograph parameters, where a steady increase of the tenacity (p) besides to a significant decrease in dough extensibility (l). Overall effect on tenacity and extensibility led to a significant increase of the curve configuration ratio (pl). Similar results were obtained by bonet et al . Where he noticed the same, with increase in go (glucose oxidase) concentration . The same was also noticed in regard to go by . But the only change was seen in the deformation energy (w) where a steady decrease was noticed in this case . This could be attributed to the addition of a percent of refined wheat flour as the raw material . As the percent of refined wheat flour reduced, the pl ratio increased which was very similar to that of control . Thus leading to better pasta quality . Whereas bonet et al . Noticed a steady increase as there was no presence of refined wheat flour and also the addition of go cause additional protein cross - links resulting in larger pl values . Colour of pasta is a key quality because of the vital impact on the point of sale . In pasta products made with semolina, the higher the value, the more desirable the product . Among l, a, and b parameters, the first two are considered more important as colour attributes . Hunter colour parameters (l, a, b) of raw samples of durum pasta (control) the other raw materials used along with the sa and the comb pasta are shown in the table 5 . The higher values are noticed in table 5, which shows increased levels of pasta quality and the presence of no adulteration in the pasta samples proving high levels of economical advantage as well as appearance . The lightness is seen lower in the all the samples, this may be attributed due to the alteration with different wheat milled products which has a lower lightness index compared to the control, thereby lower carotenoid pigments which contribute to the colour of durum pasta . Yellowness in all samples was seen lower than the control, and this is due to the same factors affecting the lightness index . It is noted that the comb3 blend shows the highest lightness, but the comb2 blend gives the highest yellowness when compared to the control . It is also noted that the influence of the additives did not cause any major difference in both indexes . But it is seen that the influence of additives has reduced the lightness but has increased the yellowness . The cooking characteristics of all the raw materials and the trails along with the comb blends compared to the control are presented in figure 3 and the photographs of raw, and cooked samples compared to the control are shown in figure 4 . High - quality pasta has a good cooking resistance and firmness, does not release amount of organic matter into the cooking water, and does not show stickiness . The cooking loss of all the comb samples is much lesser when compared to the control . The lowest cooking loss is seen in (figure 3) where it is 2.88% compared to the control, which is 4.05% . The quality of the cooked pasta can be better explained on the basis of the interactions between starch and gluten whose intensity is strongly dependant on the drying conditions, as reported by many authors . If the coagulated gluten structure lacks compactness and elasticity, the starch granules structure swell up easily during cooking and lose more soluble materials into the cooking water . Starch was the main component (63.1%) on dry basis of the cooking liquor when 100% semolina was used in spaghetti and vermicelli samples . High - temperature drying strengthens the gluten matrix, which protects starch granules from rupturing during cooking . Furthermore, high temperature drying reduces water permeability and a crake in packaging & arrangement of starch granules, contributing to reduced cooking looses and increased cooked firmness . A reconstitution study indicates that the gluten quality is the major factor determining the cooking quality . This could be very well noticed in the comb samples where gluten was added, thus decreasing the cooking loss . This is also noticed in the comb samples, where the presence of hydroxypropyl - methylcellulose (hpmc) added as an additive is noted to increase water solubility and also at high temperatures forms a gel creating a temporary network of hydrocolloid chains, thus maintaining the structure . The main criteria generally accepted to access the overall quality of the cooked pasta are based on the textural evaluation . The work done by the probes to cut the pasta cooked firmness was greater for semolina than refined flour or whole wheat flour as seen in figure 5(a). Whole wheat pasta was reported to have lower cooked firmness than traditional semolina . When cooked to optimum, firmness was seen the greatest in semolina (semolina) obtained from t. aestivum wheat but is deemed not suitable for the consumer as it fails at the other parameters . It is also noticed that the addition of additives has resulted in firmness, which is similar to that of control . This can be explained as gluten present is seen to improve the protein content, thus improving firmness of pasta . Good - quality pasta should be al dente; that is, it should have high degrees of firmness and elasticity . Comb3 pasta has moisture values higher than that of control, and at the same time, its firmness showed increased values . This would suggest that the substitution of additives to 100% semolina contributes to structural strength . In this case of comb3, this hypothesis could also be related to the low values obtained for cooking losses, indicating a well - formed structure from which small amounts of solids are released during cooking . Longer cooking times resulted in an increased water absorption that led to moisture migration into the centre of the pasta . Hence, we hypothesize that moisture migration into the centre of the pasta diminishes the resistance of the sample to cutting, since the plasticizing action of water increases the mobility of biopolymers chains . Another factor that could have affected the texture was the more prolonged exposure to heat while drying that changed the structural conformation of the protein - starch network . This phenomenon may have caused loss of rigidity in the structure with a consequent decrease in firmness . A product, even if it is highly nutritious, but does not taste good, will not be accepted in the society, thus making sensory evaluation very important and a crucial criteria in the formulation of pasta . Based on the different properties of importance, it is characterised and formulated as shown in table 6 . It was noticed that the overall score when compared to the control is slightly less compared to all the other samples . Refined wheat flour considered being the alternate for semolina as a raw material is considered to be the second control . The main aim of the present study to find a suitable alternate for the control (durum semolina) thus needs an overall score to at least at par to be acceptable . It is noticed that additives improved the sensory scores especially the mouth feel character . The increase in mouth feels score (table 6), which is one of the main attributes for pasta sensory evaluation, may also be attributed to the increased water absorption (103.8%, 94%, and 104%), respectively, for the comb blends . Comb3 mixture of semolina of t. aestivum and t. durum gives the highest overall score, which is very much similar to the control . Being very essential for the bowel movement and the digestion in the body, dietary fiber levels are much noted in the present world . Remarkable changes in the levels of fiber are noticed in the comb2 and 3 samples, where they are 4.25 and 4.1, respectively (figure 5(b)). Higher levels of dietary fiber are mostly recommended in the present world, owing to the increase in diet - conscious people and the increased digestive problems faced in today's society . The link between dietary fiber and human health is well explained by where they explain that higher levels of dietary fiber increase cancer prevention, lower risk of chronic disease and help in diabetes management and prevention . It was also noticed by that incorporation of a combination of blends of wheat bran by - products increased the dietary fiber content . Two views of sem showing the cut section and the network of the pasta are presented in figure 6(b). These micrographs are all presented in 100x and 3000x, respectively, for all of the samples . Micrographs of the control pasta samples show the protein - starch matrix to be well formed, with strong and continuous protein strands entrapping large starch granules . The starch granules within the pasta appear to be slightly swollen and regular in shape and size, perhaps indicating a level of gelatinization during the extrusion process . It is also noticed that the control has smaller pores when compared to the comb blends . This supports the higher cooking loss (4.05%) and lower cooking time (6.5 min) as boiling water can reach the deeper core faster and stay in the cavities . The addition of refined wheat flour to the semolina is seen to disrupt the continuity of the protein matrix . The protein - fiber matrix within the pasta containing a portion of refined wheat flour appears to be less developed than the control resulting in an open appearance with discrete starch granules, which is uncovered and exposed to enzymatic attack . This may be explained by the dilution of the gluten protein, thus showing reduced firmness (1.757 n) and increased water absorption (103.8%), making it a more elastic structure . The addition of semolina with refined wheat flour shows a protein starch matrix similar to that of the control . This sample is seen to have a gelatinized structure when compared to the other blends, thus forming a more compact structure, which enable reduced water absorption (92%) during cooking . Unswollen starch granules can still be found inside the durum wheat, thus reducing the water absorption during cooking due to the layer of gelatinized starch . Use of whole semolina shows a higher network of protein - starch matrix, thus resulting in a higher degree of firmness (2.43 n) and improved chewiness . The rate of starch digestion and absorption seems to be a determinant of the metabolic response to a meal . The gi is defined as the postprandial incremental glycemic area after a test meal, expressed as the percentage of the corresponding area an equi - carbohydrate portion of a reference food (glucose or white bread). A recent joint fao / who expert consultation recommended increased consumption of low - glycaemic index (gi) foods . There is increasing evidence that a low -glycaemic - index diet can be beneficial in that, it improves metabolic control of hyperlipidaemia in diabetic patients as well as in healthy subjects . The rate and extent of starch digestion instigate a number of physiological functions that have different effects on health, including reduction of the glycemic and insulinaemic responses to a food, hypocholesterolemic effects, and protective effects against colorectal cancer . It is interesting to note that the addition of refined wheat flour as a raw material to the pasta sample increased the starch released in the pasta where pure refined wheat flour as pasta shows the highest level of starch release (44.90%). This is because as refined wheat flour consists of only the endosperm of the wheat grain, thus removing the nutritious bran and germ content (wikipedia, refined wheat flour). Goi et al ., have reported a total starch content of 74 2.24 for spaghetti, but the starch content obtained for the control pasta is approximately half of the value said above . This discrepancy may probably be due to the use of raw pasta for our analysis whereas goi et al . Used the cooked samples of the pasta for their analysis . Methodological differences in determination of total starch may also be a reason for the discrepancy . There are many factors that may influence the rate of starch digestion, including the nature of starch, the starch - protein interaction, the presence of fiber and antinutrients such as lectins, phytates and enzyme inhibitors and method and time of cooking . The present study revealed that pasta could be made using different wheat - milled products, which are economically viable and also have beneficial nutritional applications . Different combinations of mixtures had been experimented to produce viable alternates where different concentrations of refined wheat flour, whole - wheat flour, and semolina of different varieties of wheat were used to prepare pasta . Pasta made from refined wheat flour was seen to have reduced cooking loss, colour, firmness and sensory score . Pasta made from a combination of t. durum (50%) semolina and t. aestivum (50%) has acceptable levels of all the parameters, but due to increase in financial requirements, it is not suitable . From all results obtained, it was seen that semolina t. durum (50%) mixed with refined wheat flour t. aestivum (50%) can be used as alternate for making pasta for use as a low glycemic index snack product with acceptable physical and sensory properties . The addition of additives to the pasta has made it almost equivalent to the standard of original 100% semolina pasta . Acceptable cooking quality parameters were obtained in the pasta samples containing a mixture of both semolina and refined wheat flour, as measured by cooked weight, cooking loss, and so forth during cooking . However the incorporation of additives in the mixture of 50% semolina (t. durum) along with 50% refined wheat flour (t. aestivum) was finally concluded as the optimal alternate, because of its almostequivalent properties to the 100% t. durum semolina pasta, thus making it a economically nutritious alternate' resulting as a staple food in developing countries.
As indicated by otto warburg many years ago and now accepted as a hallmark of cellular transformation, cancer cells entirely reprogram their metabolism to sustain hyperproliferation and growth also in particular environmental conditions . In particular, differently from normal cells, cancer cells rely mostly on glycolysis rather than oxidative phosphorylation (oxphos) for atp production [2, 3]. Tumor environment, oncogenes, and tumor suppressor mutations have an important role in this energetic shift to aerobic glycolysis [4, 5]. Another important feature of metabolic reprogramming of transformed cells is their reduced or strongly impaired mitochondrial function [3, 6]. Despite that, mitochondria cover an important role also in cancer cells, that is, through the maintenance of mitochondrial potential and oxidative equilibrium, necessary for cell viability and apoptosis control, and for the different anabolic processes that use precursors produced in this organelle such as lipid, amino acids, and nucleotides synthesis . There is a series of compounds targeting mitochondria, named mitocans, that are being tested as anticancer drugs . They usually lead to cancer cell death by inducing mitochondria destabilization with a consequent increase of reactive oxigen species (ros) and activation of apoptotic signals [7, 8]. Different classes of mitocans exist and can be classified into eight groups, more specifically hexokinase inhibitors, bcl-2 homology-3 (bh3) mimetics, thiol redox inhibitors, drugs targeting the voltage - dependent anionic channel (vdac) or the adenine nucleotide translocator (ant), agents interfering with the electron transport chain (etc), lipophilic cations targeting the inner membrane, agents interfering with the mitochondrial dna, and drugs acting on not well - defined sites . Among the compounds acting on the etc, vitamin e analogues that in particular target complex ii have been tested as anticancer agents . Complex i inhibitors have shown anticancer properties as well, for example the acetogenins, such as rollinistatin and bullatacin, and also rotenone itself, which exhibits antitumor activity in animal models . On the other hand, cancer cells for their peculiar metabolism are particularly sensitive to treatments inhibiting glycolysis and to glucose deprivation [11, 12], since in both circumstances they lose hyperproliferative ability and ultimately die [1215]. Therefore, combined treatment targeting both glycolysis and mitochondria, exploiting peculiar tumor features, may be lethal for cancer cells . In this regard it has been shown that cancer cells, like osteosarcoma cells, treated with etc inhibitors, are induced to switch over to glycolysis becoming hypersensitive to the glycolytic inhibitors . Equally, it has been shown that inhibition of glucose metabolism, for example, by using 2-deoxyglucose (2-dg), can make tumor cells more dependent on oxphos and therefore more sensitive to treatment with etc inhibitors . However, glycolytic inhibitors, like 2-dg, could be potentially toxic for tissues like the brain, retinae, and testis that use glucose as the main energy source . In addition, they are also not very potent and must be used at high concentrations . In a previous study it has been shown that treatment of cancer cells with dichloroacetate (dca), a tca cycle inducer, is able to redirect their metabolism from glycolysis to oxidative phosphorylation and hence to lead them towards apoptosis . Therefore, it has been supposed that induction of a reversion of the warburg effect coupled to a treatment able to interfere with mitochondrial activity could specifically kill cancer cells . Recently we have shown that exogenous activation of pka pathway can improve several mitochondrial parameters, leading to a warburg effect reversion, in k - ras cancer cells, where the protein kinase a (pka) pathway is generally deregulated . In fact, cancer cells treated with forskolin (fsk), an activator of adenylate cyclase, show an increase of complex i activity, an increase of mitochondrial atp production, a decrease of ros generation, and an increase of mitochondria interconnections, that may lead to survival under glucose depletion . Since nutrient deprivation widely exists in solid tumors because of the poor blood supply [21, 22], we decided to study the effects on cancer cells of glucose depletion, mimicking physiological tumor condition, instead of glycolysis inhibitors, combined with treatments with oxphos complex i inhibitors . As results we demonstrate that in low glucose availability different cancer cell lines, in a way dependent on their glycolytic metabolism, become sensitive to short treatment with low doses of complex i inhibitors as compared to optimal glucose condition . Interestingly, in such a glucose - depleted condition, we also find evidence that stimulation of mitochondrial activity by fsk can further sensitize cancer cells to complex i inhibitors by enhancing cancer cell death . Altogether our findings indicate that stimulation of respiratory chain activity, in low glucose availability, makes glycolytic cancer cells more sensitive to oxphos inhibitors . Breast cancer cells mda - mb-231, mouse fibroblasts nih3t3 normal and transformed, pancreatic cancer cells mia paca-2, and lung cancer cells a549 were routinely cultured in dulbecco's modified eagle's medium (dmem) containing 4 mm l - glutamine, 100 u / ml penicillin, and 100 mg / ml streptomycin (complete medium), supplemented with 510% fetal bovine serum (human cells) or 10% newborn calf serum (mouse cells). For the experiments cells were plated in complete growth medium . After 16 hours cells were washed twice with phosphate buffer saline (pbs) and incubated in growth medium (time 0) without glucose and sodium pyruvate, supplemented with 25 or 1 mm glucose . Treatments and analyses were performed at 48 hours (mda - mb-231 and a549) or 72 hours (nih3t3 and mia paca-2) after time 0 . All reagents for media were purchased from life technologies (carlsbad, ca, usa). (st . Louis, mo, usa). Capsaicin and piericidin a were purchased from vinci - biochem (florence, italy). Cell viable count was performed by staining cells with trypan blue 0.4% (life technologies). Propidium iodide (pi)/annexin v - fitc staining was performed using apoptosis assay kit from immunological sciences (rome, italy) and analyzed by facscan flow cytometer (becton - dickinson, franklin lakes, nj, usa) with cellquest software (becton - dickinson). Flow cytometric data were then carried out using the freely available winmdi software . For the evaluation of pi incorporation 5 10 cells were harvested and stained with 5 g / ml pi and 5 g / ml hoechst (sigma - aldrich inc .) In pbs for 15 min at r.t . After staining, cells were mounted on a microscope slide with 50% glycerol and analyzed under a nikon eclipse 90i fluorescence microscope (nikon, tokyo, japan) equipped with a b / w ccd camera (hamamatsu - coolsnap, hamamatsu corporation, hamamatsu city, japan). The images were acquired using the imaging software metamorph 7 and then visualized and processed in image j (freely available). For each sample after 12 days colonies were fixed with pbs - formaldehyde 5%, stained with crystal violet 1%, and then counted . Intracellular atp levels were measured using celltiter glo luciferin - luciferase assay (promega, madison, wi, usa) as described in . Mitochondrial potential was analyzed by staining cells with 20 nm jc-1 (5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolylcarbocyanine iodide, life technologies) for 10 minutes . After staining, flow cytometric analysis was performed acquiring fl1 (jc-1 monomers, low potential) and fl2 (jc-1 aggregates, high potential) signals . For each sample d - glucose levels in culture medium were determined using a spectrophotometric assay kit (r - biopharm, darmstadt, germany) as specified by manufacturer's datasheet . For the analysis of cleaved caspase 3 and actin b expression, cells were harvested and lysed in laemli buffer (50 mm tris - hcl ph6.8, glycerol 6%, sds 2%, -mecaptoethanol 5%, bromophenol blue 0.05%). Samples were then resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane, which was incubated overnight with antibodies for cleaved caspase 3 (cell signaling technology inc ., danvers, ma, usa; 1: 1000) and actin b (abcam, cambridge, uk; 1: 1000). Oxygen consumption was determined using seahorse xf24 extracellular flux analyzer (seahorse bioscience, north billerica, ma, usa). Cells were seeded in the 24-well xf24 cell culture plate in the culture medium containing 25 or 1 mm glucose, as described above . Where indicated, cells were also treated with fsk . Culture media were exchanged for base media (unbuffered dmem supplemented with 10 mm sodium pyruvate and 20 mm glucose for cells grown in high glucose or only 10 mm sodium pyruvate for cells grown in low glucose) 1 hour before the assay and for the duration of the experiment . Selective inhibitors were injected during the measurements to achieve final concentrations of rotenone 3 nm and piericidin a 5 nm . The baseline ocr was defined as the average of the values measured from time points 1 to 5 (045 min) during the experiments . Due to some variations in the absolute magnitude of ocr measurements in different experiments after the analysis the cells were fixed, stained with crystal violet, and dosed at spectrophotometer after colorant solubilization with acetic acid 10%; all ocr values obtained by the instrument were normalized on cell density . Mda - mb-231 human breast cancer cells, like several other cancer cells, use mainly glycolysis instead of mitochondrial respiration to generate atp and other anabolic substrates necessary for their proliferation and survival . In fact, in low glucose availability, these cells show a reduced proliferation and an increase of cell death because of their inability to maximize the use of oxphos especially for energetic use . In this scenario, we tested the ability of rotenone, an inhibitor of oxphos, to increase their sensitivity to glucose depletion . Rotenone is a natural compound that has been used to interfere with mitochondrial respiration, in particular with complex i activity, and hence to reduce intracellular atp levels especially in oxphos - dependent cell lines [25, 26]. In order to evaluate their ability to proliferate and survive under oxphos inhibition, we treated proliferating mda - mb-231 cells, grown for 48 hours in low glucose (1 mm glucose, 4 mm glutamine) or high glucose (25 mm glucose, 4 mm glutamine), with rotenone . The treatment was executed at 48 hours of culture because, despite a comparable proliferation rate in the two different glucose concentrations (figure 1(a)), in low glucose condition external medium analysis indicated that this carbon source was almost completely depleted at this time point (figure 1(b)). In addition, measurement of the basal cellular oxygen consumption rate (ocr) by seahorse xf analyzer indicated a 40% increase of cellular respiration rate in cells grown in low glucose (figure 1(c)), suggesting that mda - mb-213 cells, in absence of glucose as main substrate for glycolysis, partially shifted from glycolysis to mitochondrial respiration . Short treatment with a low concentration of rotenone (3 nm for 4 hours), known to be ineffective on normal cell mitochondria activity [2729], in glucose - depleted condition induced a reduction of cell viability, as confirmed by morphological analysis (figure 1(d), circle and floating cells) and by trypan blue viable cell count (figure 1(e)). In fact, after treatment trypan blue - positive cells increased from 10.2% to 22.3% . This effect on cell survival was also supported by clonogenic assays (figures 1(f) and 1(g)), which showed that treated cells, replated in high glucose condition (25 mm), formed less colonies (about 50% of reduction) as compared to untreated control . Such an assay shows that the short treatment is enough to reduce cancer cell ability to form a large colony and proliferate, suggesting that rotenone, inhibiting the alternative mitochondrial energetic route of these cancer cells upon glucose deprivation, heavily affects their viability . Rotenone outcome on mitochondrial activity was evaluated by determination of ocr, mainly due to mitochondrial respiration, mitochondrial potential, and intracellular atp levels . In particular, 3 nm rotenone was injected by the instrument into the cells and its effect analyzed between 30 minutes and 1 hour after the injection . As shown in figure 1(h), ocr was reduced to ~30% of the baseline rates, indicating that 3 nm rotenone is able to decrease mitochondrial respiration . Also mitochondrial potential was reduced by rotenone (figure 1(i)), confirming the direct effect of the treatment on mitochondrial function . In the same experimental setting, rotenone induced also a ~25% decrease of the intracellular atp levels (figure 1(j)). Importantly, rotenone treatment in nonlimiting glucose condition had no effect on cell survival, as confirmed by trypan blue viable cell count (figure 1(k)) and clonogenic assay (figure 1(l)). Moreover, rotenone had no effect on intracellular atp levels (figure 1(m)), suggesting that in high glucose availability atp is generated essentially by glycolysis . It has been proposed that warburg effect reversion, gained by mitochondrial reactivation, may be a promising method for promoting naturally encoded programmed cell death and hence kill cancer cells . Given that, we sought to investigate whether the combination of fsk, able to restore mitochondrial activity, and rotenone could synergistically enhance the killing of cancer cells in glucose depletion . First, we performed such an analysis on nih3t3 mouse fibroblasts (immortalized cells, normal), an oxphos - dependent cell line, and nih3t3 mouse fibroblasts expressing an oncogenic k - ras gene (transformed) [15, 24]. The latter cellular model of transformation is suitable since it presents a transcriptional profile and different metabolic features, such as the warburg effect, comparable to several human cancer cells harboring an oncogenic k - ras gene, like, for instance, mda - mb-231 cells [23, 24, 30, 31]. The cells grown for 72 hours in both initial glucose concentrations were incubated with rotenone and fsk alone or in combination . As shown in figures 2(a) and 2(b), in nonlimiting glucose condition rotenone had no effect on proliferation of both cell lines, confirming that such a low rotenone concentration does not inhibit mitochondrial respiration of normal cells (figure 2(a)) and does not induce cell death in mouse transformed cells (figure 2(b)), as previously observed in mda - mb-231 cells . On the contrary, cells grown in low glucose for 72 hours, the time point at which both cell lines have completely consumed the glucose in the culture medium, showed a different response to the treatments with rotenone and/or fsk (refer to figure 2(c) for treatments schedule). Normal cells were found to be insensitive to rotenone either alone or in combination with fsk (figure 2(d)). In contrast, transformed cells showed 17% of cell death in basal condition, 22% upon fsk treatment, 29% upon rotenone, and 42% when the two compounds were used in combination (figure 2(e)). These data indicate that normal mouse cells, relying especially on mitochondrial respiration, are less responsive to low doses of rotenone as well as to the combined treatment with fsk . On the contrary, transformed mouse cells, forced to use mitochondrial respiration by glucose deprivation or fsk treatment, become more sensitive to the complex i inhibitor . Since a previous study has indicated that mda - mb-231 cells are responsive to fsk treatment as well as mouse k - ras - transformed fibroblasts, we treated mda - mb-231 cancer cells, grown in low glucose, with rotenone and fsk alone or in combination (schedule is shown in figure 2(c)). In order to evaluate cell death upon single or combined treatments, the cells were analyzed through trypan blue viable count (figure 3(a)) or pi / annexin v staining followed by facs analysis (figure 3(b) and supplementary figure 1 in the supplementary material available online at http://dx.doi.org/10.1155/2013/243876). As shown in figures 3(a) and 3(b), both analyses indicated an increased cell death in the samples subjected to the combined treatment as compared to either untreated or rotenone - alone - treated samples . In particular, trypan blue staining indicated an increase of positive cells from 22% for rotenone alone to 33.6% in presence of fsk . Similar values were observed by pi / annexin v staining (18% rotenone versus 30% rotenone + fsk). Importantly, the combined treatment further reduced cancer cell ability to form colonies as compared to rotenone alone (figure 3(c)). The increase of cell death was associated with a reduction of about 50% of intracellular atp levels (figure 3(d)) and of around 20% of mitochondrial potential (figure 3(e)) as compared to untreated samples . To confirm a role of fsk in inducing a positive effect on mitochondrial activity, next we measured basal ocr, as previously described, in untreated or 2-to-4-hour fsk - treated cells . The interval of treatment was chosen since in our assays the cells were treated with fsk (pretreatment plus combination with oxphos inhibitors) for a maximum time of 5 hours . As shown in figure 3(f), fsk - treated samples showed an increase of around 30% of ocr as compared to untreated samples, suggesting that the formers are more respirative than the latter ones . Moreover we did not observe differences in ocr values obtained in 2 and 4 hours - treated samples . Altogether these findings indicate that, upon glucose depletion, the stimulation of respiratory chain activity makes cells more sensitive to oxphos inhibitors . To further confirm the role of mitochondrial inhibition in the cell death mechanism upon glucose depletion, and more specifically the role of complex i, we used two other inhibitors of this complex, namely piericidin a and capsaicin . Importantly piericidin a, differently from rotenone that at higher concentration may affect cell cycle [33, 34], does not interfere with the cell cycle execution . As shown in figures 4(a) and 4(b), upon 2 hours of treatment, both inhibitors, as previously observed with rotenone, did not induce cell death when added to mda - mb-231 grown in high glucose . On the contrary, their addition to glucose - depleted cells led to an increase of mda - mb-231 cell death that was much stronger in the samples treated with piericidin a (52%) (figure 4(c)) than with capsaicin (28%) (figure 4(d)). Notably, combined treatment with fsk further increased the percentage of cell death that reached a value of 80% in the sample piericidin a + fsk (figure 4(c)) and 39% in the sample capsaicin + fsk (figure 4(d)). Since cell viability was greatly affected by piericidin a, we evaluated also the potential of this molecule in combination with fsk in clonogenic assays (figure 4(e)). Obtained data indicated a significant reduction of colonies number after treatment with piericidin a, reduction that was further increased upon combination with fsk as compared to untreated and fsk - treated sample (figure 4(e)). Notably, ocr measurement indicated that piericidin a was able to decrease mitochondrial respiration (figure 4(f)) as well as previously observed with rotenone (figure 1(h)), confirming its effect on cellular mitochondrial respiration . Glucose deprivation has been shown to kill cells either by necrosis or through the mitochondrial pathway of apoptosis . Similarly, prolonged treatment with mitochondrial oxphos inhibitors also lead to necrotic cell death . In order to assess whether single or combined treatments could induce necrosis or apoptosis, we performed experiments of pi incorporation followed by microscopy analysis and of western blot . As shown in figures 5(a) and 5(b), complex i mitochondrial inhibitors (rotenone and piericidin a) caused an increase of pi incorporating cells as compared to untreated or fsk - treated samples . As expected from previous results, fsk treatment further enhanced the percentage of pi incorporating cells, supporting the notion that cells were experiencing a necrotic cell death process (figures 5(a) and 5(b)). Such a cell death mechanism was confirmed by morphology analysis indicating cell detachment (data not shown) and plasma membrane damage without nuclear condensation (figure 5(a), hoechst staining). In addition, this cell death process was not associated with activation of caspase 3 (figures 5(c) and 5(d)) and poly(adp - ribose) polymerase (parp) (data not shown), both considered apoptotic markers, as it was seen with thapsigargin treatment for 6 hours (figures 5(c) and 5(d)). To examine whether other cancer cell lines showed similar sensitivity to complex i inhibitors alone and combined with fsk, we examined the effect of the treatments on two different human cancer cell lines with a different dependence on glucose availability . Previous data, in fact, have shown that mia paca-2 (pancreatic cancer cell line) and a549 (non - small - cell lung cancer cell line) are both sensitive to glucose deprivation, in particular mia paca-2, undergoing cell death, and, in different extend, are both protected by fsk treatment because of its ability to restore complex i activity . Moreover, both cell lines express an oncogenic k - ras that has been shown to promote cell metabolic rewiring and in particular glycolysis [24, 37]. As shown in figures 6(a) and 6(b) piericidin a treatment, used at concentration of 10 nm for 8 hours, did not affect survival of both cell lines grown in 25 mm glucose, as measured by trypan blue vital staining . On the contrary, as previously observed for mda - mb-231 cells, its addition in glucose depletion led in both cell lines to an increase of cell death that was stronger in more glycolytic cells mia paca-2 (37.9%) (figure 6(c)) than in a549 (23.9%) (figure 6(d)). Notably, combined treatment with fsk further increased the percentage of cell death, reaching a value of 52.6% in mia paca-2 (figure 6(c)) and 33.4% in a549 (figure 4(d)). Interestingly, in mia paca-2 cells an increase of cell death was already observed in cells grown in low glucose as compared to high glucose (22.6% versus 10.8%), confirming their major dependence on glucose availability as compared to a549 cells . To further confirm the major sensitivity of cancer cells to inhibitors of mitochondrial function in a condition of low glucose mda - mb-231 cells are considered a cell line with a high glycolytic rate and a low level of respiration [15, 24]. For this reason we supposed that under treatment with oligomycin these cells could undergo a limited effect in high glucose and a significant effect in low glucose, since the latter condition is characterized by acute stimulation of respiration . Mda - mb-231 cells, cultured in 25 mm glucose, were treated for 1 hour with 5 m oligomycin and then analyzed . As shown in figures 7(a) and 7(b) both cell viability and total intracellular atp were not changed by the treatment, whilst a slight decrease of mitochondrial potential and a consistent decrease of colony formation ability were observed (figures 7(c) and 7(d)). On the other hand, mda - mb-231 cells grown in low glucose and treated with oligomycin showed a strong increase of cell death as indicated by trypan blue viable count (figure 7(e)). Moreover, a complete depletion of intracellular atp (figure 7(f)), associated with a partial reduction of mitochondrial potential (figure 7(g)), was observed . All these parameters were accompanied by a further decrease in the ability to form colonies as compared to cells grown in high glucose (figure 7(h)). Taken together, these data indicate that in glucose shortage glycolytic cancer cells show a stronger and forced dependence on mitochondrial respiration that make them very sensitive to inhibitors of mitochondria function . In fact, since this organelle is central both as producer of cellular atp and as central regulator of apoptosis, it may be considered a good therapeutic hit . The primary metabolic function of mitochondria is oxphos, an energy - generating process that couples the oxidation of respiratory substrates with atp production . Besides atp synthesis, mitochondria are involved in several other key metabolic processes such as oxidative decarboxylation of pyruvate, tricarboxylic acid cycle, and fatty acids oxidation . In addition, mitochondria take part in intracellular homeostasis of calcium and phosphate as well as in the balance of nad / nadh . Besides their central role in metabolic activity, more recently mitochondria have been shown to have a central role also in the cascade of events that leads to programmed cell death . In fact mitochondria represent a central checkpoint of this process by integrating various signals coming from endogenous factors (ions, metabolites, second messengers), from endogenous signaling proteins (kinases and phosphatases), and from exogenous factors (nutrients, oxygen). Therefore the strong interconnection between metabolism and apoptosis and the central role of mitochondria in both processes have led to an explosion of interest in connecting such pathways to the pathophysiology of cancer . In this report, we have decided to utilize the main metabolic alterations of cancer cells, namely hyperglycolytic phenotype (warburg effect) and mitochondria dysfunction, as targets for combined treatments aimed to specifically kill cancer cells . In fact, as shown by a number of groups, cancer cell proliferation and tumor aggressiveness correlate with an enhanced glycolysis and a low mitochondrial respiratory chain activity [3, 31], and positive or negative modulation of oxphos activity, depending on the metabolic state of cancer cells, appears to reduce tumor growth [39, 40]. Herein by using a glycolytic human breast cancer cell line, namely mda - mb-231, grown in limiting glucose availability, and some natural inhibitors of mitochondria activity, we show that complex i inhibition associated with an acute stimulation of respiration, due to glucose depletion, induces specifically necrotic cancer cell death (figures 1, 3, and 5). Notably, this finding has been observed by using three different complex i inhibitors that strongly support our results (figures 4 and 5). Moreover, we show that this cell death process, induced by the treatment with complex i inhibitors, is activated also in other three cancer cell lines, mouse k - ras transformed fibroblasts, human mia paca-2 pancreatic cancer cells, and human a549 lung adenocarcinoma cells . Importantly, such cell death mechanism is strongly dependent on glycolytic rate of the cancer cells . In fact mia paca-2 cells, known to have a higher glycolysis as compared to a549, appear to be more sensitive to the treatment with inhibitors (figure 6). Our results are interesting also because our and previously published data indicate that, at the used concentrations, rotenone (3 nm), capsaicin (100 m), and piericidin a (5 nm) have no effect on immortalized fibroblasts (figure 2), on normal pancreatic cells and on dopamine neurons, respectively . This reduced or absent effect on normal cells is an important characteristic for exploiting these compounds for cancer therapy . Regardless of the mechanism of action of the three molecules, we show that the sensitivity to them increases upon glucose depletion that reflects the dependency of the cells on glycolysis . We suppose that less glycolytic cells, such as immortalized fibroblasts or normal cells, will be also less sensitive to these treatments . Since we observed a necrotic cell death (figure 5), we attribute the synergistic effects more to atp depletion than a rapid decrease of mitochondrial potential . However we cannot exclude an increase of ros levels, as shown by other authors, as a consequence of complex i inhibition [17, 25]. In fact, it is possible that stimulation of mitochondrial activity upon glucose depletion in the deranged respiratory system of malignant cells results in an increased production of oxidants, which may overwhelm cellular antioxidant protections and lead to cell death . Further experiments exploring this point will be addressed in the future . From our studies, in particular from results obtained with fsk, an important point emerges: stimulation of mitochondrial activity and restoration of an atp generating mechanism more similar to nonmalignant cells, might be an efficient tool in anticancer strategy . In particular, shifting cellular metabolism towards mitochondrial atp production might overcome the positive effects on glycolytic pathway of oncogenes like k - ras, akt and hif-1 . The idea is not completely new, since other authors have addressed this point by redirection of pyruvate towards oxidation in the mitochondria . In fact, inhibition of pyruvate dehydrogenase kinase (pdk) by dca, or lactate dehydrogenase (ldh) by rnai, has been shown to shift metabolism from glycolysis to glucose oxidation and to strongly reduce cancer cell viability and tumor growth [18, 43]. Our results with fsk suggest a similar mechanism in which reactivation of the mitochondrial function associated with glucose depletion and mitochondrial complex i inhibition strongly affects cancer cell survival . Therefore, strategies involving the manipulation of both glycolytic and mitochondrial pathways might be useful to eradicate cancer cells . Other information in such a direction was derived also by experiments with oligomycin, an inhibitor of mitochondrial atp synthase . We show that it is able to enhance cancer cell death in low glucose as compared to high glucose (figure 7). In fact, we observed that in normal glucose conditions it does not induce a strong reduction of cell viability, although it is able to reduce the capability to form colonies . We can suppose that such a long - term effect is due to the inhibition of the reverse action of atpase that in fact is reflected in the mitochondrial potential decrease not accompanied with loss of atp . On the other hand, the stronger effect of oligomycin on mda - mb-231 cells in glucose deprivation is experimental evidence of their forced dependence on oxphos in such condition, exploitable by mitochondrial targeting therapies . Moreover, from another point of view, these data could suggest that inhibition of residual mitochondrial activity of cancer cells will further upregulate glycolysis and hence lead to their death by glucose depletion . This finding has been observed in lung carcinoma, in which oligomycin treatment upregulates glycolysis, increasing their dependence on this metabolic pathway . Taken together our results provide a rationale for the use of mitochondrial inhibitors in cancer cells exploiting cancer cell fragility versus glucose depletion . In addition they point to an energetic switch from glycolysis to oxphos as an important therapeutic approach, since normal cells appear to be resistant to such combined treatments.
Proponents of the procedure have claimed that it offers several advantages over the traditional open appendectomy through a right lower quadrant muscle splitting incision . However, the data concerning any superiority of the laparoscopic technique have not been entirely clear . To further evaluate this procedure, we examined our experience with laparoscopic and open appendectomies for acute appendicitis during a 24-month interval . Surgical technique in each case was determined by individual surgeon preference and the availability of laparoscopy equipment . Patients who were not considered candidates for laparoscopic appendectomy were excluded from analysis and included pregnant women, patients with a palpable mass in the right lower quadrant (presumably representing a large phlegmon or abscess), and patients with diffuse peritonitis . Thirty - seven patients underwent laparoscopic appendectomy, and 34 patients underwent traditional open appendectomy . The two patient groups were similar with regard to age, gender, height, weight, fever, and leukocytosis . The number of patients pathologically classified as having acute appendicitis versus gangrenous or perforated appendicitis were also similar . Open appendectomies were performed through traditional transverse, right lower quadrant, muscle splitting incisions . The base of the appendix was doubly suture ligated with 0-chromic and then cauterized to prevent lymphocele . The peritoneum and internal oblique fascia were closed with a running 0-vicryl suture as one layer separately from the external oblique fascia; scarpa's fascia was closed with a running 3 - 0 vicryl suture . Laparoscopic appendectomy was approached by a three trocar technique with the addition of a fourth trocar when necessary . Usually, a 10 mm port was placed at the umbilicus for the camera, a 12 mm port was placed in the suprapubic area, and another 10 mm port was placed in the right upper quadrant . When needed, a 5 mm or 10 mm port was placed in the left lower quadrant . The mesoappendix was transected using clips, ligatures, or an endogia stapling and cutting device (united states surgical corporation, norwalk, ct). The appendix was removed through the 12 mm port directly or after insertion into a bag . If the procedure could not be safely completed laparoscopically, the surgeon converted to an open procedure . These converted patients were included in the laparoscopy group for the subsequent analysis on an intent - to - treat basis . Surgical residents participated in nearly all cases in the roles of both surgeon and first assistant . Postoperative pain was controlled by varying parenteral and oral regimens at the discretion of the physician . Patients were discharged when they were afebrile for 24 hours and tolerating a regular diet . Measurements of patient characteristics and illness severity included age, gender, height, weight, fever, leukocytosis, and appendiceal findings by pathology . Total operating room time was measured from time of patient entrance into the room to time of exit . An unpaired student's t - test was used to assess differences between the groups . In the laparoscopy group, 31 patients had appendectomy completed laparoscopically; one had a normal appearing appendix left intact (there was a clear diagnosis of pelvic inflammatory disease), and five (14%) had conversion to open appendectomy (table 1). In four of the conversions, extensive adhesions and phlegmon precluded safe laparoscopic dissection . A fifth conversion was due to dense adhesions from prior surgery and an inadvertent enterotomy . Two conversion cases had the wounds packed open, and one laparoscopically completed case had the suprapubic wound packed open . In the open group, 11 wounds (32%) eight of these 11 wounds were closed on the third postoperative day; one was closed on the sixth postoperative day, and two patients were discharged with open wounds . Average postoperative hospitalization was 6.6 days for these 11 patients with open wounds . In the laparoscopy group, both surgery time (72 vs. 53 minutes, p<.001) and total operating room time (119 vs. 85 minutes, p<.001) were longer than in the open group (table 1). We did not observe any trend for decreasing surgical time with increasing experience in the laparoscopy group . In the open group, two patients developed wound infections: one patient developed a pelvic abscess requiring transrectal drainage, and one had a postoperative myocardial infarction which resulted in the only death of this series . In the laparoscopy group, four patients (11%) suffered postoperative morbidity (figure 1). This complication was not recognized until the postoperative period at which time the patient underwent laparotomy for primary bladder repair . Another patient suffered an enterotomy during laparoscopic dissection through extensive dense adhesions from prior surgery . An abdominal ct scan showed a 3 cm diameter pelvic fluid collection which was aspirated under radiologic guidance . The fevers resolved with antibiotic therapy . A fourth patient who had been converted to an open procedure incidence of various postoperative complications and total complications in the laparoscopic and open appendectomy groups . Analgesic and narcotic doses / postoperative hospital day were nearly identical in the laparoscopy and open groups, 2.1 vs. 2.2 analgesic doses / postoperative hospital day and 1.9 vs. 2.0 narcotic doses / postoperative hospital day, respectively (table 1). Twenty - four patients in the laparoscopy group and 23 patients in the open group were classified as having acute suppurative appendicitis (non - gangrenous, non - perforated). Mean los for the laparoscopy vs. the open group was 2.5 days versus 4.0 days (p<.01). Overall los, with analysis including patients in both groups classified as having gangrenous or perforated appendicitis, was not statistically significantly different between groups (3.7 days vs. 4.1 days, p=0.11) in the past few years, advances in laparoscopic technology have altered the surgical approach to various abdominal problems with cholecystectomy being the most prominent example . A major benefit of laparoscopy apparently derives from the reduced abdominal wall trauma as compared to traditional open procedures . However, the abdominal wall injury may not be a significant factor for every type of abdominal incision and pathologic process . Traditional open appendectomy through a muscle splitting incision generally produces a relatively small degree of abdominal wall trauma . . A more limiting factor in the postoperative course of acute appendicitis may be sequelae of the inflammatory / infectious process itself . Although patients were not truly randomized, the various patient characteristics and measurements of disease severity were equally distributed between the two groups . In attwood's series, patients were randomized between laparoscopic and open approaches but the report contained little information about the patients and the severity of illness . The longer total operating room and surgery times in the laparoscopic group were not surprising . Setting up laparoscopy equipment generally took longer than setting up traditional surgical equipment . To some degree, the frequent introduction of residents to this approach contributed to procedure length . While we observed no decrease in surgery time with increasing numbers of procedures, we still feel that our experience with this procedure is not yet enough to conquer the learning curve . As our collective experience with this approach the complication rates in our two groups were similar to those previously reported for open appendectomy . Although packed wounds in open appendectomies reduce the incidence of wound infection, they also constitute an additional element of post - operative care and patient concern . The patients requiring conversion from laparoscopic to open appendectomy were included in the laparoscopy group on an intent - to - treat basis . With further experience, we may see a drop in the number of conversion cases but it is unlikely that these will be completely eliminated . The laparoscopy group showed a tendency to earlier post - operative discharge but the difference was not statistically significant . Scott - conner reported a mean postoperative stay of 2.4 days for patients treated by laparoscopic appendectomy but excluded patients converted to an open procedure from the calculation . Mcanena reported an average postoperative stay of 4.8 days for 36 open patients and 2.2 days for 27 laparoscopic patients . The difference was reported as significant but two conversion cases appear to have been excluded from the laparoscopy group statistics . Attwood's randomized study showed significantly earlier discharge (2.5 vs. 3.8 days, p<0.01) for laparoscopic appendectomy cases . The 625 patients of pier were generally dis - charged a week after the operation . Two major factors may have lengthened the average post - operative stay of our laparoscopy group to 3.7 days . First, our statistics included the five patients who were converted to an open procedure . Our 35% incidence of gangrenous or perforated appendicitis in the laparoscopy group is greater than the 11% reported by mcanena and the 6% reported by scott - conner . Interestingly, when we excluded the gangrenous and perforated appendicitis patients, the difference in postoperative hospitalization between the two groups (2.5 vs. 4.0 days) became statistically significant (figure 2). For this subgroup, the 2.5 day postoperative hospitalization for the laparoscopy group was comparable to the 2.4 day, 2.2 day, and 2.5 day values reported by scott - conner, mcanena, and attwood . The postoperative hospitalization for the open appendectomy patients in this subgroup was similar to the 3.8 days reported by attwood . Although this sort of statistical manipulation carries limited power in a small series, the results suggest a possible relationship between disease severity, operative approach, and postoperative course . For early appendicitis with minimal peritonitis, the amount of abdominal wall trauma may play a significant role in postoperative recovery . Thus, the laparoscopic approach may result in a shorter hospitalization than the open approach . However, for advanced appendicitis, the intraperitoneal inflammation may be a more important determinant of postoperative course than the amount of abdominal wall injury from the operative approach . Postoperative length of stay in the laparoscopic versus open appendectomy groups for both acute suppurative (non - perforated, non - gangrenous) appendicitis and perforated and/or gangrenous appendicitis . The length of stay was significantly shorter for the laparoscopic appendectomy group as compared to the open appendectomy group when the appendix was not perforated or gangrenous (* p <0.01); however, in the presence of a perforated or gangrenous appendix, there was no significant difference (p = 0.11). An important issue not addressed in other reports concerns the criteria for patient discharge from the hospital . The clinical decision to send a patient home on a certain day rather than one day earlier or one day later can bias results which may be looking at only a one day difference between groups . It would be difficult to blind a surgeon with regard to operative approach so that an unbiased decision could be made regarding hospital discharge . We would advise surgeons to always remove the appendix when laparoscopically approaching patients with a preoperative diagnosis of acute appendicitis . This helps avoid future diagnostic dilemmas and prevents missing an early acute appendicitis with minimally visible inflammatory changes . One of our patients with a visibly normal appendix showed distinct microscopic acute appendicitis by pathology . We attempted to use laparoscopy as a technical approach to the appendix in patients with suspected appendicitis by traditional clinical criteria . We realize that most appendectomies are diagnostic procedures initially since there is no other confirmatory test prior to operative intervention . Laparoscopy may have utility as a diagnostic tool in a broader group of patients with lower abdominal pain but we are uncertain about the indications for use . Proponents have claimed that the laparoscopic approach allows patients to resume work or their normal lifestyle earlier than the traditional open approach but the data has not been clear . In scott - conner's report, all patients had returned to attwood's randomized study attempted to gather more detailed information by contacting patients after their postoperative clinic visit and asking about duration of pain and return to employment, sport, and full fitness . However, attwood's series also contained mostly younger patients in the laparoscopy group with a mean age of 20.8 years (range 12 to 39) in the laparoscopy group . In addition, we do not know how many of his patients had advanced appendicitis . We suspect that patients with simple appendicitis recover more quickly than patients with complicated appendicitis . In our series, we observed that laparoscopic appendectomy patients typically returned to normal activities within one to two weeks but we could not gather sufficient data for presentation . The type of work, the desire to work, the employment status, and the availability of vacation time can significantly influence a patient's progress . We also observed many motivated open appendectomy patients who returned to work in one to two weeks after surgery . Laparoscopic appendectomy reduces los as compared with the traditional open technique in patients with acute suppurative appendicitis . Mean los is not statistically different between groups when analysis includes patients with perforated or gangrenous appendicitis . The greater severity of illness in these patients likely outweighs those advantages of the laparoscopic approach which led to a decreased los in patients with uncomplicated appendicitis . Operative time for laparoscopic appendectomy was longer than for open appendectomy and is likely related to the learning curve associated with the procedure . Use of post - operative analgesia and incidence of postoperative morbidity were not statistically significantly different between groups . We agree with other authors recommending further investigation of the laparoscopic approach in the management of right lower quadrant pain . A randomized trial should be done to more clearly define the role of laparoscopy as a
The discovery of somatotropin - release inhibitory factor, or somatostatin (sst), in hypothalamic extract in the early 1970s, led to important advancements in the comprehension of the regulation of growth hormone (gh) secretion and offered the opportunity to develop drugs mimicking the actions of sst . The synthesis of the first somatostatin analog (ssa), octreotide (oct), the discovery of 5 sst receptor (sstr) subtypes, and the development of additional sstr ligands offered a great opportunity for the medical treatment of acromegaly and neuroendocrine neoplasms (nens). With an annual incidence of 5 cases/100,000 in the usa,1 nens are rare tumors composed of multipotent neuroendocrine cells able to produce, store, and secrete biologically active substances which can cause if functioning distinct clinical syndromes . Both functioning and nonfunctioning tumors can also produce symptoms due to mass effects, and even if the majority of these tumors exhibit long periods of relatively small growth, in some cases, they can show massive growth and can be associated with distant metastases . In> 50% of cases, tumors originate in the gastrointestinal system or the pancreas (gastroenteropancreatic neuroendocrine neoplasms [gep - nens]). Treatment of nens requires a multimodal approach, involving both tumor debulking and management of symptoms . Tumor diameter is one of the most important parameters in the decision - making process for nonfunctioning forms . In fact, while surgery represents the treatment of choice for larger nonfunctioning tumors, small lesions can be treated conservatively . Locally advanced and metastatic disease can also be treated with extended resections, considering tumor grading, size, ki-67 proliferation index, and the presence of extra - abdominal disease . Other approaches include pharmacological treatment (ssas, interferon, antiangiogenic agents, tyrosine kinase inhibitors, mammalian - target - of - rapamycin inhibitors, chemotherapy), radionuclide therapy, and chemo- or radioembolization . In particular, ssas, which were initially used to control hormonal syndromes associated with nens, have been successfully proposed as antiproliferative agents, able to control tumor growth.2,3 moreover, long - acting formulations of ssas have demonstrated their efficacy as antineoplastic agents in the treatment of gep - nens.4 long - acting formulations of ssas (oct lar, slow - release lan, and lan autogel) assure improved patient compliance with weekly up to monthly injections . In this review, we focus on the clinical utility of lan autogel in the treatment of gep - nens . Human sst, isolated in 1973 and soon identified as a hypothalamic inhibitor of gh,5,6 has been subsequently found in several other tissues (central nervous system, endocrine system, and gastrointestinal tract). Sst is formed by proteolytic processing of larger precursor molecules: prepro - sst and pro - sst.7 prepro - sst is a 116-aminoacid precursor which is encoded by a single gene located on chromosome 3q28, and it is processed to pro - sst (96 amino - acids). Pro - sst undergoes tissue - specific enzymatic degradation to produce 2 bioactive proteins: the predominant, but functionally less active 14-aminoacid molecule called sst-14 and the larger and more potent 28-aminoacid form sst-28 (figure 1).8 sst isoforms have endocrine, paracrine and autocrine inhibitory effects as well as on exocrine glands . The mechanisms by which sst and ssas inhibit the neuroendocrine cells are complex and poorly understood.9 once sst binds to its cell- and tissue - specific receptors, the downstream effects elicited by the ligand receptor formation are site - specific: in the central nervous system, sst acts as a neurotransmitter, while in the hypothalamic pituitary region, it acts as a neurohormone.10 sst is also known to inhibit several cellular functions . In particular, in the digestive tract, sst reduces gastrointestinal motility, inhibits gallbladder contraction, decreases portal blood flow, and suppresses the secretion of gastrointestinal hormones (insulin, glucagon, gastrin, cholecystokinin, vasoactive intestinal peptide, and secretin)11 and that of other exocrine gastrointestinal cells (gastric acid, intestinal fluid, and pancreatic enzymes).12 moreover, it inhibits proliferation of both normal and tumor cells.13 circulating sst has a short half - life (~2 minutes) because both bioactive isoforms (sst-14 and sst-28) contain multiple enzymatic cleavage sites causing a rapid degradation . This not only makes the analysis of their physiological activity difficult but also represents a serious limitation for their application in clinical practice . Synthetic ssas have been developed by reducing the polypeptide chain, leading to an increased affinity for sstrs and to a longer half - life.9 structure activity studies of sst-14 showed that the aminoacid residues phe, trp, lys, and thr, which comprise a -turn, are necessary for biological activity . Compound sms 201 - 995 (oct) exhibits a 3-fold potency in the inhibition of insulin secretion and a 19-fold potency in gh secretion inhibition compared to native sst.14 the introduction of d - phe at the n - terminal and l - thr at the c - terminal end, and the substitution of l - trp by d - trp in position 8 make the peptide resistant to degradation . Of the many hundreds of ssas synthesized, 4 are currently used in clinical settings: oct, lan, vapreotide (vap), and pasireotide (pas; som 230) (figure 2). Oct and lan (first - generation ssas) have been quickly considered first - line medical therapeutic options for the treatment of acromegaly and for the control of hormonal symptoms in patients with nens.11 oct and lan show high - affinity binding to sstr 2 and 5, with half - lives of 2 hours and <1 hour, respectively . Rebound hypersecretion of hormones does not occur.14 both drugs have a small volume of distribution and a low clearance that result in a longer duration of exposure and long - lasting biological activity compared with sst . Oct and lan are administered by multiple subcutaneous injections or by continuous subcutaneous infusion or by the intravenous route (either as a single injection or as a continuous infusion over many hours or days). The oct lar formulation has been obtained by combining oct with microspheres of carboxymethylcellulose which increase its therapeutic action to 2442 days.15 two lar / slow - release formulations have been developed: a slow - release lan obtained by combining lan with microspheres of lactide / glycolide copolymers, which allows for administration of every 728 days,16 and lan autogel which is a viscous aqueous formulation supplied in ready - to - use prefilled syringes administered every 2856 days.17 pas (som 230), a ssa developed using biodegradable polymers by a method similar to that of oct lar,18 is a multireceptor - targeted sst generated by the introduction of 4 synthetic and 2 essential amino acids of sst in a novel cyclohexapeptide structure . The biological effects of sst are mediated by its interaction with 5 sstrs (sstr 1, sstr 2, sstr 3, sstr 4, and sstr 5) belonging to the family of g - protein - coupled membrane receptors . Each receptor, encoded by genes localized on different chromosomes,19 consists of a single polypeptide chain with 7 transmembrane - spanning domains: the extracellular domain exhibits the ligand - binding sites, while the intracellular domain provides linkage to second messenger activation.20 all 5 sstrs have been identified in the central nervous system, gastrointestinal tract, endocrine glands, exocrine glands, and inflammatory and immune cells21 (figure 3). Sst-14 and sst-28 have approximately equivalent affinity for all the receptor subtypes, except for sstr 5 which has a 10-fold higher affinity for sst-28 suggesting a potentially different role for this receptor.22 the interaction between sst and its receptor subtypes activates a number of intracellular cascades, including the inhibition of adenylate cyclase activity and the activity of calcium channels, as well as stimulation of phosphotyrosine phosphatase or mapk activity.19 while all these pathways suppress secretion processes, the activation of phosphotyrosine phosphatase or mapk by sst may play a role in the regulation of cell proliferation.23,24 in addition, sstrs may affect the activity of phospholipase c, cyclic guanosine monophosphate, and phospholipase a2 all involved in signal transduction.25 however, the current understanding of sst / sstr intracellular signaling is based on in vitro models, and its in vivo relevance remains to be elucidated . The antiproliferative mechanisms of sst are different and dependent on the sstr subtype and target cell type . They involve hyperphosphorylation of the retinoblastoma gene product and g1 cell cycle arrest, but can also be mediated by sstr 3-induced apoptosis.26 moreover, sst may exert an indirect antiproliferative effect by inhibiting the release of growth factors and trophic hormones (gh, igf1, insulin, gastrin, epidermal growth factor) both from neoplastic cells and from the surrounding tumor matrix.9 the latter mechanism also involves the antiproliferative effects on tumor angiogenesis which plays a critical role in tumor progression . Sst shows antiangiogenic properties by inhibiting the production and release of pro - angiogenic factors as well as expression of their receptors . In particular, in the pancreatic cancer cell line pc-3, sstr 2 expression correlates with expression of vegf and matrix metalloproteinase-2.27 as well as the normal cell lines in sst - target tissues, most tumors originating from these tissues express a high density of sstr . This is the case of nens deriving from the neuroendocrine cells of the gastrointestinal and bronchopulmonary systems, as well as of pituitary tumors, medulloblastomas, medullary thyroid carcinomas, and adenocarcinomas of the breast, ovary, and colon.7,28 on the other hand, poorly differentiated or undifferentiated tumors express sstr at a lower density than their corresponding well - differentiated neoplasias.28 the synthetic ssas oct, lan, and vap bind preferentially to sstr 2 and sstr 5, and with moderate affinity to sstr 3 and low affinity to sstr 1 and sstr 4;29 in contrast, the multireceptor ligand pas (som 230) binds with high affinity to subtypes sstr 1, sstr 2, sstr 3, and sstr 5.30 compared with oct, pas displays a 40-, 30-, and 5-fold higher binding affinity for sstr 5, sstr 1, and sstr 3, respectively, and a 2.5-time lower binding affinity for sstr 2 . Moreover, pas has a 106-fold higher affinity for sstr 5 in comparison with lan.29 both oct and lan have potent activity against gep - nens and inhibit cell proliferation by several mechanisms . A direct antitumor effect may result from the activation of sstrs on tumor cells leading to modulation of intracellular signaling pathways . Immunohistochemistry and autoradiography reveal that sstr proteins are highly expressed in gastrinomas, insulinomas, and carcinoid tumors, and their metastases . The majority of the tumors express sstr 2, followed by sstr 1, sstr 5, and sstr 3, while sstr 4 is expressed in a minority of cases.31 the frequency and pattern of expression of each subtype can, however, vary in different tumor types and in each patient.32 indeed, sstrs are expressed in lower density in undifferentiated gep - nens than well - differentiated tumors . The largest expression of sstr 2 on pancreatic endocrine or carcinoid tumors is the reason for the successful clinical application of oct and lan in controlling symptoms related to hormonal hypersecretion.33 furthermore, ssas may also produce an indirect antitumor effect by inhibiting mitogenic growth factors (such as igf) and by inhibiting tumor angiogenesis through interaction with sstrs on endothelial cells and monocytes . In immortalized human dermal microvascular endothelial cells, expression of vegf and vegf receptor-2 and vegf release are inhibited by sstr 1 agonists.34 human sst, isolated in 1973 and soon identified as a hypothalamic inhibitor of gh,5,6 has been subsequently found in several other tissues (central nervous system, endocrine system, and gastrointestinal tract). Sst is formed by proteolytic processing of larger precursor molecules: prepro - sst and pro - sst.7 prepro - sst is a 116-aminoacid precursor which is encoded by a single gene located on chromosome 3q28, and it is processed to pro - sst (96 amino - acids). Pro - sst undergoes tissue - specific enzymatic degradation to produce 2 bioactive proteins: the predominant, but functionally less active 14-aminoacid molecule called sst-14 and the larger and more potent 28-aminoacid form sst-28 (figure 1).8 sst isoforms have endocrine, paracrine and autocrine inhibitory effects as well as on exocrine glands . The mechanisms by which sst and ssas inhibit the neuroendocrine cells are complex and poorly understood.9 once sst binds to its cell- and tissue - specific receptors, the downstream effects elicited by the ligand receptor formation are site - specific: in the central nervous system, sst acts as a neurotransmitter, while in the hypothalamic pituitary region, it acts as a neurohormone.10 sst is also known to inhibit several cellular functions . In particular, in the digestive tract, sst reduces gastrointestinal motility, inhibits gallbladder contraction, decreases portal blood flow, and suppresses the secretion of gastrointestinal hormones (insulin, glucagon, gastrin, cholecystokinin, vasoactive intestinal peptide, and secretin)11 and that of other exocrine gastrointestinal cells (gastric acid, intestinal fluid, and pancreatic enzymes).12 moreover, it inhibits proliferation of both normal and tumor cells.13 circulating sst has a short half - life (~2 minutes) because both bioactive isoforms (sst-14 and sst-28) contain multiple enzymatic cleavage sites causing a rapid degradation . This not only makes the analysis of their physiological activity difficult but also represents a serious limitation for their application in clinical practice . Synthetic ssas have been developed by reducing the polypeptide chain, leading to an increased affinity for sstrs and to a longer half - life.9 structure activity studies of sst-14 showed that the aminoacid residues phe, trp, lys, and thr, which comprise a -turn, are necessary for biological activity . Compound sms 201 - 995 (oct) exhibits a 3-fold potency in the inhibition of insulin secretion and a 19-fold potency in gh secretion inhibition compared to native sst.14 the introduction of d - phe at the n - terminal and l - thr at the c - terminal end, and the substitution of l - trp by d - trp in position 8 make the peptide resistant to degradation . Of the many hundreds of ssas synthesized, 4 are currently used in clinical settings: oct, lan, vapreotide (vap), and pasireotide (pas; som 230) (figure 2). Oct and lan (first - generation ssas) have been quickly considered first - line medical therapeutic options for the treatment of acromegaly and for the control of hormonal symptoms in patients with nens.11 oct and lan show high - affinity binding to sstr 2 and 5, with half - lives of 2 hours and <1 hour, respectively . Rebound hypersecretion of hormones does not occur.14 both drugs have a small volume of distribution and a low clearance that result in a longer duration of exposure and long - lasting biological activity compared with sst . Oct and lan are administered by multiple subcutaneous injections or by continuous subcutaneous infusion or by the intravenous route (either as a single injection or as a continuous infusion over many hours or days). The oct lar formulation has been obtained by combining oct with microspheres of carboxymethylcellulose which increase its therapeutic action to 2442 days.15 two lar / slow - release formulations have been developed: a slow - release lan obtained by combining lan with microspheres of lactide / glycolide copolymers, which allows for administration of every 728 days,16 and lan autogel which is a viscous aqueous formulation supplied in ready - to - use prefilled syringes administered every 2856 days.17 pas (som 230), a ssa developed using biodegradable polymers by a method similar to that of oct lar,18 is a multireceptor - targeted sst generated by the introduction of 4 synthetic and 2 essential amino acids of sst in a novel cyclohexapeptide structure . The biological effects of sst are mediated by its interaction with 5 sstrs (sstr 1, sstr 2, sstr 3, sstr 4, and sstr 5) belonging to the family of g - protein - coupled membrane receptors . Each receptor, encoded by genes localized on different chromosomes,19 consists of a single polypeptide chain with 7 transmembrane - spanning domains: the extracellular domain exhibits the ligand - binding sites, while the intracellular domain provides linkage to second messenger activation.20 all 5 sstrs have been identified in the central nervous system, gastrointestinal tract, endocrine glands, exocrine glands, and inflammatory and immune cells21 (figure 3). Sst-14 and sst-28 have approximately equivalent affinity for all the receptor subtypes, except for sstr 5 which has a 10-fold higher affinity for sst-28 suggesting a potentially different role for this receptor.22 the interaction between sst and its receptor subtypes activates a number of intracellular cascades, including the inhibition of adenylate cyclase activity and the activity of calcium channels, as well as stimulation of phosphotyrosine phosphatase or mapk activity.19 while all these pathways suppress secretion processes, the activation of phosphotyrosine phosphatase or mapk by sst may play a role in the regulation of cell proliferation.23,24 in addition, sstrs may affect the activity of phospholipase c, cyclic guanosine monophosphate, and phospholipase a2 all involved in signal transduction.25 however, the current understanding of sst / sstr intracellular signaling is based on in vitro models, and its in vivo relevance remains to be elucidated . The antiproliferative mechanisms of sst are different and dependent on the sstr subtype and target cell type . They involve hyperphosphorylation of the retinoblastoma gene product and g1 cell cycle arrest, but can also be mediated by sstr 3-induced apoptosis.26 moreover, sst may exert an indirect antiproliferative effect by inhibiting the release of growth factors and trophic hormones (gh, igf1, insulin, gastrin, epidermal growth factor) both from neoplastic cells and from the surrounding tumor matrix.9 the latter mechanism also involves the antiproliferative effects on tumor angiogenesis which plays a critical role in tumor progression . Sst shows antiangiogenic properties by inhibiting the production and release of pro - angiogenic factors as well as expression of their receptors . In particular, in the pancreatic cancer cell line pc-3, sstr 2 expression correlates with expression of vegf and matrix metalloproteinase-2.27 as well as the normal cell lines in sst - target tissues, most tumors originating from these tissues express a high density of sstr . This is the case of nens deriving from the neuroendocrine cells of the gastrointestinal and bronchopulmonary systems, as well as of pituitary tumors, medulloblastomas, medullary thyroid carcinomas, and adenocarcinomas of the breast, ovary, and colon.7,28 on the other hand, poorly differentiated or undifferentiated tumors express sstr at a lower density than their corresponding well - differentiated neoplasias.28 the synthetic ssas oct, lan, and vap bind preferentially to sstr 2 and sstr 5, and with moderate affinity to sstr 3 and low affinity to sstr 1 and sstr 4;29 in contrast, the multireceptor ligand pas (som 230) binds with high affinity to subtypes sstr 1, sstr 2, sstr 3, and sstr 5.30 compared with oct, pas displays a 40-, 30-, and 5-fold higher binding affinity for sstr 5, sstr 1, and sstr 3, respectively, and a 2.5-time lower binding affinity for sstr 2 . Moreover, pas has a 106-fold higher affinity for sstr 5 in comparison with lan.29 both oct and lan have potent activity against gep - nens and inhibit cell proliferation by several mechanisms . A direct antitumor effect may result from the activation of sstrs on tumor cells leading to modulation of intracellular signaling pathways . Immunohistochemistry and autoradiography reveal that sstr proteins are highly expressed in gastrinomas, insulinomas, and carcinoid tumors, and their metastases . The majority of the tumors express sstr 2, followed by sstr 1, sstr 5, and sstr 3, while sstr 4 is expressed in a minority of cases.31 the frequency and pattern of expression of each subtype can, however, vary in different tumor types and in each patient.32 indeed, sstrs are expressed in lower density in undifferentiated gep - nens than well - differentiated tumors . The largest expression of sstr 2 on pancreatic endocrine or carcinoid tumors is the reason for the successful clinical application of oct and lan in controlling symptoms related to hormonal hypersecretion.33 furthermore, ssas may also produce an indirect antitumor effect by inhibiting mitogenic growth factors (such as igf) and by inhibiting tumor angiogenesis through interaction with sstrs on endothelial cells and monocytes . In immortalized human dermal microvascular endothelial cells, expression of vegf and vegf receptor-2 and vegf release are inhibited by sstr 1 agonists.34 literature regarding gep - nens has considerably increased during the past 2030 years, with changes in classifications, grading systems, and proposed treatments . Over the years, classification systems have aimed at classifying nens on the basis of their differentiation and grade . The grading system currently used for the classification of all gep - nens is based on the ki-67 proliferation index or mitotic count.35 grading includes site - specific tumor node metastasis staging, referring to the extent of tumor spread.36 current and previous nen classifications are reported in table 1.37,38 clinical manifestations of gep - nens are very heterogeneous and cover a wide spectrum: from remaining asymptomatic for several years to causing obstructive symptoms (such as abdominal pain, nausea, vomiting, and cholestasis), and from presence of metastases at the time of diagnosis to presenting signs and symptoms due to hormonal hypersecretion . In most cases, because of vagueness and nonspecificity of symptoms, the diagnosis is delayed (310 years on average), with an increased risk of developing metastases.39 in cases of functioning gep - nens, a specific syndrome develops due to hormonal hypersecretion . Moreover, gep - nen cells, which have neuroendocrine differentiation, express both specific neuroendocrine markers such as chromogranin a (cga)40 and synaptophysin, and less specific markers including cd56 and neuron - specific enolase (nse).41 although these biomarkers are not associated with specific syndromes, they can be monitored in both functional and nonfunctional nens in relation to disease progression and response to treatment . In particular, the use of cga is recommended in clinical practice, while the utility of serum nse as a marker of tumor aggressiveness needs to be evaluated by further studies before it can be recommended for routine monitoring . The 5-hydroxyindoleacetic acid (5-hiaa) is the main urinary metabolite of human serotonin, and its determination in 24-hour urine collection has a sensitivity of over 90% and a specificity of 90% for advanced carcinoid syndrome (cs).42 various blood serotonin assays have been proposed, but their actual accuracy has not been established and serotonin determination is not recommended in clinical practice . Serum gastrin determination is crucial in the diagnosis of gastrinoma and related syndrome (zollinger ellison syndrome). Simultaneous measurement of gastric ph is needed to rule out secondary hypergastrinemia due to other causes . For example, in achlorhydria, pernicious anemia, or atrophic gastritis, high gastrin levels are usually associated with high ph values, while elevated serum gastrin levels combined with gastric ph <2 are virtually diagnostic of zollinger the occurrence of symptomatic hypoglycemia with non - suppressed endogenous insulin levels is suspicious for insulinoma . Other specific markers include serum glucagon concentrations for the diagnosis of glucagonoma, associated with a characteristic clinical syndrome (diabetes mellitus and cutaneous manifestations, such as migratory necrolytic erythema, nail dystrophies, and stomatitis), and vasointestinal peptide (vip) for vip - secreting tumors which cause the verner morrison syndrome, characterized by watery diarrhea, hypokalemia, achlorhydria, weight loss, metabolic acidosis, hypercalcemia, glucose intolerance, and flushing . Different from the hypersecreting nens, which can present with specific endocrine syndromes, tumors which do not secrete biologically active substances may be present for years without ever displaying signs or symptoms, except for vague abdominal pain . The most important clinical manifestations of nens are related to mechanical complications (pain, obstruction, and bleeding) or to the bioactive factors secreted . The cs is characterized by signs and symptoms associated with hypersecretion of vasoactive substances by nens (serotonin, histamine, tachykinins, and prostaglandins). The symptoms of cs include cutaneous flushing (which occurs in 84% of patients), gastrointestinal hypermotility and diarrhea, heart disease, bronchial constriction, myopathy, and an abnormal increase in skin pigmentation.39 lan autogel is available at doses of 60 mg, 90 mg, or 120 mg.17 the recommended dose for the treatment of gep - nens is 120 mg administered every 4 weeks by deep subcutaneous injection allowing to reach steady - state concentrations after 45 injections . Even if the use in the geriatric population is associated with differences in pharmacokinetics, no dose adjustment is required because of the wide therapeutic window of lan . On the other hand, there is no experience with lan in the pediatric population, in which its use should be avoided . Since in preclinical studies lan has shown embryocidal effects, in the absence of adequate and well - controlled reproductive studies in humans, its use in pregnancy should be considered with particular care for the potential risk to the fetus . Since lan may cause a reduction in heart rate, patients affected by underlying cardiac conditions should have their heart rate monitored prior to starting lan . In a group of 81 patients affected by gep - nens treated with lan autogel, the incidence of heart rate <60 bpm was 23% (vs 16% in placebo group), while the incidence of episodes of heart rate <50 bpm as well as adverse event of bradycardia was 1% in each group . This finding can be explained by the activity of the bulbospinal neurons in the rostral ventrolateral medulla (rvlm), which are known to be critical for the maintenance of sympathetic vasomotor tone and normal cardiovascular reflex function . In particular, rvlm presympathetic neurons that express sstr 2a are essential for maintaining and potentially generating sympathetic vasomotor tone.43 in rats, microinjection of either sst or lan into the rvlm causes a dose - dependent sympathoinhibition, hypotension, and bradycardia that is blocked by the sstr 2 antagonist.43 preclinical and clinical pharmacological studies show that lan, such as sst and other ssas, inhibits the secretion of insulin and glucagon . Hence, patients treated with lan may experience hypoglycemia or hyperglycemia . For this reason, glucose levels should be monitored during treatment with lan, especially in diabetic patients who may require adjustments in their antihyperglycemic therapy.17 in the gastrointestinal system, lan significantly reduces the levels of pancreatic polypeptide, motilin, and gastric - inhibitory peptide, as well as postprandial gastrin secretion, without affecting secretin . Moreover, lan may reduce the intestinal absorption of drugs and may reduce gallbladder motility leading to gall stone formation.17 lan clearance is reduced by 30% in patients with moderate - to - severe hepatic impairment, but the effects of lan in patients with hepatic failure have not been studied . At the dose of 120 mg, the clearance of lan is not modified in patients with mild - to - moderate renal impairment, while patients with severe renal impairment have not been studied . Lan autogel has been approved for the long - term treatment of patients with acromegaly and for the treatment of gep - nens in order to delay disease progression in patients with g1 or a subset of g2 (equivalent to ki-67 <10%), unresectable, locally advanced or metastatic disease.17 the efficacy of lan autogel has been shown by studies demonstrating a benefit in progression - free survival (pfs), even if there is no evidence of an overall survival benefit (table 2). Moreover, lan autogel is widely recognized as effective in controlling tumor - related symptoms in the majority of patients affected by gep - nens (table 2). The rationale of using ssas as medical therapy in patients with nens is based on the expression of sstrs on the cell surfaces of the majority of these tumors . Lan and oct, the most used ssas in this context, bind with high affinity to receptor subtypes 2 and 5, inhibiting the signal - transmission pathways, causing a reduction in secretion of hormone and amine which can ameliorate tumor - related syndromes and stabilize tumor growth . Moreover, compared with the earlier formulation of ssas, newer long - acting formulations reduce the number of injections required, increasing patients compliance . Since the introduction of lan autogel in the clinical practice, several studies have compared the therapeutic equivalence between lan autogel formulation (injected every 4 weeks at a dose of 60 mg, 90 mg, or 120 mg) and lan microparticles (injected every 714 days at the dose of 30 mg, or every 1428 days at the dose of 60 mg) in gep - nens . In a phase iii randomized clinical trial, involving 46 patients who completed the study, lan autogel (120 mg/6 weeks) demonstrated the same efficacy of lan microparticles (30 mg/3 weeks) in terms of reduction of tumor markers and tumor size, offering the possibility to use a more delayed formulation of ssa.44 the efficacy, safety, and tolerability of lan autogel have been evaluated in metastatic, well - differentiated nens in an italian retrospective evaluation performed by bianchi et al.45 the study included 23 patients affected by metastatic nens, and in ~65% of cases, tumor was localized in the gastrointestinal system . Functional tumors were 43.5% . In this clinical study, lan autogel 120 mg, given once a month by deep subcutaneous injection for at least 24 months, was well tolerated and induced long - lasting responses in terms of clinical symptoms . A partial response in terms of tumor reduction was observed only in 2 out of 5 lung tumors, but not in gep - nens, which remained either stable or showed progression (table 2). Prospective evaluations investigating the antiproliferative effects of lan have showed an effective tumor stabilization and a pfs> 12 months in patients with progressive nens ineligible for surgery or chemotherapy (table 2).46 the clarinet study represents a fundamental contribution to the evaluation of efficacy of lan autogel in gep - nens (table 2). This randomized, double - blind, placebo - controlled, 96-week study assessed the effects of lan autogel 120 mg / monthly in patients with advanced, well - differentiated, nonfunctioning, sstr - positive, g1 or g2 gep - nens and documented disease - progression status . Tumors originated in the pancreas, midgut, hindgut, or unknown primary location . Compared to placebo, lan autogel at a dose of 120 mg / monthly was associated with significant increase in pfs and a 53% reduction in the risk of tumor progression.47 quality of life did not differ significantly between treatment groups . Moreover, in patients with baseline levels of cga above the upper limit of the normal range, lan autogel induced a significantly greater reduction compared to placebo . The overall incidence of adverse events was similar between lan autogel and placebo (~90%), but half the patients treated with lan autogel experienced drug - related adverse events (diarrhea, hyperglycemia, or cholelithiasis). The decrease of cga levels during treatment with lan autogel from baseline was associated with a significant reduction of the hazard of disease progression, confirming the utility of this marker in the clinical follow - up of patients with nens.48 recent data from the open - label extension of the clarinet study confirm that lan autogel maintains favorable risk / benefit profile, providing new evidence of the lan antitumor benefits in indolent and progressive gep - nens (table 2).49 the ki-67 proliferation index represents a good marker in predicting tumor response to ssas treatment (table 2). In patients with well - differentiated gep - nens, a ki-67 proliferation index of up to 5%, stable disease prior to treatment, and low - to - moderate hepatic tumor involvement (25%) have been associated with tumor control during lan autogel treatment.50 the antiproliferative effects of long - acting ssas according to ki-67 index have been recently evaluated by an italian multicenter observational study using both oct lar 30 mg/28 days and lan 120 mg/28 days.51 objective response and tumor stability were not significantly different between g1 and g2 nens, and between locoregional disease and distant metastases . Interestingly, the clinical benefit (improvement of symptoms) was significantly greater in patients with locoregional disease than in those with distant metastases as well as in patients with gep - nens than those with other primary tumors . Although pfs was longer in g1 than g2 nens, the difference was not significant . However, in the subgroup of nens with ki-67 <5%, the pfs was significantly longer compared to nens with ki-67 5%, consistent with previous data.46 furthermore, while pfs was not different between gep and thoracic nens, it was longer in gep and thoracic nens compared to those with unknown primary tumors.51 some functional nens release peptides and amines which produce a characteristic set of symptoms of the cs . This syndrome occurs in ~10% of patients with metastatic nens, and it is most prevalent in those with nens of the small intestine (~20%). It is caused by the release of serotonin, which is no longer metabolized in the liver, and other substances, such as tachykinins, prostaglandins, and bradykinins.52 the predominant signs and symptoms of cs are flushing (90%), diarrhea (70%), and abdominal pain (40%); less frequent events are lacrimation, profuse sweating, telangiectasias, cardiac fibrosis, and cutaneous pellagra - like manifestations due to lack of niacin.39 these can be very distressing for patients and have a negative impact on their quality of life . Ssas are currently considered the standard of care for symptom control in cs, demonstrating a significant reduction of flushing and diarrhea since the first day of treatment . Moreover, a reduction of cga and 5-hiaa can be detected (table 2).53 a recent 16-week, randomized, double - blind, phase iii trial involving 115 patients evaluated the response of cs symptoms to treatment with lan autogel 120 mg/4 weeks . In this study, patients had access to short - acting oct as rescue medication . Results showed that the proportion of patients requiring oct rescue was significantly lower in the lan - treated group than in the placebo (table 2).54 satisfaction with diarrhea control and flushing control and a good impact on the quality of life have also been reported in an open - label observational study (table 2).55 moreover, the once - monthly dosing regimen of lan is associated with improved patient adherence and patient perception of lan injection.56,57 although in the majority of patients with metastatic carcinoids and pancreatic endocrine tumors treatment with ssas induces a rapid improvement of clinical symptomatology related to hormonal hypersecretion, most patients can develop desensitization within weeks to months.58,59 the potential mechanisms responsible for this phenomenon, as well as for the considerable variability in the duration of the responses to medical therapy, are not known at present . Potential mechanisms of resistance to ssas therapy in patients with sst - positive tumors are the possibility of receptor downregulation as well as reduction in the number and/or affinity of sstrs . Moreover, a decrease in responsiveness due to receptor uncoupling from second messenger activation can determine desensitization . Other potential causes could be a non - homogeneous expression of sstrs in tumors, outgrowth of sst - negative cell clones, absence of sstr subtypes with high affinity for ssas, tachyphylaxis of the inhibitory effect of ssas on indirect tumor growth - promoting mechanisms, and mutations in sstr genes leading to the absence of functional receptor proteins.7 to date, ssas represent the main symptomatic therapeutic approach for the management of nens . In this context, there are no differences between lan microparticulate and lan autogel in their ability to control tumor growth and tumor hypersecretion . Their long - acting formulation, which allows up to once - monthly administration regimens, increases the compliance of patients with nens . Although several studies have been conducted since the approval of lan autogel, their conclusions are often difficult to compare due to differences in study design, eligibility criteria, and study end points . Nonetheless, the majority of the studies indicate that the effects of lan autogel are mainly on improving symptoms and stabilization of the disease progress, and even if a benefit in pfs has been demonstrated, there is no evidence of an overall survival benefit . This may partly be explained by the fact that nens are frequently tumors showing slow progression and an indolent behavior . Therefore, more data must be derived by the extension of studies that have already been published . In addition, the molecular bases of tumor response to ssas are still unclear, although the application of proteomics to this field has led to promising results.60 finally, the ongoing drug research has shown that lan above a critical assembly concentration of 20 mm spontaneously forms hollow nanotubes when solubilized in pure water.61,62 moreover, a recent study evaluated the utility of a self nano - emulsifying delivery system for model peptide lan which provided a protective effect toward thiol disulfide exchange reactions and can be useful to overcome sulfhydryl barrier of the gastrointestinal tract.63 therefore, lan is an interesting self - assembly model that can provide insights on how these mechanisms can be controlled, and applied to nanotechnology and drug delivery.64
The review and meta - analysis focusing on optimal glycemic control in neurocritical care (ncc) patients by kramer and colleagues in this issue of critical care provide a contemporary and comprehensive overview of randomized controlled trials (rcts) that apply different glycemic control strategies in this challenging intensive care unit (icu) population . Optimal glycemic control in critical care (cc) patients, in general, and ncc patients, in particular, has evolved dramatically over the past 15 years and remains under active investigation and debate about the ideal target range and impact of dysglycemia (high, low, and variable glucose levels) on outcome in the heterogeneous icu population . Prior to 2001, clinicians frequently applied a somewhat' permissive' glycemic control approach in cc and ncc patients since hyperglycemia was considered a physiological response to stress or insult . Starting about a decade ago, the target range for glycemic control was the achievement of euglycemia (80 to 110 mg / dl) by using intense insulin therapy as an infusion in the hopes of reducing icu and hospital morbidity and mortality . Subsequently, the latter approach was shown to result in an increased, but varying, incidence of hypoglycemia . The incidence appears to vary by study, patient population, glucose goal, and intensity of insulin infused . However, hypoglycemia develops also in some critically ill patients in the absence of infused insulin . Whether there is an unfavorable cause - and - effect impact on outcome in critically ill patients who become hypoglycemic remains under scrutiny . Currently, a key issue with tight glycemic control (target range of 80 to 110 mg / dl) achieved by using intensive insulin therapy in ncc patients is the report of higher rates of hypoglycemia and lack of benefit on outcome [5 - 7]. Kramer and colleagues confirm these data but provide solid evidence about the relationship between hyperglycemia and worsened neurological outcome after acute brain damage . In their review and meta - analysis, the primary outcomes were mortality at 6 months and level of neurological recovery / function (according to the quantitative functional scale used by the individual study). Secondary outcomes included hypoglycemia (defined by different thresholds ranging from 40 to 80 mg / dl), nosocomial pneumonia, and other nosocomial infections . Articles to be retrieved and reviewed were searched by using three criteria: intensive glycemic control, ncc, and clinical trials (only rcts were considered eligible). In total, 23 studies were analyzed . Primary pooled outcomes were extracted from 16 studies that involved 1,248 patients . Reported mortality was similar in patients treated with intensive and conventional glycemic targets (26% versus 27%, relative risk (rr) 0.99, 95% confidence interval (ci) 0.83 to 1.17, p = 0.89). However, poor neurological outcome was less frequent in patients who received intensive glycemic management (58% versus 68%, rr 0.91, hypoglycemia was markedly greater among treated patients (30% versus 14%, rr 3.10, 95% ci 1.54 to 6.23, p = 0.002). No effects were detected in the incidence of nosocomial pneumonia, and other nosocomial infections were infrequently reported, and thus a conclusive statistical analysis was not possible . The results of this review and meta - analysis confirm that tight blood glucose control in ncc patients does not reduce mortality but increases the rate of hypoglycemia . The new message from this review it that, in patients treated with an' active' glucose control strategy with insulin infusion to maintain a blood glucose concentration (bgc) of less than 180 mg / dl, in comparison with those in whom' loose' glycemia control was allowed (bgc of greater than 200 mg / dl before the start of insulin infusion), there is an improvement in neurological outcome . Of relevance, the authors reported that information on nutritional status and nutritional support was reported poorly in the original articles but that' in most of the cases, tube feeding appeared to have been initiated as soon as possible' . In conclusion, optimal glucose control in ncc patients should include an active therapeutic strategy . Tight blood glucose control (with bgc target range of 80 to 110 mg / dl) exposes ncc patients to an increased risk of iatrogenic hypoglycemia . Patients with hypoglycemia (bgc values of less than 80 mg / dl) have increased mortality and the potential for worsened long - term functional status . On the other hand, patients with acute brain damage and permissive hyperglycemia with bgc values of greater than 180 mg / dl have a worsened neurological outcome . During the last decade, it is now clear that this physiological variable cannot be overlooked and deserves committed clinical attention that is based on what we have learned . We now know that the time frame for manipulation of bgc is not as strict as that for hemodynamic variables and that sudden changes in bgc are potentially as dangerous as extreme bgc values . We know that intense insulin infusion used to control glucose must be monitored appropriately and administered along with adequate enteral or parenteral nutrition and that iatrogenic hypoglycemia must be avoided . We also know that currently available clinical experience and technology are often derived from chronic management of patients with diabetes mellitus but that the unique characteristics of cc and ncc patients require new understandings and applications of technology . This requires more information on the impact of glucose homeostasis and control in specific ncc patient subgroups: traumatic brain injury, ischemic / hemorrhagic stroke, neurooncologic pathology, subarachnoid hemorrhage, and brain injury severity [9 - 11]. Of note is the study by schlenk and colleagues, who used brain microdialysis in patients with subarachnoid hemorrhage to demonstrate the association of a target bgc of less than 110 mg / dl with increased acute brain metabolic derangements . Mathematical models to describe and tailor an individual patient's glucose sensitivity, the changes in glucose sensitivity over time and the relationship between changes in insulin sensitivity and the insulin / nutrition infusion protocol (amount of calories, access used for nutrition, amount and timing of insulin infusion) used in the individual patient . 3 . Dedicated engineering technology, including continuous glucose monitoring devices with the application of closed loop insulin / nutrition infusion systems [13 - 15]. Greater understanding of the relationship between peripherally measured glucose, glucose in the healthy brain, and glucose in the injured or ischemic brain . In accordance with the conclusions of kramer and colleagues, we recommend that insulin infusion for glucose control in ncc patients be aimed at a' moderate' target range (110 to 180 mg / dl). In addition, we would recommend an adequate nutrition support of ncc patients before and during insulin infusion, the avoidance of insulin boluses, and the use of continuous insulin infusions initially at low dose, titrated to individual sensitivity with the application of a standardized and easily applied glycemic monitoring protocol . Bgc: blood glucose concentration; cc: critical care; ci: confidence interval; icu: intensive care unit; ncc: neurocritical care; rct: randomized controlled trial; rr: relative risk.
The pelvis is an important body part that connects the trunk and the lower limbs, transferring the movement of the lower limbs to the trunk1 . The pelvis tilts anteriorly and posteriorly under the action of muscles during flexion and extension of the hip joint2 . Two major muscles that cause anterior and posterior tilting of the pelvis are the rectus abdominis and the rectus femoris . These two muscles secure the stability of the pelvis through coordination during hip joint flexion1 . For this reason, leg raises in the supine position are used as a trunk stabilization exercise to prevent unnecessary pelvic movements3 . Therefore, to increase activation of deep trunk muscles, various leg raising exercises are being studied by researchers6 . However, the relationship between muscles that directly interfere with anterior and posterior tilting of the pelvis to stabilize the position of the pelvis remains unclear at different angles during leg raises . Therefore, this study investigated the characteristics of the rectus abdominis and rectus femoris muscle that directly interfere with the anterior and posterior tilting of the pelvis during leg raises to clarify the relationship between leg and pelvic movement . The subjects of this study were 20 helthy adults (10 males and 10 females). When selecting the participants, those with present or past malformation or neurological disease of the leg joints were excluded . All the subjects voluntarily participated in this study and signed an informed consent from, which stated that they could withdraw from the experiment at anytime of their choosing . Moreover, this study complied with the ethical principles of the declaration of helsinki . The average age, height, and weight of the participants were 21.551.43 years old, 166.759.30 cm, and 57.710.63 kg, respectively . In this study, usa) was used to measure muscle activation . For emg signal processing, the myoresearch xp master edition 1.06 the sampling rate of emg signals was set to 1,024 hz, and data were filtered with a 20 to 500 hz bandpass filter and a 60 hz notch filter . The electrodes (iwc - dts, 9113a - dts) were ag / agcl electrodes with adhesive backing including hypoallergenic gel . The diameter of the conducting area was 1 cm and the distance between the electrodes was 2 cm . The electrode attachment sites were depilated with a razor, and horny substance was removed with sandpaper . The activities of each muscle were measured for five seconds in the anatomical position and converted to root mean square value . The level of muscle activation during exercise was expressed as% rvc, relative to the average muscle contraction (100%) in the middle one second of a three - second measurement; i.e. Ignoring the first and last second of the measurement . For precision of the raise, an arch with a radius adjusted to the leg length of each participant was created using a plastic rope, and the subjects raised their legs following the plastic arch from 0 to 60 of hip flexion . The measured data were analyzed using one - way anova to investigate the effect of leg raise angle on muscle activity . 17.0, and p values less than 0.05 were considered significant for all cases . Statistically significant differences in the muscle activities of the rectus femoris and rectus abdominis were found among each division of the leg raise (p <0.05) (table 1table 1.comparison of muscle activation and ratio in each division of the leg raise015 division1530 division3045 division4560 divisionrf * (% rvc)1,361.8175.84,988.0596.86,031.9617.25,663.0566.8ra * (% rvc)192.340.1567.1120.2789.8174.61,159.1268.6ratio of ra / rf * (%) 13.15.612.33.813.13.420.45.7rf: rectus femoris; ra: rectus abdominis; ratio of ra / rf: ra / rf100 . The values with different superscripts (,) in the same column are significantly different (p<0.05) according to tukey s test . ). The ratio of the rectus abdominis and rectus femoris increased as the angle of hip flexion increased (p <0.05) (table 1). Rf: rectus femoris; ra: rectus abdominis; ratio of ra / rf: ra / rf100 . The values with different superscripts (,) in the same column are significantly different (p<0.05) according to tukey s test . This study was conducted to determine the characteristics of the muscles around the pelvis during the performance of leg raises . It was found that rectus abdominis activity increased more sharply than rectus femoris activity 4560 of a leg raise . This seems to be the result of posterior tilting of the pelvis to secure space between the pelvis and the femur for hip flexion during leg raises . Thus, the rectus femoris is used more during 045 hip flexion, to induce anterior tilting and hip flexion of the pelvis, whereas rectus abdominis activation is used for hip flexion at 45 and greater, to induce hip flexion accompanied by posterior tilting of the pelvis . Therefore, lumbo - pelvic rhythm through the motion of the lower limbs starts to occur when the hip joint is in flexion of 45 or greater . This result supports previous studies, which have reported that movements of the femur and pelvis are related to the two joint muscles . Since the leg raise of the present study was performed by healthy adults without hamstring muscle shortening, it explains normal kinematics, not the influence of hamstring muscle tension . In terms of kinematics, it was shown that raising the leg to greater than or equal to 45 induces pelvic motion in normal adults . Patients with spinal disc herniation feel pain when raising the leg to greater than or equal to 45 because force is transferred to the lumbar spine . Researchers suspect a herniated intervertebral disc when the angle is greater than or equal to 60 during the straight leg raise test7, 8 . The results of the present study confirm the results of research about the positive range of the existing slr test . It seems necessary to conduct further studies about the motions of the femur, pelvis, and lumbar spine to clarify the rhythms that occur in the human body.
Oral submucous fibrosis (osmf) is a chronic debilitating disease and a potentially malignant disorder of the oral cavity . The incidence of oral squamous cell carcinoma has been estimated to be 7.6% in osmf patients after a follow up period of 17 years . Areca - associated oral squamous cell carcinoma (oscc) is the 3 most common malignancy in developing countries . Osmf is associated exclusively with areca chewing and is believed to be a homeostatic disorder of extracellular matrix (ecm). Epidemiological evidences strongly indicate the association of the betel quid (bq) habit and osmf . The areca nut (betel nut) component of bq, especially an alkaloid called arecoline, plays a major role in the pathogenesis of osmf by causing an abnormal increase in the collagen production . The synthesis of collagens is influenced by a variety of mediators, prominent mediator being transforming growth factor - beta (tgf-). Tgf-1, in particular, seems to be the one that plays a major role in wound repair and fibrosis . It causes the deposition of extra cellular matrix (ecm) by increasing the synthesis of matrix proteins like collagen and decreasing its degradation by stimulating various inhibitory mechanisms . Tgf- has been found to strongly promote the expression of lox both at the mrna and protein levels in various cell lines . Areca nuts have been shown to have a high copper content and chewing areca nuts for 5 - 30 min significantly increases soluble copper levels in oral fluids . This increased level of soluble copper could act as an important factor in osmf by stimulating fibrogenesis through upregulation of lox activity . Previous studies have specified the upregulation of lox expression in normal oral keratinocytes by areca nut extract and its oncogenesis for oscc . There are seven single nucleotide polymorphism (snp) sites in lox coding region, including c225 g, g409c, g473a, c476a, g816a, t924 g and a1135 g . Among all snp sites in lox coding region, it causes a change of arg at residue 158 to gln (lox arg158gln). The present study investigated the presence of lox g473a polymorphism in group 1 (osmf patients), group 2 (betel quid chewers without osmf) and group 3 (healthy individuals). The study was conducted in department of oral medicine and radiology, from january 2011-june 2012 . The subjects for the study were selected by employing convenience sampling method reporting to the department of oral medicine and radiology . A total of 60 patients were taken for the study, which was divided as follows: group 1 - 20 osmf patients (clinically and histopathologically diagnosed) with betel quid chewing habit, group 2 - 20 betel quid chewers without osmf [table 1] and group 3 - 20 healthy individuals without osmf and betel quid chewing habit . Distribution of study samples based on duration of betel quid chewing habit patients with clinically and histopathologically diagnosed osmfpatients with betel quid chewing habit without clinical evidence of osmf who were undergoing 3 molar surgeryhealthy individuals without betel quid chewing habit and having normal oral mucosa who were undergoing 3 molar surgeryabove patients who are willing to participate in the study . Patients with clinically and histopathologically diagnosed osmf patients with betel quid chewing habit without clinical evidence of osmf who were undergoing 3 molar surgery healthy individuals without betel quid chewing habit and having normal oral mucosa who were undergoing 3 molar surgery above patients who are willing to participate in the study . Patients on regular analgesics or anti - inflammatory drugs, which are known to interfere with lysyl oxidase activitypatient who already received or on treatment for osmf . Patients on regular analgesics or anti - inflammatory drugs, which are known to interfere with lysyl oxidase activity patient who already received or on treatment for osmf . A detailed case history was taken with an emphasis on the information regarding the habits like betel quid, tobacco chewing, smoking and alcohol consumption . The mean duration of betel quid chewing habit in group 1, group 2 and group 3 was 8.55 years, 8.45 years and 8.50 years, respectively [table 2]. In group 1, 13 patients had the habit of chewing betel quid 1 - 10 times / day, 6 patients had the habit of chewing betel quid 11 - 15 times / day and 1 patient had the habit of chewing betel quid more than 15 times / day . In group 2, 14 patients had the habit of chewing betel quid 1 - 10 times / day, 6 patients had the habit of chewing betel quid 11 - 15 times / day . Distribution of study samples based on frequency of betel quid chewing habit per day the study protocol was explained and written informed consent was obtained from all the patients . A wedge biopsy was performed on the patients with clinical suspicion of osmf and the tissue was divided into 2 halves (one for the purpose of histopathological diagnosis and the other to assess lox polymorphism). For group 2 and group 3, samples were obtained from patients undergoing 3 molar extraction for the analysis of lox gene polymorphism . Amplicons containing the lox gene were obtained by polymerase chain reaction (pcr) reaction with primers: f-5-cactggttccaagctggcta-3, r-5-ggaagtagccagtgccgtat-3. Genotyping was performed using pcr - based restriction fragment length polymorphism (pcr - rflp) analysis . 20 lt of dna was loaded along with this 100 bp ladder was loaded and allowed it to run for 30 mins, the bands formed were observed under gel documentation system . The pcr amplicons were subjected to dna sequencing using abi - prlsm dye terminator at eurofins genomics india pvt ltd bangalore . Patients with clinically and histopathologically diagnosed osmfpatients with betel quid chewing habit without clinical evidence of osmf who were undergoing 3 molar surgeryhealthy individuals without betel quid chewing habit and having normal oral mucosa who were undergoing 3 molar surgeryabove patients who are willing to participate in the study . Patients with clinically and histopathologically diagnosed osmf patients with betel quid chewing habit without clinical evidence of osmf who were undergoing 3 molar surgery healthy individuals without betel quid chewing habit and having normal oral mucosa who were undergoing 3 molar surgery above patients who are willing to participate in the study . Patients on regular analgesics or anti - inflammatory drugs, which are known to interfere with lysyl oxidase activitypatient who already received or on treatment for osmf . Patients on regular analgesics or anti - inflammatory drugs, which are known to interfere with lysyl oxidase activity patient who already received or on treatment for osmf . A detailed case history was taken with an emphasis on the information regarding the habits like betel quid, tobacco chewing, smoking and alcohol consumption . Detailed clinical examination was performed on all the patients . The mean duration of betel quid chewing habit in group 1, group 2 and group 3 was 8.55 years, 8.45 years and 8.50 years, respectively [table 2]. In group 1, 13 patients had the habit of chewing betel quid 1 - 10 times / day, 6 patients had the habit of chewing betel quid 11 - 15 times / day and 1 patient had the habit of chewing betel quid more than 15 times / day . In group 2, 14 patients had the habit of chewing betel quid 1 - 10 times / day, 6 patients had the habit of chewing betel quid 11 - 15 times / day . Distribution of study samples based on frequency of betel quid chewing habit per day the study protocol was explained and written informed consent was obtained from all the patients . A wedge biopsy was performed on the patients with clinical suspicion of osmf and the tissue was divided into 2 halves (one for the purpose of histopathological diagnosis and the other to assess lox polymorphism). For group 2 and group 3, samples were obtained from patients undergoing 3 molar extraction for the analysis of lox gene polymorphism . Amplicons containing the lox gene were obtained by polymerase chain reaction (pcr) reaction with primers: f-5-cactggttccaagctggcta-3, r-5-ggaagtagccagtgccgtat-3. Genotyping was performed using pcr - based restriction fragment length polymorphism (pcr - rflp) analysis . 20 lt of dna was loaded along with this 100 bp ladder was loaded and allowed it to run for 30 mins, the bands formed were observed under gel documentation system . The pcr amplicons were subjected to dna sequencing using abi - prlsm dye terminator at eurofins genomics india pvt ltd bangalore . The isolated dna was made to run in an agarose gel electrophoresis for 30 min . The bands were observed in the gel documentation system [figure 1]. Ratio of a260/a280 more than 2 ng/l were treated with proteinase k and subjected to pcr basis . Further, the amplified pcr products were subjected to automated dna sequencer to assess lox g473a polymorphism . Dna sequencing results did not show lox g473a polymorphism in any of the 3 groups [figure 2]. Gel electrophoresis showing dna bands electrophoregram showing ctgcgg instead of ctgcag (box) indicating absence of g to a polymorphism the present study aimed at investigating the presence of lox g473a polymorphism in group 1 (osmf patients), group 2 (betel quid chewers without osmf) and group 3 (healthy individuals). According to the current literature, only one study has been reported to find the association between lox g473a polymorphisms and osmf which was conducted on taiwan population . The gene sequencing results did not show lox g473a polymorphism in any of the 3 groups . Despite the implication of lox in many diseases including inflammation and inflammatory diseases; fibrosis of distinct organs and fibrotic disorders; and cancer promotion and progression, there are only sparse reports of any mutations or epigenetic alterations in the lox gene . Shieh et al ., (2009) conducted a study on taiwan population to find the association between lox gene polymorphism and osmf in older male areca chewers . The presence of g to a polymorphism at nucleotide at 473, caused a non - conservative arg158gln change in the lox amino acid sequence . There was a borderline statistical significant difference in arg158gln genotype between control and osmf patients . However, the g / a + a / a of lox arg158gln in osmf patients older than 50 years was statistically significantly higher than controls below 50 years . In the present study, most of the patients were less than 40 years old, which could be one of the reasons for absence of g to a polymorphism at nucleotide at 473 . In study conducted by shieh et al.,(2009) on taiwan population, the mean duration of areca chewing habit in patients less than 50 years and more than 50 years was 15 years and 21 years, respectively . In the present study, the mean duration of areca chewing habit in group 1 (osmf) was 8.5 years, which could also be one of the reasons for absence of g to a polymorphism at nucleotide at 473 . Shieh et al ., (2007) identified an upregulation of lox and loxl2 mrna expression in areca - associated oscc tissues and cell lines relative to their normal counterparts . Other previous investigations that focused mostly on breast carcinomas, identified that lox can induce migration and invasion of malignant breast epithelial cells . Shieh et al ., (2009) in his study included sample size of 83 osmf patients and 216 areca chewers without osmf as control . In the present study, the absence of lox gene polymorphism in the present study could also be attributed to the smaller sample size . Another possible explanation for the variation in the lox gene polymorphism between taiwan population and indian population could be the difference in genetic makeup . Lox is a cuproenzyme that is essential for stabilization of extracellular matrixes, specifically the enzymatic cross - linking of collagen and elastin . Hence local factors such as composition of the quid, consistency of quid, duration and frequency of habit and whether saliva is expelled or swallowed after chewing, can affect the uptake of copper into the oral epithelium . These factors may explain the marked variations seen in the copper levels within the osmf group which in turn may affect the regulation of lox gene and its polymorphism in osmf patients . Lox coding region contains seven single nucleotide polymorphism (snp) sites that includes c225 g, g409c, g473a, c476a, g816a, t924 g and a1135 g . Among all the snp sites in lox coding region although lox is implicated in many fibrotic disorders, there are only sparse reports of any mutations or epigenetic alterations in the lox gene . The study showed that only elder osmf patients had significant lox gene polymorphism . In our study, lox gene polymorphism was not observed in osmf patients which could be attributed to the difference in the genetic makeup between taiwan and indian population and on the factors like sample size, frequency and duration of habit, composition and consistency of the quid, which can affect the uptake of copper into the oral epithelium in turn affecting the regulation of lox gene and its polymorphism . Hence, further studies are required to assess the presence of lox gene polymorphism in osmf.
Immunoglobulin a (iga) nephropathy is characterized by mesangial iga deposits and expansion of glomerular mesangial matrix with proliferation of mesangial cells . Its clinical manifestation is extremely variable, ranging from asymptomatic microscopic hematuria to rapidly progressive renal failure . It is considered as the most common primary glomerulonephritis worldwide and 36% of adult patients with iga nephropathy (igan) progress to end - stage renal failure within 20 years in china . However, the pathogenesis of igan is obscure and its current therapy remains unsatisfactory . Activation of inflammatory and immune system is one of the important causal factors in the pathogenesis of chronic renal disease . It is becoming clear that toll - like receptors (tlrs) are directly involved in the pathogenesis of chronic kidney diseases related to inflammatory responses . Tlrs are evolutionarily conserved receptors that bind to pathogens and initiate inflammatory responses which might trigger renal injuries . In humans, tlrs are expressed on a wide range of cell types including renal mesangial cells and tubular epithelial cells . Among the eleven human tlrs, tlr4 has been demonstrated to be related to mesangial cell injury and renal fibrosis by induction of proinflammatory cytokines, profibrotic molecules, and transforming growth factor-1 in chronic kidney diseases, including igan, renal ischemia - reperfusion injury, acute kidney injury, and transplantation rejection . In vitro experiments have confirmed that tlr4 expression is increased in circulating mononuclear cells of patients with igan and tlr4 signaling is implicated in the activation of iga - stimulated mesangial cells . However, at present, there are few in vivo researches concerning tlr4 and igan . Coppo et al . Revealed that the upregulation of tlr4 in circulating mononuclear cells of patients with igan was associated with heavy microscopic hematuria and proteinuria . He et al . Observed that the expression of tlr4 message ribonucleic acid (mrna) and protein in renal tissue was significantly increased in igan rats established by oral and intravenous immunization with bovine serum albumin (bsa) for 12 weeks . Although these researches mentioned above suggested that the expression of tlr4 was increased in the serum or kidney of patients or rats with igan, they could not demonstrate what role tlr4 might play in the progression of igan . Peroxisome proliferator - activated receptor- (ppar-) is a member of the nuclear hormone receptor family which expresses in many tissues including the kidney . In addition to regulating the metabolism of glucose and lipid, ppar- also exerts anti - inflammatory effects on kidney diseases including igan . Our groups and others have noted that thiazolidinediones, a ppar- agonist, could attenuate inflammatory responses through the angiotensin ii signaling pathway by suppressing the activation of angiotensin receptor 1, extracellular regulated protein kinases, and nuclear factor - kappa b (nf-b) in the in vitro experiments . As it has been proved, nf-b is the key factor of the downstream of tlr4 signaling pathway . It has been reported that pioglitazone exerted its anti - inflammatory effect through suppression of the expression and activity of tlr4 in many in vitro experiments of other disease models . So far, the interaction between ppar- and tlr4 and the plausible mechanism in igan have not been fully studied . The current study was conducted to assess whether tlr4 signaling pathway was involved in the pathogenesis and progression of igan in vivo . We hypothesized that the ppar- agonist exerted its role on alleviating the severity of igan by interfering with tlr4-dependent signaling pathway and impressed the increase of inflammatory mediators . With this aim, we evaluated the renal expression of inflammatory cytokine interleukin-1 beta (il-1), and tlr4 mrna and protein in rats before and after the establishment of igan and the changes of these factors after treated with tlr4 inhibitor, toll - like receptor 4 inhibitor (tak242). Moreover, we tested the alterations of renal tlr4 mrna and protein in rats after gavaged by pioglitazone with a focus on the interactive roles between tlr4 and ppar- and how this interaction on the severity of experimental igan . Bovine gamma globulin (bgg) was purchased from sigma chemical co. (st . Louis, mo, usa). Antibodies were rabbit anti - rat tlr4, rabbit anti - rat il-1 (all from santa cruz biotechnology, santa cruz, ca, usa), and goat anti - rat iga (lifespan biosciences, seattle, ca, usa). All procedures performed in studies involving animals were in accordance with the ethical standards of the ethics committee of huadong hospital for animal studies and the national institute of health guidelines for the care and use of laboratory animals . A total of 54 male, 3-week - old lewis rats weighing 45 5 g were purchased from weitonglihua animal technical co., ltd ., in beijing, china, and housed in an specific pathogen - free room at the animal experimental center of fudan university with a room temperature of 25c and a 12-h light - dark cycle . The animals were provided standard rodent chow and water to drink ad libitum for 1 week before the initiation of the study . Because of the different treating time of tak242 and pioglitazone, the animals were sacrificed at different time intervals . Hence, 54 lewis rats were randomly divided into six groups with the method of simple randomization: controltak242, igantak242, tak242, controlpio, iganpio, and pio groups . Experimental igan was induced in 4-week - old lewis rats by continuous oral immunization with 0.1% bgg in 6 mmol / l hcl as drinking water for 9 weeks, followed by intravenous injection of 1 mg bgg (dissolved in 0.3 ml 6 mmol / l hcl) into the tail vein daily for 3 successive days . Tak242, dissolved in 20% lipovenoes at 3 mgkgd (7.5 ml / kg), was injected into rats with igan for 8 days after completing the model - creation (tak242 group). Experimental igan rats receiving intravenous injection of the same amount of lipovenoes for 8 days by tail veins were named igantak242 group . Controltak242 group was normal rats who drank 6 mmol / l hcl for 9 weeks, and then injected with 1 mg 6 mmol / l hcl into the tail vein daily for three successive days followed by injection with 20% lipovenoes at 7.5 mlkgd for 8 days . Pioglitazone, dissolved in saline at 1 mg / ml, was administered to rats after the model - creation by intragastric injection at 10 mgkgd for 4 weeks until the rats were killed (pio group). Rats of experimental igan receiving intragastric injection of saline at 10 mlkgd for 4 weeks were named iganpio group and controlpio group referred to normal rats who drank 6 mmol / l hcl for 9 weeks, and then were given intravenous injection of 1 mg 6 mmol / l hcl into the tail vein daily for three successive days followed by intragastric injection of saline at 10 mlkgd for 4 weeks . Albumin - to - creatinine ratio (acr) was used to express the degree of albuminuria . The urine was centrifuged at 600 revolutions per minute for 5 min and the sediment was examined microscopically for erythrocytes . A diagnostic criterion for hematuria was more than 10 red cells on average per high - power field . Serum creatinine (scr) and blood urea nitrogen (bun) were measured at the clinical laboratory at huadong hospital affiliated to fudan university . Blood samples were drawn through the abdominal aorta after intraperitoneal anesthesia with 2% pentobarbital sodium to detect serum il-1 by enzyme - linked immunosorbent assay kits (multisciences biotech co., ltd ., the cortex from half of the kidney was fixed in 4% paraformaldehyde and paraffin - embedded for histological observation . The other half was snap frozen in liquid nitrogen and stored at 80c for total rna and protein extraction . Three - micrometer thick paraffin - embedded kidney sections were deparaffinized with xylene and then rehydrated through a descending gradient of ethanol . The sections were stained with h and e. glomerular damage and mesangial hypercellularity were determined by examining 25 glomeruli in each sample . Immunofluorescence examination of the paraffin - embedded renal sections was performed by staining with fluorescing isothiocyanate - labeled, specific antibodies against rat iga (1:16) at 4c overnight . The anti - rat iga antibody was visualized as bright green color using the dako envision plus system (dako, carpinteria, ca, usa). The nephrologist who examined the specimens was unaware of the group assignments of the individual animals . Total rna was extracted from renal tissues using the trizol reagent kit (life technologies, usa) according to the method described in the manufacturer's protocol . Thereafter, 1 ng of total rna was reverse transcribed using the primescript reverse transcription kit and random primers (takara biotechnology co., ltd ., real - time polymerase chain reaction amplification was carried out in an abi prism 7500 sequence detection system . Primer sequences for rat tlr4 are forward 5-tgacagacctcaggcagattgt-3, reverse 5-aatagtgcaatcgatagaaggaaca-3; il-1 forward 5-ccgtggcacattctggtca-3, reverse 5-gctgtgcactggtccaaattc-3; and -actin forward 5-agattactgccctggctcctag-3, reverse 5-catcgtactcctgcttgctga-3. -actin was used as an endogenous control . Protein extracted from the renal tissues was analyzed by western blotting for tlr4 and il-1 in the following manners . After homogenization, the solution was centrifuged at 10,800 g at 4c for 5 min . Protein was separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane . The membrane was incubated with bsa, and then with following primary antibodies including rabbit antibody to il-1 and tlr4 . Detection was used by enhanced chemiluminescence, and the light signals were exposed to x - ray film . Data were analyzed using spss software version 20 (ibm, new york, usa). Differences between groups were analyzed by one - way analysis of variances (anova). Bovine gamma globulin (bgg) was purchased from sigma chemical co. (st . Louis, mo, usa). Antibodies were rabbit anti - rat tlr4, rabbit anti - rat il-1 (all from santa cruz biotechnology, santa cruz, ca, usa), and goat anti - rat iga (lifespan biosciences, seattle, ca, usa). All procedures performed in studies involving animals were in accordance with the ethical standards of the ethics committee of huadong hospital for animal studies and the national institute of health guidelines for the care and use of laboratory animals . A total of 54 male, 3-week - old lewis rats weighing 45 5 g were purchased from weitonglihua animal technical co., ltd ., in beijing, china, and housed in an specific pathogen - free room at the animal experimental center of fudan university with a room temperature of 25c and a 12-h light - dark cycle . The animals were provided standard rodent chow and water to drink ad libitum for 1 week before the initiation of the study . Because of the different treating time of tak242 and pioglitazone, the animals were sacrificed at different time intervals . Hence, 54 lewis rats were randomly divided into six groups with the method of simple randomization: controltak242, igantak242, tak242, controlpio, iganpio, and pio groups . Experimental igan was induced in 4-week - old lewis rats by continuous oral immunization with 0.1% bgg in 6 mmol / l hcl as drinking water for 9 weeks, followed by intravenous injection of 1 mg bgg (dissolved in 0.3 ml 6 mmol / l hcl) into the tail vein daily for 3 successive days . Tak242, dissolved in 20% lipovenoes at 3 mgkgd (7.5 ml / kg), was injected into rats with igan for 8 days after completing the model - creation (tak242 group). Experimental igan rats receiving intravenous injection of the same amount of lipovenoes for 8 days by tail veins were named igantak242 group . Controltak242 group was normal rats who drank 6 mmol / l hcl for 9 weeks, and then injected with 1 mg 6 mmol / l hcl into the tail vein daily for three successive days followed by injection with 20% lipovenoes at 7.5 mlkgd for 8 days . Pioglitazone, dissolved in saline at 1 mg / ml, was administered to rats after the model - creation by intragastric injection at 10 mgkgd for 4 weeks until the rats were killed (pio group). Rats of experimental igan receiving intragastric injection of saline at 10 mlkgd for 4 weeks were named iganpio group and controlpio group referred to normal rats who drank 6 mmol / l hcl for 9 weeks, and then were given intravenous injection of 1 mg 6 mmol / l hcl into the tail vein daily for three successive days followed by intragastric injection of saline at 10 mlkgd for 4 weeks . Albumin - to - creatinine ratio (acr) was used to express the degree of albuminuria . The urine was centrifuged at 600 revolutions per minute for 5 min and the sediment was examined microscopically for erythrocytes . A diagnostic criterion for hematuria was more than 10 red cells on average per high - power field . Serum creatinine (scr) and blood urea nitrogen (bun) were measured at the clinical laboratory at huadong hospital affiliated to fudan university . Blood samples were drawn through the abdominal aorta after intraperitoneal anesthesia with 2% pentobarbital sodium to detect serum il-1 by enzyme - linked immunosorbent assay kits (multisciences biotech co., ltd ., the cortex from half of the kidney was fixed in 4% paraformaldehyde and paraffin - embedded for histological observation . The other half was snap frozen in liquid nitrogen and stored at 80c for total rna and protein extraction . Three - micrometer thick paraffin - embedded kidney sections were deparaffinized with xylene and then rehydrated through a descending gradient of ethanol . The sections were stained with h and e. glomerular damage and mesangial hypercellularity were determined by examining 25 glomeruli in each sample . Immunofluorescence examination of the paraffin - embedded renal sections was performed by staining with fluorescing isothiocyanate - labeled, specific antibodies against rat iga (1:16) at 4c overnight . The anti - rat iga antibody was visualized as bright green color using the dako envision plus system (dako, carpinteria, ca, usa). The nephrologist who examined the specimens was unaware of the group assignments of the individual animals . Total rna was extracted from renal tissues using the trizol reagent kit (life technologies, usa) according to the method described in the manufacturer's protocol . Thereafter, 1 ng of total rna was reverse transcribed using the primescript reverse transcription kit and random primers (takara biotechnology co., ltd ., real - time polymerase chain reaction amplification was carried out in an abi prism 7500 sequence detection system . Primer sequences for rat tlr4 are forward 5-tgacagacctcaggcagattgt-3, reverse 5-aatagtgcaatcgatagaaggaaca-3; il-1 forward 5-ccgtggcacattctggtca-3, reverse 5-gctgtgcactggtccaaattc-3; and -actin forward 5-agattactgccctggctcctag-3, reverse 5-catcgtactcctgcttgctga-3. -actin was used as an endogenous control . Protein extracted from the renal tissues was analyzed by western blotting for tlr4 and il-1 in the following manners . After homogenization, the solution was centrifuged at 10,800 g at 4c for 5 min . Protein was separated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane . The membrane was incubated with bsa, and then with following primary antibodies including rabbit antibody to il-1 and tlr4 . Detection was used by enhanced chemiluminescence, and the light signals were exposed to x - ray film . Data were analyzed using spss software version 20 (ibm, new york, usa). Differences between groups were analyzed by one - way analysis of variances (anova). As shown in figure 1a and 1b, urinary acr and serum il-1 level were significantly increased in igantak242 rats compared to controltak242 rats (4.45 1.33 mg / mmol vs. 2.89 0.96 mg / mmol, p <0.05; 48.28 13.49 furthermore, treatment with tak242 significantly reversed the elevations of acr and serum il-1 (1.34 0.33 mg / mmol vs. 4.45 1.33 mg / mmol, p <0.05; 32.43 7.42 pg / ml vs. 48.28 13.49 pg / ml, p <0.05). Similarly, urinary acr was significantly increased in iganpio rats compared to controlpio rats (1.72 0.41 mg / mmol vs. 1.27 0.15 mg / mmol, p <0.05), which was reversed by pioglitazone (1.13 0.44 mg / mmol vs. 1.72 0.41 mg / mmol, p <0.05) [figure 1c]. Compared to iganpio group, there was a tendency of reduction of serum il-1 levels in pio group, but the difference did not reach the statistical difference (39.06 17.92 pg / ml, p> 0.05) [figure 1d]. However, urine from normal rats, igan rats, rats received treatment with tak242 or pioglitazone all revealed sporadic hematuria and the difference among groups did not reach the statistical significance . There was no significant difference in bun or scr between various groups (data not shown). (d) serum concentration of il-1 of all groups detected by elisa kits . * compared to controltak242, p <0.05; compared to igantak242, p <0.05; compared to controlpio, p <0.05; compared to iganpio, p <0.05 . Acr: urinary albumin - to - creatinine ratio; igan: immunoglobulin a nephropathy; pio: pioglitazone; tak242: toll - like receptor 4 inhibitor; il-1: interleukin-1 beta; elisa: enzyme - linked immunosorbent assay . Figure 2a indicates that there is hardly no clear green fluorescence seen in the glomeruli of controltak242 and controlpio groups . In contrast, bright mass - like green fluorescence was found in the glomeruli of igantak242 and iganpio rats . Morphological changes observed by immunofluorescence and h and e staining (n=9 in each group). (a) contrastive immunofluorescence of iga deposition in the glomeruli of kidney sections from normal rats and rats with iga nephropathy (450). (b) representative h and e staining of glomeruli in kidney sections from rat of each group (400). (c) glomerular cell number expressed as cells per glomerular cross - section of all groups stained by he . * compared to controltak242, p <0.05; compared to igantak242, p <0.05; compared to controlpio, p <0.05; compared to iganpio, p <0.05 . Igan: immunoglobulin a nephropathy; pio: pioglitazone; tak242: toll - like receptor 4 inhibitor . The kidney sections were stained with h and e. renal pathologic structure of controltak242 and controlpio groups was found without any glomerular or tubular injuries . The histological changes of rats in igantak242 and iganpio groups were both characterized by glomerular hypercellularity, moderate proliferation of the mesangial cells, and hyperplasia of mesangial matrix . The changes mentioned above were apparently ameliorated in rats of tak242 and pio groups [figure 2b]. The number of glomerular cells of cross section in experimental rats was higher than those in normal rats (51.39 3.58 vs.41.24 2.24, p <0.01). Compared to igantak242 group, the number of glomerular cells of cross section was much lower in tak242 group (35.47 3.38 vs.51.39 3.58, p <0.01). In addition, there was a significant decrease of glomerular cells in pio group compared to iganpio group (45.53 6.37 vs.50.12 8.43, p <0.05) [figure 2c]. We studied the expression of il-1 and tlr4 mrna as well as protein of each sample from renal tissues . Our findings illustrated that tlr4, il-1 mrna, and protein in igantak242 group were dramatically higher than those in controltak242 group (tlr4 mrna: 1.36 0.54 vs. 1.03 0.24, p <0.05; il-1 mrna: 1.38 0.62 vs. 0.88 0.37, p <0.01; tlr4 protein: 0.81 0.18 vs. 0.30 0.07, p <0.01; il-1 protein: 1.10 0.33 vs. 0.74 0.19, p <0.05). In contrast, the levels of tlr4, il-1 mrna, and protein in tak242 group were much lower than those in igantak242 group (tlr4 mrna: 0.92 0.26 vs. 1.36 0.54, p <0.01; il-1 mrna: 0.95 0.45 vs. 1.38 0.62, p <0.01; tlr4 protein: 0.43 0.30 vs. 0.81 0.18, p <0.01; il-1 protein: 0.60 0.33 vs. 1.10 0.33, p <0.01). Likewise, there was a significant increase of tlr4, il-1 mrna, and protein in iganpio group compared to controlpio group (tlr4 mrna: 1.72 0.45 vs. 1.58 0.37, p <0.05; il-1 mrna: 1.53 0.19 vs. 1.32 0.37, p <0.05; tlr4 protein: 0.21 0.05 vs. 0.16 0.06, p <0.05; il-1 protein: 0.66 0.16 vs. 0.46 0.21, p <0.05). In addition, the increase was reversed in pio group: the expression of tlr4, il-1 mrna, and protein in pio group was dramatically decreased compared to iganpio group (tlr4 mrna: 1.22 0.28 vs. 1.72 0.45, p <0.05; il-1 mrna: 1.27 0.41 vs. 1.53 0.19, p <0.01; tlr4 protein: 0.12 0.03 vs. 0.21 0.05, p <0.05; il-1 protein: 0.37 0.20 vs. 0.66 0.16, p <0.05) [figure 3a and 3b]. Expression of il-1 and tlr4 in renal tissues of all groups of rats (n=9 in each group). (a) the levels of il-1 and tlr4 in the kidney tissues determined by reverse transcription polymerase chain reaction . (b) the levels of il-1 and tlr4 in the kidney tissues determined by western blots . Compared to controltak242, p <0.05; compared to igantak242, p <0.05; compared to controlpio, p <0.05; compared to iganpio, p <0.05 . Igan: immunoglobulin a nephropathy; pio: pioglitazone; tak242: toll - like receptor 4 inhibitor; il-1: interleukin-1 beta; mrna: message ribonucleic acid; tlr4: toll - like receptor 4; gapdh: glyceraldehyde-3-phosphate dehydrogenase . As shown in figure 1a and 1b, urinary acr and serum il-1 level were significantly increased in igantak242 rats compared to controltak242 rats (4.45 1.33 mg / mmol vs. 2.89 0.96 mg / mmol, p <0.05; 48.28 13.49 furthermore, treatment with tak242 significantly reversed the elevations of acr and serum il-1 (1.34 0.33 mg / mmol vs. 4.45 1.33 mg / mmol, p <0.05; 32.43 7.42 pg / ml vs. 48.28 13.49 pg / ml, p <0.05). Similarly, urinary acr was significantly increased in iganpio rats compared to controlpio rats (1.72 0.41 mg / mmol vs. 1.27 0.15 mg / mmol, p <0.05), which was reversed by pioglitazone (1.13 0.44 mg / mmol vs. 1.72 0.41 mg / mmol, p <0.05) [figure 1c]. Compared to iganpio group, there was a tendency of reduction of serum il-1 levels in pio group, but the difference did not reach the statistical difference (39.06 17.92 pg / ml, p> 0.05) [figure 1d]. However, urine from normal rats, igan rats, rats received treatment with tak242 or pioglitazone all revealed sporadic hematuria and the difference among groups did not reach the statistical significance . There was no significant difference in bun or scr between various groups (data not shown). (d) serum concentration of il-1 of all groups detected by elisa kits . * compared to controltak242, p <0.05; compared to igantak242, p <0.05; compared to controlpio, p <0.05; compared to iganpio, p <0.05 . Acr: urinary albumin - to - creatinine ratio; igan: immunoglobulin a nephropathy; pio: pioglitazone; tak242: toll - like receptor 4 inhibitor; il-1: interleukin-1 beta; elisa: enzyme - linked immunosorbent assay . Figure 2a indicates that there is hardly no clear green fluorescence seen in the glomeruli of controltak242 and controlpio groups . In contrast, bright mass - like green fluorescence was found in the glomeruli of igantak242 and iganpio rats . Morphological changes observed by immunofluorescence and h and e staining (n=9 in each group). (a) contrastive immunofluorescence of iga deposition in the glomeruli of kidney sections from normal rats and rats with iga nephropathy (450). (b) representative h and e staining of glomeruli in kidney sections from rat of each group (400). (c) glomerular cell number expressed as cells per glomerular cross - section of all groups stained by he . * compared to controltak242, p <0.05; compared to igantak242, p <0.05; compared to controlpio, p <0.05; compared to iganpio, p <0.05 . Igan: immunoglobulin a nephropathy; pio: pioglitazone; tak242: toll - like receptor 4 inhibitor . The kidney sections were stained with h and e. renal pathologic structure of controltak242 and controlpio groups was found without any glomerular or tubular injuries . The histological changes of rats in igantak242 and iganpio groups were both characterized by glomerular hypercellularity, moderate proliferation of the mesangial cells, and hyperplasia of mesangial matrix . The changes mentioned above were apparently ameliorated in rats of tak242 and pio groups [figure 2b]. The number of glomerular cells of cross section in experimental rats was higher than those in normal rats (51.39 3.58 vs.41.24 2.24, p <0.01). Compared to igantak242 group, the number of glomerular cells of cross section was much lower in tak242 group (35.47 3.38 vs.51.39 3.58, p <0.01). In addition, there was a significant decrease of glomerular cells in pio group compared to iganpio group (45.53 6.37 vs.50.12 8.43, p <0.05) [figure 2c]. We studied the expression of il-1 and tlr4 mrna as well as protein of each sample from renal tissues . Our findings illustrated that tlr4, il-1 mrna, and protein in igantak242 group were dramatically higher than those in controltak242 group (tlr4 mrna: 1.36 0.54 vs. 1.03 0.24, p <0.05; il-1 mrna: 1.38 0.62 vs. 0.88 0.37, p <0.01; tlr4 protein: 0.81 0.18 vs. 0.30 0.07, p <0.01; il-1 protein: 1.10 0.33 vs. 0.74 0.19, p <0.05). In contrast, the levels of tlr4, il-1 mrna, and protein in tak242 group were much lower than those in igantak242 group (tlr4 mrna: 0.92 0.26 vs. 1.36 0.54, p <0.01; il-1 mrna: 0.95 0.45 vs. 1.38 0.62, p <0.01; tlr4 protein: 0.43 0.30 vs. 0.81 0.18, p <0.01; il-1 protein: 0.60 0.33 vs. 1.10 0.33, p <0.01). Likewise, there was a significant increase of tlr4, il-1 mrna, and protein in iganpio group compared to controlpio group (tlr4 mrna: 1.72 0.45 vs. 1.58 0.37, p <0.05; il-1 mrna: 1.53 0.19 vs. 1.32 0.37, p <0.05; tlr4 protein: 0.21 0.05 vs. 0.16 0.06, p <0.05; il-1 protein: 0.66 0.16 vs. 0.46 0.21, p <0.05). In addition, the increase was reversed in pio group: the expression of tlr4, il-1 mrna, and protein in pio group was dramatically decreased compared to iganpio group (tlr4 mrna: 1.22 0.28 vs. 1.72 0.45, p <0.05; il-1 mrna: 1.27 0.41 vs. 1.53 0.19, p <0.01; tlr4 protein: 0.12 0.03 vs. 0.21 0.05, p <0.05; il-1 protein: 0.37 0.20 vs. 0.66 0.16, p <0.05) [figure 3a and 3b]. Expression of il-1 and tlr4 in renal tissues of all groups of rats (n=9 in each group). (a) the levels of il-1 and tlr4 in the kidney tissues determined by reverse transcription polymerase chain reaction . (b) the levels of il-1 and tlr4 in the kidney tissues determined by western blots . Compared to controltak242, p <0.05; compared to igantak242, p <0.05; compared to controlpio, p <0.05; compared to iganpio, p <0.05 . Igan: immunoglobulin a nephropathy; pio: pioglitazone; tak242: toll - like receptor 4 inhibitor; il-1: interleukin-1 beta; mrna: message ribonucleic acid; tlr4: toll - like receptor 4; gapdh: glyceraldehyde-3-phosphate dehydrogenase . The present study provided evidence that inflammatory responses and tlr4 signaling pathway are involved in the progression of igan . Pioglitazone exerted its anti - inflammatory effects by interfering with tlr4 signaling pathway and reducing inflammatory cytokines to alleviate the progression of igan . Research of igan has been handicapped due to the lack of a good animal model of igan for many years . Since rifai et al . Established igan model with balb / c mice for the first time in 1979, many scholars have made various igan models based on the known pathogenesis . However, there were wide gaps in pathologic changes between the animal models and human igan . Recently, the ddy strain of mouse was used as a spontaneous animal model for human igan . However, these mice showed mild proteinuria without hematuria at more than 40 weeks of age and the incidence of igan was highly variable . Some investigators used bsa gavage and hypodermic injection of staphylococcal enterotoxin b plus lipopolysaccharides (lps) to establish igan models . However, lps itself is a high potent agonist of tlr4 which is apt to confound the interpretation of the subsequent inflammatory pathway . In 1983, for the first time made igan models with balb / c mice by orally immunizing bgg . Only part of the mice showed proteinuria and hematuria was not obvious . In 1992, gesualdo et al . Induced igan in different rat strains by oral bgg immunization and found that mesangial iga deposition was most severe in lewis rats . Due to the mild histopathology even after prolonged observation of this model, lai et al . Improved the model with lewis rats by immunization of bgg and followed by unilateral nephrectomy . This model had diffuse glomerular iga deposition, mesangial hypercellularity, high acr and obvious hematuria . Although unilateral nephrectomy hastened the development of renal abnormalities, it may lead to acute kidney injury and other injuries not characteristic to igan which could confound the findings . Taken together, our establishment of igan models with lewis rats by oral immunization of bgg for 9 weeks and then injection of bgg through tail vein for 3 successive days might be able to avoid those disadvantages mentioned above . Although our models did not show obvious hematuria which was consistent with other studies, immunofluorescent assay showed granular and massive iga depositions and pathological observation revealed diffuse proliferation of mesangial cells and matrix . Furthermore, our models showed obvious proteinuria . Above all, igan rat model were successfully established and can be used to reflect the pathophysiological findings in human igan . Il-1 is one of the most powerful moderators of inflammation and is closely involved in the development of mesangial cell proliferation and extracellular matrix production . In igan patients, il-1 has been shown to be produced locally in the glomeruli and interstitial . In our study, induction of igan is associated with increased renal expression of il-1 mrna and protein as well as serum il-1 which suggests that inflammatory responses participate in the pathogenesis of igan . There has been circumstantial evidence for the involvement of tlrs, notably tlr4, in the pathogenesis of renal diseases . However, the reports about the effects of tlr4 on igan in vivo are incompletely elucidated . In our study, tak242, a selective inhibitor of tlr4 intracellular signaling pathway, had been used in the current study to unravel whether the mechanisms of inflammatory response in igan were tlr4 dependent and, in such a case, to ascertain the specific role of tlr4 . We found that the expression of tlr4 mrna and protein significantly was decreased in rats treated with tak242 . This finding further supported the role of activated tlr4 signaling pathway in the pathophysiology of igan . In keeping with our findings, the study conducted by kwon et al . Proved that in igan patients, the expression of renal tlr4 mrna was significantly elevated . The transcriptional level of tlr4 mrna in circulating mononuclear cells was significantly higher in patients with igan than those in healthy controls . What's more, the increased expression of tlr4 in circulating mononuclear cells was significantly correlated with proteinuria or phases of clinical activity in patients with igan . Binding with their ligands, tlrs initiate an intracellular signaling cascade, activate protein kinases, release cytokines, and enhance the expression of transcription factors which results in the generation of mediators including adhesion molecules, chemokines, cytokines, and inflammatory responses . The nuclear translocation of the nf-b favored the hyperplasia of b - cell and therefore increased iga synthesis . Tlrs have been conformed to be involved in the switch from igm to iga production in b - cells in experimental animals . In addition, tlr4 was involved in the injuries of mesangial cells by induction of proinflammatory cytokines in igan and was constitutively expressed in podocytes to mediate glomerular injuries by modulating the expression of chemokines . All of the results mentioned above suggested that renal tlr4 signaling pathway may be a therapeutic target for the alleviation of igan . Recently, increasing attention has been paid to the role of ppar- agonists in regulating inflammatory responses in kidney diseases . However, there were few reports on whether ppar- agonists exerted anti - inflammatory effects by interfering with tlr4 signaling pathway to improve igan in vivo . In the present study, activation of ppar- pathway with pioglitazone this confirmed that tlr4 pathway was involved in the renal protective effects of ppar- in vivo . Our findings are consistent with that of dasu et al, who demonstrated that pioglitazone decreased the expression of tlr4 mrna and protein in a concentration - dependent manner and lowered the activity of nf-b, il-1, tnf - alpha, and other inflammatory factors in human monocytes . In addition, in peritoneal macrophages of db / db mice, pioglitazone markedly suppressed the expression of myd88 (myeloid differentiation factor 88) and trif (tir - domain - containing adaptor inducing interferon-) protein in the cytoplasm and reduced the phosphorylation of interleukin receptor - associated kinase and p38, which subsequently inhibited the activation of tlr4 signaling pathway and nf-b . Ji et al . Revealed that ppar- agonist, rosiglitazone, could concentration dependently downregulate the expression of tlr4 mrna and protein in vascular smooth muscle cells of sprague dawley rats . The beneficial effects of rosiglitazone on inflammation were mediated through interference with tlr4 and its downstream signaling components such as trif, interferon regulatory factor 3 (irf3), and inducible protein-10 . First, we do not provide additional evidence that the alterations of tlr4 mrna and protein as well as inflammatory factors after using tlr4 agonist . Second, our research is lack of data using specific ppar- antagonists for igan rats . It is unclear whether pioglitazone - induced anti - inflammatory effects are direct or indirect in in vivo experiment . One possible scenario is that pioglitazone as a ligand binds to the ppar- receptor and directly binds to the ppre of il-1 gene itself or genes which codes for its upstream mediators and inactivates them such as tlr4 . Another possible scenario is whether pioglitazone itself or its metabolites are the active compound responsible for anti - inflammatory effects . In vitro researches with renal cells are recommended to dissect the precise mechanism of pioglitazone - induced anti - inflammatory effects in tlr4 signaling pathway in igan . In summary, our study indicated that tlr4 and inflammatory responses played a key role in the progression of igan . Pioglitazone improved biochemical parameters, ameliorated renal histological damage, and alleviated iga deposits in glomeruli, at least in part, by negatively regulating the expression of tlr4 and blunted the activation of its downstream signal pathways involved in the expression of inflammatory factors . It suggested that either drug antagonizing tlr4 or ppar- agonists could have potently beneficial effects in the treatment of igan . This study was supported by a grant from the national natural science foundation of china (no . This study was supported by a grant from the national natural science foundation of china (no.
Cancer cachexia is a condition of progressive muscle wasting that develops as a secondary condition in response to tumour growth . While cachexia develops in a wide range of pathologies including anorexia nervosa, acquired immunodeficiency syndrome, amyotrophic lateral sclerosis, congestive heart failure and various malignant cancers, cancer cachexia has been reported to develop at a faster rate than any other cachectic condition . Approximately half of all patients with cancer experience cachexia and almost a third of mortalities are estimated to result from cachexia . Tumour growth induces a specific catabolic response in skeletal muscle that causes the accelerated loss of protein, characterised by the significant loss of body weight . Animal tumour models have been used to investigate cancer cachexia, developing up to 30% loss in body weight after tumour cell implantation . Nmri mice implanted with murine adenocarcinoma (mac) 16 present with solid tumour growth and cachexia, as have been shown in rats bearing the yoshida ah-130 hepatoma, characteristic of the human cancer cachectic condition . An abundance of evidence exists for the induction of the ubiquitin (ub)-proteasome proteolytic pathway in the development of cancer cachexia; however, evidence of protein oxidation in cancer cachectic patients suggests the involvement of reactive oxygen species (ros). Studies in both humans and animals with cancer cachexia have shown the presence of oxidised proteins . Furthermore, cancer patients have been shown to present with elevated levels of serum ros and lower antioxidant levels, indicative of a pro - oxidative shift . Similarly, increased levels of ros have been shown in cachectic tumour - bearing rats, with no compensatory response from antioxidant enzymes . Although the catabolic mechanism leading to the severe muscle wasting associated with cancer development remains relatively unknown, there is evidence to suggest a role for ros . Nicotinamide adenine dinucleotide phosphate (nadph) oxidase (nox) is a multicomponent enzyme that when activated catalyzes the production of superoxide anion (o2). The nox2 enzyme consists of five subunits segregated into membrane bound, nox2 (gp91) and p22, and cytosolic, p67, p47 and p40, units . On stimulation, the protein components of the nox2 enzyme assemble, forming the active oxidase and the production of o2 . It has been apparent in recent years that non - phagocytes possess homologues to nox2, and that these nox enzymes may activate in a similar manner to the well - established phagocyte nox2 enzyme . Currently, there are five known nox homologues (nox1 - 5) found to function in a variety of tissues, with the expression of both nox2 and nox4 enzyme subunits in skeletal muscle tissue, although its function in this particular cell system remains undefined . Despite the unknown functionality of the nox enzymes in skeletal muscle tissue, o2 generated from nox the abundance of evidence for nox enzyme - generated o2 in disease, establishes its potential role in the catabolic skeletal muscle wasting in cancer cachexia . It is well - known that ros are generated in skeletal muscle as an essential by - product of cellular metabolism and oxidative enzymes . Due to the high metabolic activity and oxidative capacity of skeletal muscle, the antioxidant system is a crucial component for the maintenance of cellular oxidative homeostasis . With the knowledge that ros are likely catabolic factors in the development of cancer cachexia, antioxidant enzymes are of equal importance for their crucial role in the maintenance of cellular oxidative homeostasis and the protection against harmful ros . O2 is dismutated by the o2 specific antioxidant enzyme, superoxide dismutase (sod). Three distinct forms of sod exist in the intracellular cytoplasmic compartment (sod1), mitochondria (sod2) and extracellular matrix (sod3). The sod enzymes catalyse the conversion of o2 to h2o2, which is further converted by catalase and glutathione peroxidise (gpx) scavenging . A depression in these antioxidant enzyme systems can consequently lead to an imbalance in cellular oxidation and oxidative tissue damage, which has been shown particularly in response to disease . Endogenous antioxidants such as sod and gpx are low in cancer patients presenting with high levels of ros, indicating the importance of tight regulation from endogenous antioxidants . The ub - proteasome proteolytic pathway has been established in cancer - induced cachexia . In addition, tumour - derived factors such as tnf- and proteolysis - inducing factor (pif) are well recognised in the cancer cachectic condition . What is yet to be elucidated, however, are the downstream mediators of this complex system, in response to tumour growth and development of cancer cachexia . A study by russell et al . Proposed a cachectic pathway of increased ub gene expression, downstream of nox - generated ros production, establishing a potential role for the nox enzymes in mediating skeletal muscle atrophy through the ub - proteasome proteolytic pathway . Therefore, this study aims to investigate the o2 generating nox and antioxidant enzyme systems in a well - established animal model of cancer cachexia . In particular, this study aims to determine o2 levels and nox enzyme system expression (nox2, nox4, p22, p40, p47, p67 and rac1) in the skeletal muscle of cachectic (mac16) tumour - bearing mice compared to non - cachectic (mac13) tumour - bearing mice . Furthermore, this study aims to determine levels of the primary antioxidants for o2 and h2o2 dismutation (sod1, sod2, sod3, catalase and gpx), in the skeletal muscle of tumour - bearing cachectic mice (mac16) compared to tumour - bearing non - cachectic (mac13) mice . All experimental procedures were carried out with approval from the victoria university animal ethics committee (aeeth 07/05). Two weight - matched groups of female balb / c nu / nu mice were maintained under controlled environmental conditions, 12-h light / dark cycle, 21c 2c, 30% humidity, in conventional cages with ad libitum access to standard chow and water throughout the course of the study . As has previously been described, donor mice were used to establish and maintain a tumour line, which consistently produced tumour growth (mac13) (n = 3) and tumour growth with cachexia (mac16) (n = 4). Tumour fragments grown and maintained in donor mice were dissected and re - implanted subcutaneously into the flank of recipient mice with frequent monitoring and recording of body weight and tumour size . With cachectic weight loss evident at approximately 912 days post implantation, mice were anaesthetised, using pentobarbital sodium (70 mg / kg), for tissue collection before weight loss exceeded 25% of original body weight or before tumour growth exceeded 1,000 mm . Skeletal muscle tissue was collected, weighed and immediately snap - frozen in liquid nitrogen and stored at 80c for later use . Dhe (5 m) was applied to quadriceps cross - sections (5 m) and incubated in a light - protected oven at 37c for 30min . The sections were washed with pbs to remove excess dhe, and fluorescence was assessed by way of fluorescence microscopy (axiocam hbo 50/ac, zeiss, germany). Ethidium fluorescence density was detected from the whole section with mcid imaging software (imaging research inc, australia) and expressed as arbitrary units of fluorescence . Rna was extracted from frozen quadriceps muscle using tri reagent (molecular research centre), according to the manufacturer's protocol . Total rna concentration was determined spectrophotometrically at 260 nm . Prior to reverse transcription (rt), all rna samples were dnase - treated (promega), and first - strand cdna was generated from 1-g rna using amv rt (promega). Pre - designed taqman gene expression assays (applied biosystems) were used containing specific primers and probes for the genes of interest . Real - time pcr was performed using applied biosystems 7500 detection system and pcrs were performed using taqman gene expression master mix (applied biosystems). To compensate for variations in input rna amounts and efficiency of reverse transcription, gapdh mrna was quantified and all results were normalised to these values . Protein was extracted from frozen quadriceps muscle homogenised in ice - cold radio - immunoprecipitation assay buffer containing tris hcl (50 mmol / l; ph 7.4), nacl (150 mmol / l), np-40 (1%), sodium deoxycholate (0.5%) and sds (0.1%) and centrifuging at 13,000g for 15 min at 4c, to remove insoluble material . The protein concentration was determined by the bradford method (bio - rad) and equal amounts of protein were separated by sds - page and transferred to polyvinylidene difluoride membranes . The membranes were blocked for 2 h at room temperature in tris - buffered saline containing tris hcl (20 mm; ph 7.6) nacl (137 mm) and tween 20 (0.1%) with 5% bsa and probed with primary antibodies for either nox2 (gp91), p40, p67 or gapdh (1:200; santa cruz biotechnology) overnight at 4c . Antibody binding was detected using horseradish peroxidase - conjugated secondary antibody (1:50,000; santa cruz biotechnology). The protein bands were detected by supersignal west dura chemiluminescence reagents (thermo scientific). The las 4000 imaging system (fujifilm life science, usa) was used to visualize protein bands, and densitometry was performed with multigauge software (fujifilm life science, usa). To compensate for variation in protein loading, the relative density of immunoreactive bands were normalised to the density of the corresponding bands for gapdh . Spectrophotometric assay kits were used to measure sod (cayman-706002), catalase (cayman-707002) and gpx (cayman- 703102) activity and hydrogen peroxide (cayman-600050) levels, in muscle homogenates . Frozen muscle pieces (100 mg) were placed in ice - cold hepes buffer (20 mm) containing egta (1 mm), mannitol (210 mm) and sucrose (70 mm) and adjusted to a ph of 7.2 (10 ml / g). Muscle aliquots were homogenised in buffer, using a glass on glass homogeniser, and centrifuged at 1,500g for 5 min at 4c to remove insoluble connective tissue . For the detection of sod1 and sod2, cytosolic and mitochondrial fractions the supernatant was centrifuged at 10,000g for 5 min at 4c, and the resulting supernatant, containing the cytosolic fraction, was collected for sod1 enzyme analysis . The remaining pellet containing the mitochondrial fraction was resuspended and homogenised in ice - cold hepes buffer (20 mm) for sod2 enzyme analysis . The amount of enzyme activity and hydrogen peroxide levels were calculated and standardised for protein using the bradford method (bio - rad). Differences were determined by one - way anova with tukey hsd as posthoc to determine significant differences between groups, and results were considered statistically significant if p values were equal to or <0.05 . All experimental procedures were carried out with approval from the victoria university animal ethics committee (aeeth 07/05). Two weight - matched groups of female balb / c nu / nu mice were maintained under controlled environmental conditions, 12-h light / dark cycle, 21c 2c, 30% humidity, in conventional cages with ad libitum access to standard chow and water throughout the course of the study . As has previously been described, donor mice were used to establish and maintain a tumour line, which consistently produced tumour growth (mac13) (n = 3) and tumour growth with cachexia (mac16) (n = 4). Tumour fragments grown and maintained in donor mice were dissected and re - implanted subcutaneously into the flank of recipient mice with frequent monitoring and recording of body weight and tumour size . With cachectic weight loss evident at approximately 912 days post implantation, mice were anaesthetised, using pentobarbital sodium (70 mg / kg), for tissue collection before weight loss exceeded 25% of original body weight or before tumour growth exceeded 1,000 mm . Skeletal muscle tissue was collected, weighed and immediately snap - frozen in liquid nitrogen and stored at 80c for later use . Dhe (5 m) was applied to quadriceps cross - sections (5 m) and incubated in a light - protected oven at 37c for 30min . The sections were washed with pbs to remove excess dhe, and fluorescence was assessed by way of fluorescence microscopy (axiocam hbo 50/ac, zeiss, germany). Ethidium fluorescence density was detected from the whole section with mcid imaging software (imaging research inc, australia) and expressed as arbitrary units of fluorescence . Rna was extracted from frozen quadriceps muscle using tri reagent (molecular research centre), according to the manufacturer's protocol . Total rna concentration was determined spectrophotometrically at 260 nm . Prior to reverse transcription (rt), all rna samples were dnase - treated (promega), and first - strand cdna was generated from 1-g rna using amv rt (promega). Pre - designed taqman gene expression assays (applied biosystems) were used containing specific primers and probes for the genes of interest . Real - time pcr was performed using applied biosystems 7500 detection system and pcrs were performed using taqman gene expression master mix (applied biosystems). To compensate for variations in input rna amounts and efficiency of reverse transcription, gapdh mrna was quantified and all results were normalised to these values . Protein was extracted from frozen quadriceps muscle homogenised in ice - cold radio - immunoprecipitation assay buffer containing tris hcl (50 mmol / l; ph 7.4), nacl (150 mmol / l), np-40 (1%), sodium deoxycholate (0.5%) and sds (0.1%) and centrifuging at 13,000g for 15 min at 4c, to remove insoluble material . The protein concentration was determined by the bradford method (bio - rad) and equal amounts of protein were separated by sds - page and transferred to polyvinylidene difluoride membranes . The membranes were blocked for 2 h at room temperature in tris - buffered saline containing tris hcl (20 mm; ph 7.6) nacl (137 mm) and tween 20 (0.1%) with 5% bsa and probed with primary antibodies for either nox2 (gp91), p40, p67 or gapdh (1:200; santa cruz biotechnology) overnight at 4c . Antibody binding was detected using horseradish peroxidase - conjugated secondary antibody (1:50,000; santa cruz biotechnology). The protein bands were detected by supersignal west dura chemiluminescence reagents (thermo scientific). The las 4000 imaging system (fujifilm life science, usa) was used to visualize protein bands, and densitometry was performed with multigauge software (fujifilm life science, usa). To compensate for variation in protein loading, the relative density of immunoreactive bands were normalised to the density of the corresponding bands for gapdh . Spectrophotometric assay kits were used to measure sod (cayman-706002), catalase (cayman-707002) and gpx (cayman- 703102) activity and hydrogen peroxide (cayman-600050) levels, in muscle homogenates . Frozen muscle pieces (100 mg) were placed in ice - cold hepes buffer (20 mm) containing egta (1 mm), mannitol (210 mm) and sucrose (70 mm) and adjusted to a ph of 7.2 (10 ml / g). Muscle aliquots were homogenised in buffer, using a glass on glass homogeniser, and centrifuged at 1,500g for 5 min at 4c to remove insoluble connective tissue . For the detection of sod1 and sod2, cytosolic and mitochondrial fractions the supernatant was centrifuged at 10,000g for 5 min at 4c, and the resulting supernatant, containing the cytosolic fraction, was collected for sod1 enzyme analysis . The remaining pellet containing the mitochondrial fraction was resuspended and homogenised in ice - cold hepes buffer (20 mm) for sod2 enzyme analysis . The amount of enzyme activity and hydrogen peroxide levels were calculated and standardised for protein using the bradford method (bio - rad). Differences were determined by one - way anova with tukey hsd as posthoc to determine significant differences between groups, and results were considered statistically significant if p values were equal to or <0.05 . Female balb / c nu / nu mice were implanted with the mac13 cell line (n = 12) that had previously developed a tumour in the same mouse model at approximately 912 days post implantation (n = 3) and where the animal did not lose weight . Another group of female balb / c nu / nu mice were implanted with the mac16 cell line (n = 16) that had previously developed a tumour in the same mouse model at approximately 912 days post implantation (n = 4) and lost approximately 1525% of their original body weight were used in this study . Mouse body weight and skeletal muscle weights were recorded at tissue collection and evaluated as a measure of body mass and skeletal muscle cachexia for all mice . Body weight and quadricep weights were significantly less in cachectic (mac16 tumour - bearing mice) when compared to mice with cancer alone (bearing mac13 tumours) (p <0.001; p = 0.003, table 1). To account for any differences in calorie intake between the groups, food intake was recorded throughout the course of the study and showed no differences between cachectic and cancer mice (table 1).table 1mean body weight, quadriceps weight and food intake of mice with cancer and cancer cachexiaweightcancercachexiabody weight (g)22.3 0.317.1 0.3quadriceps (mg)117.0 4.290.1 2.1food intake (g)3.6 0.33.6 0.5the values represent the mean semp <0.003, statistically significant differences mean body weight, quadriceps weight and food intake of mice with cancer and cancer cachexia the values represent the mean sem p <0.003, statistically significant differences o2 levels were measured by histological dhe fluorescence in skeletal muscle sections from mice with cancer and cachexia . Skeletal muscle o2 levels were significantly higher in cachectic mice (9.36 2.67 au) when compared to mice with cancer (6.47 0.77 au) alone (p = 0.001, fig the values represent the mean sem . Statistically significant difference * p = 0.001 . A o2 levels detected by dihydroethidium (dhe) the values represent the mean sem . Statistically significant difference * p = 0.001 . B histological dhe fluorescence in the skeletal muscle of mice with cancer and cancer cachexia o2 levels in the skeletal muscle of mice with cancer and cancer cachexia . Statistically significant difference * p = 0.001 . A o2 levels detected by dihydroethidium (dhe) the values represent the mean sem . Statistically significant difference * p = 0.001 . B histological dhe fluorescence in the skeletal muscle of mice with cancer and cancer cachexia the mrna level of the nox2 and nox4 enzyme systems previously shown to express in skeletal muscle to generate o2 were measured in mice with cancer and cancer cachexia . The mrna of the regulatory and catalytic nox2 enzyme subunits nox2, p40 and p67 were lower in the skeletal muscle of cachectic mice when compared to mice with cancer alone (p = 0.031; p <however, the mrna expression of the additional nox enzyme subunits, nox4, p22 and p47 and rac1 were similar in skeletal muscle from mice bearing with cancer and cancer cachexia (table 2). The mrna level of sod antioxidant enzymes responsible for the dismutation of o2 were measured in mice with cancer and cancer cachexia . The mrna level of the sod1 and sod2 were significantly lower in the skeletal muscle from cachectic mice when compared to mice with cancer alone (p = 0.007; p <0.001, table 2). Similarly, the mrna level of the h2o2 scavenging antioxidant enzyme gpx was lower in skeletal muscle from cachectic mice compared to mice with cancer alone (p <0.001, table 2). However, the antioxidant enzymes sod3 and catalase had similar mrna levels in both groups (table 2).table 2gene expression in skeletal muscle of mice with cancer and cancer cachexiacancercachexiagene expression (au) nox211.8 2.35.0 1.1 * p22phox88.7 2.975.5 10.5 p40phox21.4 2.87.9 1.6 * p47phox5.7 1.27.3 1.4 p67phox367 92.715.6 3.3 * nox44.3 0.68.5 2.7 rac13.6 1.17.5 1.6 sod1350 36.5120 37.8 * sod2122 8.059.4 5.4 * sod32.6 0.31.8 0.3 gpx23.5 2.312.8 1.2 * catalase49.3 7.343.2 5.6the values represent the mean semp <0.003, statistically significant differences gene expression in skeletal muscle of mice with cancer and cancer cachexia the values represent the mean sem p <0.003, statistically significant differences changes in the mrna levels of the key nox2 enzyme subunits nox2, p40 and p67lead to further analysis of protein expression . The protein expression of the regulatory and catalytic nox2 enzyme subunits nox2 and p40 were lower in cachectic mice when compared to mice with cancer alone (p = 0.021; p <0.012, fig . 2). However, the protein expression of p67 was similar in skeletal muscle from mice in both groups (fig . 2protein expression levels of nox2 enzyme subunits in the skeletal muscle of mice with cancer and cancer cachexia . Statistically significant differences * p <0.05 . A nox2, bp40phox, cp67pho protein expression levels of nox2 enzyme subunits in the skeletal muscle of mice with cancer and cancer cachexia . Statistically significant differences * p <0.05 . A nox2, bp40phox, cp67pho to validate changes in mrna and protein levels, sod1, sod2, catalase and gpx activity levels were measured in skeletal muscle of mice with cancer and cancer cachexia . Sod1 and gpx activities were significantly lower in skeletal muscle from cachectic mice (sod1 9.9 0.9 u / mg protein, gpx 0.74 0.22 mmol / min / mg protein) when compared to mice with cancer alone (sod1 11.2 0.8 u / mg protein, gpx 1.48 0.08 mmol / min / mg protein) (p <0.003, table 3). However, levels of sod2 and catalase activity were similar in both groups of (table 3). H2o2 levels were similar in skeletal muscle from cachectic mice and mice with cancer alone (table 3).table 3enzyme activity and h2o2 levels in skeletal muscle of mice with cancer and cancer cachexiacancercachexiasod1 (u / mg protein)23.3 0.817.8 0.9sod2 (u / mg protein)11.2 0.89.9 0.8catalase (mmol / min / mg protein)4.1 0.43.5 0.2gpx (mmol / min / mg protein)1.48 0.080.74 0.22h2o2 (m)1.11 0.071.15 0.04the values represent the mean semp <0.003, statistically significant differences enzyme activity and h2o2 levels in skeletal muscle of mice with cancer and cancer cachexia the values represent the mean sem p <0.003, statistically significant differences female balb / c nu / nu mice were implanted with the mac13 cell line (n = 12) that had previously developed a tumour in the same mouse model at approximately 912 days post implantation (n = 3) and where the animal did not lose weight . Another group of female balb / c nu / nu mice were implanted with the mac16 cell line (n = 16) that had previously developed a tumour in the same mouse model at approximately 912 days post implantation (n = 4) and lost approximately 1525% of their original body weight were used in this study . Mouse body weight and skeletal muscle weights were recorded at tissue collection and evaluated as a measure of body mass and skeletal muscle cachexia for all mice . Body weight and quadricep weights were significantly less in cachectic (mac16 tumour - bearing mice) when compared to mice with cancer alone (bearing mac13 tumours) (p <0.001; p = 0.003, table 1). To account for any differences in calorie intake between the groups, food intake was recorded throughout the course of the study and showed no differences between cachectic and cancer mice (table 1).table 1mean body weight, quadriceps weight and food intake of mice with cancer and cancer cachexiaweightcancercachexiabody weight (g)22.3 0.317.1 0.3quadriceps (mg)117.0 4.290.1 2.1food intake (g)3.6 0.33.6 0.5the values represent the mean semp <0.003, statistically significant differences mean body weight, quadriceps weight and food intake of mice with cancer and cancer cachexia the values represent the mean sem p <0.003, statistically significant differences o2 levels were measured by histological dhe fluorescence in skeletal muscle sections from mice with cancer and cachexia . Skeletal muscle o2 levels were significantly higher in cachectic mice (9.36 2.67 au) when compared to mice with cancer (6.47 0.77 au) alone (p = 0.001, fig . The values represent the mean sem . Statistically significant difference * p = 0.001 . A o2 levels detected by dihydroethidium (dhe) fluorescence expressed as arbitrary units of fluorescence . B histological dhe fluorescence in the skeletal muscle of mice with cancer and cancer cachexia o2 levels in the skeletal muscle of mice with cancer and cancer cachexia . The values represent the mean sem . Statistically significant difference * p = 0.001 . A o2 levels detected by dihydroethidium (dhe) fluorescence expressed as arbitrary units of fluorescence . The values represent the mean sem . Statistically significant difference * p = 0.001 . The mrna level of the nox2 and nox4 enzyme systems previously shown to express in skeletal muscle to generate o2 were measured in mice with cancer and cancer cachexia . The mrna of the regulatory and catalytic nox2 enzyme subunits nox2, p40 and p67 were lower in the skeletal muscle of cachectic mice when compared to mice with cancer alone (p = 0.031; p <however, the mrna expression of the additional nox enzyme subunits, nox4, p22 and p47 and rac1 were similar in skeletal muscle from mice bearing with cancer and cancer cachexia (table 2). The mrna level of sod antioxidant enzymes responsible for the dismutation of o2 were measured in mice with cancer and cancer cachexia . The mrna level of the sod1 and sod2 were significantly lower in the skeletal muscle from cachectic mice when compared to mice with cancer alone (p = 0.007; p <0.001, table 2). Similarly, the mrna level of the h2o2 scavenging antioxidant enzyme gpx was lower in skeletal muscle from cachectic mice compared to mice with cancer alone (p <0.001, table 2). However, the antioxidant enzymes sod3 and catalase had similar mrna levels in both groups (table 2).table 2gene expression in skeletal muscle of mice with cancer and cancer cachexiacancercachexiagene expression (au) nox211.8 2.35.0 1.1 * p22phox88.7 2.975.5 10.5 p40phox21.4 2.87.9 1.6 * p47phox5.7 1.27.3 1.4 p67phox367 92.715.6 3.3 * nox44.3 0.68.5 2.7 rac13.6 1.17.5 1.6 sod1350 36.5120 37.8 * sod2122 8.059.4 5.4 * sod32.6 0.31.8 0.3 gpx23.5 2.312.8 1.2 * catalase49.3 7.343.2 5.6the values represent the mean semp <0.003, statistically significant differences gene expression in skeletal muscle of mice with cancer and cancer cachexia the values represent the mean sem p <0.003, statistically significant differences changes in the mrna levels of the key nox2 enzyme subunits nox2, p40 and p67lead to further analysis of protein expression . The protein expression of the regulatory and catalytic nox2 enzyme subunits nox2 and p40 were lower in cachectic mice when compared to mice with cancer alone (p = 0.021; p <0.012, fig . 2). However, the protein expression of p67 was similar in skeletal muscle from mice in both groups (fig . 2).fig . 2protein expression levels of nox2 enzyme subunits in the skeletal muscle of mice with cancer and cancer cachexia . Statistically significant differences * p <0.05 . A nox2, bp40phox, cp67pho protein expression levels of nox2 enzyme subunits in the skeletal muscle of mice with cancer and cancer cachexia . Statistically significant differences * p <0.05 . A nox2, bp40phox, cp67pho to validate changes in mrna and protein levels, sod1, sod2, catalase and gpx activity levels were measured in skeletal muscle of mice with cancer and cancer cachexia . Sod1 and gpx activities were significantly lower in skeletal muscle from cachectic mice (sod1 9.9 0.9 u / mg protein, gpx 0.74 0.22 mmol / min / mg protein) when compared to mice with cancer alone (sod1 11.2 0.8 u / mg protein, gpx 1.48 0.08 mmol / min / mg protein) (p <0.003, table 3). However, levels of sod2 and catalase activity were similar in both groups of (table 3). H2o2 levels were similar in skeletal muscle from cachectic mice and mice with cancer alone (table 3).table 3enzyme activity and h2o2 levels in skeletal muscle of mice with cancer and cancer cachexiacancercachexiasod1 (u / mg protein)23.3 0.817.8 0.9sod2 (u / mg protein)11.2 0.89.9 0.8catalase (mmol / min / mg protein)4.1 0.43.5 0.2gpx (mmol / min / mg protein)1.48 0.080.74 0.22h2o2 (m)1.11 0.071.15 0.04the values represent the mean semp <0.003, statistically significant differences enzyme activity and h2o2 levels in skeletal muscle of mice with cancer and cancer cachexia the values represent the mean sem p <0.003, statistically significant differences evidence of this was found with increased levels of o2 detected by dhe fluorescence in the skeletal muscle of cachectic mice, when compared to non - cachectic mice . However, the nox enzyme systems do not appear to be the source of increased o2 in cancer - induced skeletal muscle cachexia . In fact, we observed a decrease in the expression of the nox2 enzyme subunits, nox2, p40 and p67 in cachectic skeletal muscle . Although this study would imply an additional source of o2 production in cancer - induced skeletal muscle cachexia, what may be of greater importance is the decrease in antioxidant enzyme function, primarily responsible for o2 dismutation . While this study may be indicative of a lack of compensation by sod1 and gpx, it may give evidence for their depression, in response to cancer induction and lack of dismutation of even normal cellular o2 production . The decrease in antioxidant gene expression and activity that function as part of the cellular defence system, for the elimination of o2 and h2o2, may indicate antioxidant dysfunction in cachectic skeletal muscle, rather than an increase in ros production . Cancer induces changes in skeletal muscle that lead to an imbalance in protein synthesis and degradation, resulting in muscle protein loss and function . Interestingly, not all cancer patients develop this secondary condition, and it is this phenomena that has made the development of cancer cachexia relatively undefined [1, 2]. In order to investigate this complex condition, we investigated a model that utilises two similar mac models, only one of which induces the secondary muscle wasting condition of cancer cachexia . The significant decline in body weight (1525%) and skeletal muscle mass that was observed in mice bearing the mac16 tumour, demonstrated the development of cancer - induced skeletal muscle cachexia . While the mac16 cancer cachectic model is well - established in nmri mice, this study mimics the cachectic condition in mac16 tumour - bearing nude mice . This model of cancer and cancer cachexia establishes a direct comparison between a cancer control and cancer cachectic mouse model . Contrasting studies have implicated the importance of calorie restriction in the development of cancer cachexia; however, our study, like others, did not observe any change in food intake from cachectic mice . This study therefore suggests a more complex catabolic - mediated response to protein degeneration, in the development of cancer cachexia . The physical changes in skeletal muscle were mirrored by significant cellular oxidative changes in response to mac16 induction and cachectic development . In particular, the significant increase in o2 levels observed in cachectic skeletal muscle indicates that o2 is implicated in the cancer cachectic condition . Previous studies have proposed a central mediating role for ros in the development of cancer cachexia [5, 18]; however, the source of ros in this process remains to be elucidated . The knowledge that the nox enzyme system functions primarily to produce o2, recognises nox - generated o2 as more than a by - product of cellular metabolism, but rather a product of a regulated response to stimuli for a physiological purpose . This function of nox makes this enzyme system the potential source of o2, vulnerable to changes associated with the cancer condition and development of cachexia . To our knowledge, this study is the first to investigate the nox enzymes in cancer cachectic skeletal muscle . Interestingly, the nox2 enzyme has consistently been shown to increase in degenerative conditions; however, this study does not support an increase in the nox2 enzyme in cancer cachexia . It is evident, however, from this study and others, that the absence of a functioning antioxidant enzyme system to dismutate o2 is present in cancer cachectic skeletal muscle . Cellular oxidative stress has been implicated in skeletal muscle wasting conditions with marked attenuation following antioxidant induction . Despite the increase in o2- in cachectic skeletal muscle that is indicative of a need for sod activity furthermore, our sod gene expression data would appear to suggest that the inability for sod to compensate for the increase in o2 is regulated at the gene level . Alterations in this important protective system can lead to oxidative imbalance, and induce critical changes to cellular structure and function . Antioxidant enzymes are valuable indicators of ros production as well as changes in cellular redox signalling and evidence of cell functionality . The consequences of sod antioxidant enzyme modifications have been demonstrated in studies investigating sod knockout models . Muller et al . Demonstrated an age - dependent loss of muscle mass in mice lacking sod1, as well as a significant decrease in their average life span . Furthermore, sun et al . Investigated the effects of sod1 and sod2 overexpression in drosophila that demonstrated a decrease in cumulative oxidative damage and increased metabolic potential, with an increased life span by up to 37% and 75%, respectively . These models demonstrate a crucial role for sod1 shown through its significant contribution to oxidative damage and muscle degeneration . It is also not surprising that with a decline in o2 dismutation by sod, to convert o2 to h2o2, the gene expression and activity of the h2o2 scavenging antioxidant enzyme gpx would also decrease . Consequently, these important cellular systems would indeed contribute to significant changes in redox - sensitive signalling pathways in cancer cachexia . Sod, not only has a crucial role in eliminating o2 accumulation, but also plays an important role in intracellular redox - sensitive signalling and regulation of oxidative systems . As ros have been described as important mediators of redox - sensitive intracellular signalling, so too are the antioxidants that regulate them . It is well - known that with the generation of intracellular o2, sod1 functions to dismute o2 to h2o2 . H2o2 in particular, has been shown to be involved in numerous signalling cascades, and therefore, its cellular regulation, via sod, has the potential to influence a number of important cellular pathways . The changes in sod1 function would indeed have critical consequences to cellular function that is most certainly redox - related . A circulatory protein, pif was first described as a causative agent in cachexia when it was discovered in mice expressing the mac16 tumour . As this glycoprotein was initially discovered in the sera of mice bearing the cachectic mac16 tumour, but not in the sera of the mice bearing the non - cachectic mac13 tumours, it was regarded as an important factor in the development of cancer cachexia . The accelerated loss of skeletal muscle protein in cachexia has been attributed to ub - proteasome pathway activation [15, 26], and pif has been shown to induce this pathway of skeletal muscle atrophy . Although the pif / ub pathway indeed plays an important role in the development of cancer cachexia, a number of additional mechanisms are most likely involved . Preproteasomal mechanisms, mediators, receptor binding, signalling pathways and activation of specific transcription factors are all important considerations and ros have been implicated in these cellular processes . Collectively, the results of this study suggest influential signalling involving o2 and antioxidant enzymes in cancer cachectic skeletal muscle . However, the signalling pathway(s) leading to the decrease in the nox2 subunits and antioxidant enzyme expression, together with decreased antioxidant enzyme activity in cachectic skeletal muscle remains undefined . In response to ros, cells activate the expression of a number of genes via transcription factor regulation, leading to modifications in the gene expression of important proteins, including antioxidant enzymes and those involved in muscle protein synthesis and regeneration . Furthermore, it is possible to speculate that the downregulation of the nox2 enzyme system is a compensatory response to o2 accumulation, induced primarily by sod and gpx antioxidant system dysfunction and is therefore a regulated response to o2 build - up in the cellular system . However, with the knowledge that ros can cause damage to cellular proteins, it is possible that the decrease in the nox2 enzyme subunits and antioxidant enzymes is a result of protein oxidation . With this in mind, it would be important to investigate these oxidative systems and the oxidative status of cachectic skeletal muscle during the progression of the disease . It is evident from cachectic studies that show the presence of oxidised proteins and attenuation following antioxidant administration that ros play an important role in the development of cachexia . Although the exact mechanisms are poorly defined, experimental research in the nox enzyme systems have indicated a number of important roles for ros, in addition to direct oxidative tissue damage, for its involvement in redox - sensitive signalling pathways . While studies have implicated the involvement of ros in the pathogenesis of cachexia and have suggested a role for nox, this study suggests that the increase in o2 in cachectic skeletal muscle is a result of antioxidant dysfunction . However, what remains unclear is whether this result is a regulated response in the cellular system or an important contributor to the changes observed in skeletal muscle physiology . Furthermore, with the well - established role for the ub - proteasome pathway in cancer cachexia, the increase in o2 levels in our study provides evidence of a signalling role for o2 in this pathway of skeletal muscle atrophy in cancer cachexia . While these results, along with the additional findings of this study indeed demonstrate complex changes in cachectic skeletal muscle, this multifactoral condition coupled with a multifunctional system, further demonstrates the complexity of skeletal muscle response(s) to cancer induction . It is therefore important to understand further the role that these oxidative and antioxidative systems play in the skeletal muscle system and development of cancer cachexia.
Pig - keeping and pork consumption in eastern and southern africa (esa) are rising rapidly as demand for pork increases and rural and peri - urban families discover pig farming to be profitable and cost effective . In uganda, the establishment of piggeries and increased pig production by rural farmers is encouraged by government and forms a part of central government agricultural planning . In some instances, local governments are supplying piglets to rural families in order to promote an alternative source of income . Pigs are considered low - input livestock which, as accomplished scavengers, can grow to market size on minimal feed inputs from the farmer . Humans serve as the definitive host of the zoonotic helminth parasite taenia solium, harbouring the adult tapeworm (infection termed taeniosis), which is acquired through the consumption of under - cooked pork (the larval cysticerci in the consumed pork develop into adult tapeworms in the human gut). Pigs acquire larval t. solium infection (infection termed cysticercosis) by ingestion of t. solium eggs passed in human faeces (i.e., coprophagy). Humans may also be infected with cysticercosis from ingestion of t. solium eggs through faecal - oral contamination . If cysts form in human brain tissue, a neurological disorder known as neurocysticercosis results; this commonly manifests as epileptic seizures . Cysticercosis is acquired in settings with poor household and/or personal hygiene and thus can be acquired by both pork eaters and nonpork eaters; t. solium is emerging as a serious agricultural and public health problem in the esa region . In eastern africa, the disease has been reported in tanzania, kenya, uganda, burundi [8, 9] and rwanda, though it is known to be substantially under - reported in both humans and pigs . Epidemiological field research is required for improving our understanding of both the prevalence and risk factors of this parasitic disease, while also investigating the transmission dynamics [11, 12]. The aim of the present work is to determine the prevalence of cysticercosis among domestic pigs by b158/b60 ag - elisa in the districts of kamuli and kaliro, south - east uganda, to provide baseline information on the level of exposure in this region . Anecdotally, kamuli is known to be a source of pig meat for the burgeoning demand of urban kampala, the capital city, and consumption of pork meat amongst the local population is also extensive . There have been no recent epidemiological studies on human taeniasis, human cysticercosis or porcine cysticercosis in uganda; assessing the infection rate in the pig population has been shown to be a good indicator of t. solium risk to humans . Kamuli and kaliro districts in uganda were chosen for the study as pig keeping is known to have become popular in these districts . Kaliro was formally part of kamuli district (until 2005 when it became a district in its own right). The study area had an estimated pig population of 25,860, as determined from the 2003 - 2004 agricultural census conducted by the district veterinary office (dvo) with a spatial distribution as shown in figure 1 . The pigs can best be described as the local breeds; the percentages of the cross breeds cannot be estimated with certainty since there are no breeding records kept . Previous studies elsewhere in east africa have found a prevalence of cysticercosis infection among pigs in some areas to be around 20% [2, 6]; no such studies have previously been published for our study area . The sample size for a cross - sectional survey to estimate prevalence was calculated based on random sampling of pigs and inflated to account for the uncertainty and multi - stage design . With an expected prevalence of 20%, an accepted error of 5% and 95% confidence interval, the minimum number to sample was 246 pigs, which, doubled, was 492, rounded to 500 . We aimed to sample pigs in every parish and the 500 samples were allocated to the overall total of 129 parishes (which are within 21 sub - counties). Sampling was proportionally allocated to each parish, depending on the pig population of that parish, so that parishes with more pigs had a higher proportion of samples . Animal health workers provided a complete list of homesteads in each parish from which homes were selected randomly and pigs to sample were also randomly chosen during the home to home visits . Pigs were bled from the cranial vena cava and blood collected into plain vacutainer tubes and then allowed to clot at ambient temperature and later centrifuged to separate the serum which was extracted and stored at 20c until use . The age of the pigs sampled was recorded and a total of 528 homesteads were visited for this purpose in the two districts . The pig husbandry system was assessed by direct observation and discussions with the pig owners . In each village visited during the home to home pig survey, the availability of pit latrines and information on deworming of pigs by the veterinary staff in the area was captured at every fifth homestead . The b158/b60 ag - elisa for the detection of circulating antigens of t. solium cysticerci was conducted as described by brandt et al ., and modified by dorny et al . . The test was carried out at the school of veterinary medicine, university of zambia, lusaka (a regional reference laboratory for cysticercosis). Two monoclonal antibodies (moab) sourced from the institute of tropical medicine (itm), antwerp, belgium were used . The first was moab b158c11a10 diluted at 3 g / ml in carbonate buffer (0.06 m, ph 9.6) and used for coating while the second biotinylated moab b60h8a4 diluted at 2.5 g / ml in phosphate buffered saline - tween 20 (pbs - t20) + 1% new born calf serum (nbcs) was used as detector antibody . The incubations were carried out at 37c on a shaker during 30 minutes for the coating of the first moab and during 15 minutes for all sub - sequent steps . Blocking was done by adding 150 l of pbs - t20 + 1% nbcs per well . Without washing the plate, 100 l of pre - treated sera at a dilution of 1/4 all steps [addition of second moab, streptavidine and orthophenylene diamine (opd)] were done after washing the plates five times with pbs - t20 . This was followed by addition of 100 l of biotinylated moab b60h8a4 diluted at 3.2 g / ml in pbs - t20/1% nbcs . Hundred microlitres of streptavidin - horseradish peroxidase (jackson immunoresearch lab, inc .) Diluted at 1/10,000 in pbs - t20/1% nbcs was added to act as conjugate . Thereafter, 100 l of the opd solution and h2o2 was added and incubated without shaking in the dark at room temperature . To stop the reaction, 50 l of 4n h2so4 the plates were read using an elisa reader at 492 nm . To determine the cut - off, the optical density (od) of each serum sample was compared with a series of 8 reference negative serum samples (of ugandan pigs) at a probability level of p = .001 . 161 of the samples were collected in the new kaliro district, with the remainder (352) in kamuli . For the purposes of this analysis overall, 74% of pig owners reported that their home had access to a pit latrine, and 18.2% had previously de - wormed their pigs, indicating a relatively low level of veterinary intervention involving the pig population . Of the 513 samples, 480 were screened with the b158/b60 (inadequate volume of serum available in 33 samples). Forty - one pig samples were positive for cysticercosis antigen (see table 1), or 8.5% (95% ci 611%), with no significant differences by age group (= 1.355, df = 2, p = .508). Positive parishes are shown in figure 2; 36 of the 129 (28%) parishes in the study area showed at least 1 b158/b60 ag - elisa positive pig among the randomly selected individuals sampled from the total population . Free range management was found to be the most common method of pig husbandry, based on observations and farmer reports, with the tethering of pigs usually only taking place when neighbours protested against the free roaming of the animals . Although there are previous studies on porcine cysticercosis in uganda [18, 19] this is the most recent, albeit preliminary, field survey for porcine cysticercosis in the country, and has investigated the prevalence of antigen - positive pigs across an entire rural district of south - east uganda . The district under study has seen, in line with many other parts of uganda, large increases in pig production over the past few years; pigs are relatively cheap and easy to keep in rural areas, where their husbandry has been actively encouraged . The study region is also thought to be a source for pig meat consumed in the capital city, kampala pork consumption has been estimated at 5,000 kg per year in pork - roasting centres of kampala city alone (cw, pers . 8.5% of 480 pigs screened were seropositive for the parasite by b158/b60 ag - elisa . Preliminary data from other studies conducted in 2008 (fao tcp / uga/3104 project; in prep .) Indicate similar results by lingual examination, which may indicate a rising prevalence since lingual examination is less sensitive and if the samples from the tcp are subjected to the b158/b60 ag - elisa, a higher prevalence might be expected . The results generated in this study showed a lower prevalence than in other studies in esa in recent years with 23% recorded using ag - elisa in zambia; 17% recorded using lingual examination in tanzania and 4050% recorded using ag - elisa in eastern cape province, south africa . These differences are, in themselves, intriguing; latrine provision was reportedly higher in this study than in zambia, for example,, possibly limiting pig access to infectious material . Although in zambia, latrine provision in itself was not found to be a risk factor for cysticercosis in pigs, the free range method of pig husbandry in the current study implies that those homes not using latrines may be a major risk facilitating access by pigs to infectious material . Cysticercosis infection patterns are known to vary spatially even in fairly restricted areas, and infection in pigs is a function of both human contamination of the environment and pig proximity to a human t. solium tapeworm carrier . This preliminary study should thus serve as a stimulant for the generation of larger and more comprehensive datasets on this neglected zoonotic disease in uganda; a particular focus of future work should be on the adult worm and antibody prevalence in humans, as well as risk factors for infection in both pigs and humans . In this regard, further surveys of pigs, seroprevalence surveys in humans and an understanding of cysticercosis - related epilepsy are required, together with risk - factor studies where human infection is found . This will enable a better understanding of the scale of the cysticercosis problem and consequent evidence - based decisions on appropriate methods for its control.
Many patients with bipolar disorder experience hypomania for periods less than the minimum episode length criterion of 4 days in the dsm - iv [14]. There is a need to increase focus on this brief hypomania since even subsyndromal symptoms may impair functioning and diminish the quality of life [57]. Additionally, some researchers feel that a shorter length criterion would better reflect the entire spectrum of bipolar disorder, and improve detection of bipolarity in patients with depressive episodes [2, 3, 810]. The criteria used to define an episode of hypomania are under revision as part of the ongoing dsm - v review process . Within this context, the primary objective of this study was to characterize brief hypomania lasting less than 4 days that is experienced by patients who meet the dsm - iv criteria for bipolar disorder . The secondary objective was to determine if the patients who experienced an episode of hypomania only when using a shortened criterion were a distinct subgroup from those who met the 4-day criterion during the study period . We have previously investigated the impact of changing the length criterion for an episode of hypomania from 4 days to 2 using daily self - reported mood ratings from 203 patients who were diagnosed with bipolar disorder using the dsm - iv criteria . As the minimum length criterion for an episode of hypomania decreased, a large increase was found in both the number of patients with episodes and the number of episodes . This study repeated the prior analysis using a larger sample and including a length criterion of a single day . The inclusion criteria were a diagnosis of bipolar disorder by dsm - iv criteria, age 18 years or older, currently receiving pharmacological treatment and a willingness to record mood daily for 5 months using computer software in their native language . All participants volunteered, provided informed written consent, and received treatment as usual throughout the study . The naturalistic approach with minimal exclusion criteria the diagnosis of bipolar disorder was made by the prescribing psychiatrist in a clinical interview . Patients from our prior study were included in this analysis, although some provided additional data . Patients provided a daily mood rating using the previously validated chronorecord software that was installed on a home computer [13, 14]. A 100-unit visual analogue scale was used to rate mood between the most extreme mania and depression the patient ever experienced . Based upon the validation studies comparing the self - ratings with clinician ratings on the hamilton depression rating scale (hamd) and the young mania rating scale (ymrs) [13, 14], a mood entry less than 40 was considered depression, 4060 euthymia, and greater than 60 hypomania / mania . The self - ratings of mania reflect activation levels for either euphoric or dysphoric mood . The demographic characteristics, mood ratings, and psychotropic medications taken by the patients were obtained . To be considered using a medication, a patient had to take any dose of the drug for at least 50% of the days . Episodes of hypomania and depression were determined using a published algorithm to calculate episodes from daily self - reported mood data based on the dsm - iv criteria . Episodes of hypomania were also calculated while varying the minimum duration between 3, 2, and 1 days . The demographic characteristics and medications taken were compared between patients with bipolar i and bipolar ii disorder, using the pearson 2-sided test for distributions and the independent sample 2-sided t test for mean values . For the subgroup analysis, the demographic characteristics and medications taken were compared between patients with one or more episode of hypomania using the 4-day length criterion, and those with one or more episode of hypomania only if a shorter criterion was used (4-day vs. 3-day length, 4-day vs. 2-day length, and 4 day vs. 1-day length). Data were available from 410 patients, 247 with a diagnosis of bipolar i disorder, 146 with a diagnosis of bipolar ii disorder, and 17 with a diagnosis of bipolar nos . Since the group with a diagnosis of bipolar nos was so small, they were excluded from this analysis leaving 393 patients . The 393 patients returned 75,284 days of data (mean 191.6 days). Of the 393 patients 266 (68%) were recruited from a university mood clinic, and 127 (32%) from a private practice . The demographic characteristics of those with bp i and bp ii disorder are shown in table 1 . Patients with bipolar i disorder had more hospitalizations (2.8 vs. 1.4, p = 0.002), took antidepressants less frequently (45 vs. 63%, p <0.001), lamotrigine less frequently (33 vs. 45%, p = 0.018), and lithium more frequently (32 vs. 21%, p = 0.018). No other significant differences in demographic characteristics were found between patients with bipolar i and bipolar ii disorder.table 1comparison of demographics of patients with diagnosis of bipolar i or bipolar ii disorderdiagnosisbipolar i (n = 247)bipolar ii (n = 146)total (n = 393)degrees of freedom (df)page (mean years, sd)39.4 (11.2)38.2 (11.4)39.0 (11.3)299.10.299age of onset (mean years, sd)22.6 (9.7)20.9 (10.9)22.0 (10.2)240.40.133years of illness (mean years, sd)16.9 (11.1)17.7 (12.3)17.2 (11.5)241.70.535hospitalizations (mean n, sd)2.8 (4.2)1.4 (3.2)2.3 (3.9)326.10.001gender10.059 female (n,%) 160 (65)108 (74)268 (68) male (n,%) 87 (35)38 (26)125 (32)education level20.274 high school (n,%) 33 (14)12 (9)45 (12) some college (n,%) 75 (33)45 (33)120 (33) college graduate (n,%) 121 (53)79 (58)200 (55)employment status20.079 disabled (n,%) 66 (29)25 (19)91 (25) working full - time (n,%) 102 (44)72 (56)174 (49) other (n,%) 61 (27)33 (25)94 (26)marital status20.370 married (n,%) 107 (46)66 (50)173 (47) single (n,%) 90 (39)52 (39)142 (39) divorced (n,%) 37 (15)14 (11)51 (14)number of daily medications (mean n, sd)2.8 (1.6)2.6 (1.5)2.7 (1.6)322.80.330taking mood stabilizer (n,%) 204 (83)111 (76)315 (80)10.115taking lithium (n,%) 78 (32)30 (21)108 (28)10.018taking valproate (n,%) 49 (20)32 (22)81 (21)10.622taking lamotrigine (n,%) 82 (33)66 (45)148 (38)10.018taking antidepressant (n,%) 110 (45)92 (63)202 (51)1<0.001taking antipsychotic (n,%) 114 (46)57 (39)171 (44)10.169taking benzodiazepine (n,%) 51 (21)35 (24)86 (22)10.441taking sleep medication (n,%) 18 (7)7 (5)25 (6)10.328student s t test, equal variances not assumedpearson chi - square test comparison of demographics of patients with diagnosis of bipolar i or bipolar ii disorder student s t test, equal variances not assumed pearson chi - square test the impact of changing the minimum length requirement for an episode of hypomania is shown in detail in table 2 . When the minimum episode length was decreased from 4 to 2 days, the percent of days spent in a hypomanic episode by each patient nearly doubled (4.58.2%), and the number of patients experiencing a hypomanic episode nearly doubled (102190). When the minimum episode length was decreased to 1 day, the percent of days spent in a hypomanic episode by each patient more than doubled (4.510.6%), and the number of patients experiencing a hypomanic episode was more than 21/2 times greater (102271).table 2impact of changing the minimum episode length for hypomania for patients with bipolar disorderhypomanic episode minimum length% change4 days3 days2 days1 dayn%n%n%n%42 day41 dayall patients (n = 393) mean percent days in hypomanic episode4.55.98.210.682136 mean percent days in depressed episode8.28.08.07.3211 number of hypomanic episodes3054918632,164183610 number of depressed episodes303293289264513 number of patients with hypomanic episodes1022614537190482716986166 number of patients with depressed episodes1132911429114291082814all hypomanic days (n = 6,188) hypomanic days in hypomanic episode2,699443,356544,169676,18810054129 hypomanic days not in hypomanic episode3,489562,832462,019330042100all depressed days (n = 15,699) depressed days in depressed episode5,988385,901385,839375,5783627 depressed days not in depressed episode9,711629,798629,8606310,1216424all hypomanic days bipolar i (n = 3,772) hypomanic days in hypomanic episode1,650442,046542,531673,77210053126 hypomanic days not in hypomanic episode2,122561,726461,241330042100all hypomanic days bipolar ii (n = 2,416) hypomanic days in hypomanic episode1,049431,310541,638682,41610056130 hypomanic days not in hypomanic episode1,367571,10646778320043100 impact of changing the minimum episode length for hypomania for patients with bipolar disorder with a 4-day length requirement, 56% of reported days of hypomania and 62% of days of depression occurred outside of an episode . When the length requirement was decreased to 2 days, 33% of hypomanic days occurred outside of an episode . With a 1-day length requirement, there was a 13% decrease in depressed episodes for all patients, as the occurrence of brief hypomanic interrupted depressed episodes based on the algorithm . As the episode length criterion decreased, the pattern of change was very similar for patients with bipolar i and bipolar ii disorder . There was no significant difference in the percent of days of hypomania reported outside of an episode between patients with bipolar i and bipolar ii disorder whether the minimum length was 4 days (p = 0.173), 3 days (p = 0.212), or 2 days (p = 0.404). When the minimum length was 1 day, all hypomania was included in an episode . With a 4-day length criterion, with a 2-day length criterion, there were 88 additional patients, and with a 1-day length, 169 additional patients . The comparison of the demographic characteristics of the 102 patients who experienced at least one episode of hypomania with a 4-day length, with the additional patients who experienced a hypomanic episode as the length criterion was shortened is shown in table 3 . As the length criterion decreased, a significantly smaller percentage of patients who had a hypomanic episode were taking antipsychotics (4-day vs. 2-day, 53 vs. 38%, p = 0.033; and 4-day vs. 1-day, 53 vs. 39%, p = 0.026).table 3demographics of patients with 4 day hypomanic episodes compared to additional patients with 3, 2, and 1 day hypomanic episodes4 day hypomanic episode3 day hypomanic episode2 day hypomanic episode1 day hypomanic episodepatients (n = 102)additional patients (n = 43)dfpadditional patients (n = 88)dfpadditional patients (n = 169)dfpage (mean years, sd)38.9 (11.9)40.4 (9.0)103.10.40039.5 (11.0)187.10.68739.8 (11.3)204.90.524age of onset (mean years, sd)23.8 (11.3)20.1 (10.3)79.90.06820.6 (10.9)169.20.05421.4 (10.7)188.70.102years of illness (mean years, sd)15.5 (10.7)20.0 (10.8)73.10.03219.0 (12.2)158.40.05018.4 (12.6)221.10.054hospitalizations (mean n, sd)2.8 (5.0)2.5 (3.1)120.40.5481.8 (2.5)137.20.0791.9 (2.8)125.10.075diagnosis20.27910.65010.925 bipolar i (n,%) 64 (63)31 (72)58 (66)107 (63) bipolar ii (n,%) 38 (37)12 (28)30 (34)62 (37)gender10.42310.70510.570 female (n,%) 71 (70)27 (63)59 (67)112 (66) male (n,%) 31 (30)16 (37)29 (33)57 (34)education level20.54420.73620.423 high school (n,%) 13 (14)3 (8)9 (11)18 (11) some college (n,%) 35 (38)15 (37)29 (35)51 (32) college graduate (n,%) 45 (48)22 (55)44 (54)91 (57)employment status20.16920.03020.103 disabled (n,%) 21 (24)11 (28)20 (25)37 (23) working full - time (n,%) 51 (59)17 (42)33 (41)75 (48) other (n,%) 15 (17)12 (30)27 (34)46 (29)marital status20.92820.92820.927 married (n,%) 45 (48)20 (50)36 (46)78 (49) single (n,%) 34 (37)15 (37)30 (38)59 (37) divorced (n,%) 14 (15)5 (13)13 (16)21 (13)number of daily medications (mean n, sd)2.8 (1.6)2.7 (1.4)86.80.7692.6 (1.6)184.70.3102.6 (1.5)206.30.183taking any mood stabilizer (n,%) 80 (78)36 (84)10.46769 (78)10.997133 (79)10.959taking lithium (n,%) 30 (29)11 (26)10.64020 (23)10.29740 (24)10.295taking valproate (n,%) 24 (24)11 (26)10.79220 (23)10.89634 (20)10.507taking lamotrigine (n,%) 34 (33)14 (33)10.92829 (33)10.95664 (38)10.451taking antidepressant (n,%) 50 (49)23 (54)10.62344 (50)10.89386 (51)10.766taking antipsychotic (n,%) 54 (53)17 (40)10.14033 (38)10.03366 (39)10.026taking benzodiazepine (n,%) 27 (27)11 (26)10.91120 (23)10.55135 (21)10.274taking sleep medication (n,%) 10 (10)3 (7)10.5865 (6)10.2938 (5)10.104student s t test, equal variances not assumedpearson chi - square test demographics of patients with 4 day hypomanic episodes compared to additional patients with 3, 2, and 1 day hypomanic episodes student s t test, equal variances not assumed pearson chi - square test hypomania lasting less than 4 days occurred frequently in this study of patients diagnosed with bipolar disorder using the dsm - iv criteria . As the minimum length criterion for an episode of hypomania decreased, there was a large increase in both the number of patients experiencing an episode and in the number of episodes . When comparing a 2-day minimum length to a 4-day minimum length, there were almost twice as many patients with an episode of hypomania, and about twice as many episodes . These results were very similar to our prior findings, with the current sample about double in size . The majority of symptomatic days occurred outside of a dsm - iv episode, which agrees with prior longitudinal studies of bipolar disorder [1618]. With a 4-day minimum length, more than half of the days of hypomania occurred outside of an episode . Even with a 2-day minimum length, one - third of all hypomania remained outside of an episode . Single days of hypomania were so frequent that with a length criterion of 1-day, 271 patients (69%) experienced an episode . For any episode length, the percent of hypomanic days occurring outside of an episode did not differ between bipolar i and ii disorder . Additionally, about 60% of the reported days of depression occurred outside of an episode regardless of the length criterion for hypomania . In prior research based on clinician interviews, and including patients with depressive disorders, most hypomania lasted for 13 days [1, 2, 8, 9, 19], which is consistent with the current findings . The high frequency of brief hypomania in this sample suggests that if the length criterion for an episode of hypomania is decreased, the frequency criterion should be increased, as frequency may be an important dimensional aspect of brief hypomania . The current study cannot address whether decreasing the minimum length criterion for an episode of hypomania would improve the accuracy of the diagnosis of bipolar disorder . While most of the hypomania reported in this study was brief and self - limited, in a sample of patients who are all receiving treatment, the high frequency of hypomanic symptoms occurring outside of a dsm - iv episode is troublesome . Although some patients, especially with bipolar ii disorder, report improved functioning when experiencing mild hypomania [20, 21], subsyndromal or residual symptoms of mania are associated with an increased risk of relapse [22, 23]. Furthermore, cumulative morbidity in bipolar disorder, even from mild symptoms, may be associated with more functional impairment than the total number of episodes [12, 24, 25]. Hypomania is associated with a high risk for divorce, and over a lifetime, an increased use of health services, an increased need for social welfare, and an increase in suicidal behaviors . Clinicians should probe patients with bipolar disorder for brief hypomanic episodes, and assess whether these are interfering with everyday functioning . One clinical benefit of daily charting may be to improve the detection and monitoring of subsyndromal mood symptoms . As found in our earlier study, there was no significant difference in the occurrence of 1-day hypomania between patients with bipolar i and bipolar ii disorders . Additionally, there were no significant demographic differences between patients who met the 4-day length criterion and those who only experienced a hypomanic episode with a shorter length criterion . It does not appear that either experiencing brief hypomanic episodes or single days of hypomania are useful parameters for distinguishing subgroups within a sample of patients who meet the dsm - iv criteria for bipolar disorder ., the patients in this sample varied in disease severity, phase of illness and medications taken . All mood ratings used in this analysis were self - reported, and the chronorecord mood rating does not measure the specific components of hypomania / mania or depression . While a chronorecord rating of mania / hypomania best reflects activation levels, this is not a specific measure of overactivity which may be a core feature of hypomania [2, 8, 26]. Additionally, clinical diagnosis of a hypomanic episode traditionally emphasizes behavioral manifestations and functional impairment . However, the demographic profile of the patients who use chronorecord is similar to that in other studies of bipolar disorder . This study did not include patients with bipolar disorder who were not receiving treatment, and patients who never experience hypomania that lasts for 4 days . Finally, this study did not address the issues of differentiating brief hypomania from normal mood elevation, or bipolar disorder from unipolar depression . In conclusion, decreasing the episode length criterion for hypomania will significantly increase the number of patients with episodes and the aggregate number of episodes.
India currently has surpassed china as having the largest number of suicides in the world, a situation made even more alarming by the unusually large proportion of youth - including young women - that complete suicide in the country . Suicide patterns in rural central india have been described in some studies; however, patterns across different tribal and ethnic groups have been insufficiently researched to date . This paper describes the profile of suicide attempters presenting to a rural secondary - level hospital in a predominantly tribal area in central india, with the aim to identify the areas of potential future research toward preventive strategies . Padhar hospital is a 200-bed, multispecialty, lutheran mission hospital in the betul district of the state of madhya pradesh . The psychiatry department at padhar is currently the only full - time mental health facility in a radius of 200 km . The department provides outpatient and inpatient care for psychiatric disorders and also runs a community mental health project, project shifa, covering 75 surrounding villages . It also provides consultation - liaison services to other departments, including for all patients who survive after presenting to the hospital with deliberate self - harm . The population served is multi - ethnic, predominantly consisting of tribal populations such as the gonds as well as hindi - speakers, and a few large minorities such as a second - generation bengali refugee population from bangladesh . Padhar hospital is a 200-bed, multispecialty, lutheran mission hospital in the betul district of the state of madhya pradesh . The psychiatry department at padhar is currently the only full - time mental health facility in a radius of 200 km . The department provides outpatient and inpatient care for psychiatric disorders and also runs a community mental health project, project shifa, covering 75 surrounding villages . It also provides consultation - liaison services to other departments, including for all patients who survive after presenting to the hospital with deliberate self - harm . The population served is multi - ethnic, predominantly consisting of tribal populations such as the gonds as well as hindi - speakers, and a few large minorities such as a second - generation bengali refugee population from bangladesh . A retrospective chart review was done of all patients who were admitted in padhar hospital between june 2014 and april 2016 with presenting complaints of deliberate self - harm and survived till they underwent inpatient psychiatric consultation . Although 82 patients met inclusion criteria, 2 patients were excluded as their charts could not be traced . Data were collected from the charts using a structured data collection sheet that included both demographic and clinical details . Forty - five (56%) of our patients were male, and 35 (44%) were female . Apart from ten patients (12%) of other ethnicity (including seven bengalis), the overwhelming majority were members of the local hindi - speaking or gondi tribal populations . Seventy - six patients (95%) were hindus, with two each from christian and muslim backgrounds . Fifty - five patients (69%) were from rural agrarian families while 25 (31%) were from urban or semi - urban backgrounds . The overwhelming majority of the patients were young, with 74 (93%) of our patients 41 males and 33 females below the age of 45 years . As regards marital status, 23 (51%) of the males were single and 21 (47%) were married while one was a widower . This contrasted somewhat with the female patients, only 12 (34%) of whom were single whereas 22 (63%) were married and one was a widow . A surprising finding was that very few only five (6%) lived alone; all the rest lived with family members . Only 11 (14%) patients had a previous history of suicide attempt . As regards psychiatric syndromes, 53 (67%) patients 34 (76%) males and 19 (54%) females had psychiatric syndromes that were identified during the psychiatric consultations . Among these 34 males with psychiatric syndromes, the most common condition was alcohol use disorders with 22 (49%), followed by nine (20%) with anxiety and other neurotic disorders, seven (16%) with personality disorders, three (7%) with depression, three (7%) with delirium, and one each with psychosis, compromised intelligence, school refusal, and headache syndromes . The picture was considerably different among the 19 females with psychiatric syndromes, with depression topping the list with seven (20%) followed by five (14%) with personality disorders, four (11%) with alcohol use disorders, three (9%) with delirium, and three (9%) with anxiety and other neurotic disorders . Only 11 (14%) patients in all had nonpsychiatric chronic medical illnesses . As regards the nature of the suicide attempts, only 11 (14%) were planned attempts, the vast majority of attempts 69 (86%) were thus impulsive in nature . Only 17 (21%) patients could be described as having high intentionality at the time of the attempt, and a mere four (5%) patients had active suicidal ideas at the time of the psychiatric consultation . Apart from a single hanging attempt, the modalities of the attempts were of low lethality: 68 (85%) were ingestions of agricultural poisons, seven (9%) were drug overdoses, and three (4%) were self - mutilations . Fifty - eight (73%) patients had a clear antecedent precipitating event; 41 (71%) of these were interpersonal disputes (which were mostly with family members). In addition, ten (13%) of the attempts (nine males and one female) were made under the influence of alcohol, often with the patient having little to no recollection of the actual circumstances surrounding the attempt . Regarding background psychosocial stressors, surprisingly, few five (6%) reported financial issues as contributory; in contrast to this as many as 36 (45%) reported background interpersonal conflicts . All patients reviewed underwent brief individual and family counseling, with a focus on crisis intervention, support and education about suicidal risks and precautions, along with a verbal no - suicide contract and education about contact mechanisms in case of emergencies . Among the 53 (66%) patients with diagnosed psychiatric syndromes, a total of 37 (70%) patients were prescribed various psychotropic drugs . In addition, 57 (71%) patients were advised or offered regular outpatient psychotherapy . However, only 22 (28%) patients followed up in the psychiatry opd at least once after discharge from the hospital, and one patient was recorded as having died in hospital after the psychiatric consultation . Only three (4%) forty - five (56%) of our patients were male, and 35 (44%) were female . Apart from ten patients (12%) of other ethnicity (including seven bengalis), the overwhelming majority were members of the local hindi - speaking or gondi tribal populations . Seventy - six patients (95%) were hindus, with two each from christian and muslim backgrounds . Fifty - five patients (69%) were from rural agrarian families while 25 (31%) were from urban or semi - urban backgrounds . The overwhelming majority of the patients were young, with 74 (93%) of our patients 41 males and 33 females below the age of 45 years . As regards marital status, 23 (51%) of the males were single and 21 (47%) were married while one was a widower . This contrasted somewhat with the female patients, only 12 (34%) of whom were single whereas 22 (63%) were married and one was a widow . A surprising finding was that very few only five (6%) lived alone; all the rest lived with family members . Only 11 (14%) patients had a previous history of suicide attempt . As regards psychiatric syndromes, 53 (67%) patients 34 (76%) males and 19 (54%) females had psychiatric syndromes that were identified during the psychiatric consultations . Among these 34 males with psychiatric syndromes, the most common condition was alcohol use disorders with 22 (49%), followed by nine (20%) with anxiety and other neurotic disorders, seven (16%) with personality disorders, three (7%) with depression, three (7%) with delirium, and one each with psychosis, compromised intelligence, school refusal, and headache syndromes . The picture was considerably different among the 19 females with psychiatric syndromes, with depression topping the list with seven (20%) followed by five (14%) with personality disorders, four (11%) with alcohol use disorders, three (9%) with delirium, and three (9%) with anxiety and other neurotic disorders . Only 11 (14%) patients in all had nonpsychiatric chronic medical illnesses . As regards the nature of the suicide attempts, only 11 (14%) were planned attempts, the vast majority of attempts 69 (86%) were thus impulsive in nature . Only 17 (21%) patients could be described as having high intentionality at the time of the attempt, and a mere four (5%) patients had active suicidal ideas at the time of the psychiatric consultation . Apart from a single hanging attempt, the modalities of the attempts were of low lethality: 68 (85%) were ingestions of agricultural poisons, seven (9%) were drug overdoses, and three (4%) were self - mutilations . Only one patient had presented with attempts in more than one modality . Fifty - eight (73%) patients had a clear antecedent precipitating event; 41 (71%) of these were interpersonal disputes (which were mostly with family members). In addition, ten (13%) of the attempts (nine males and one female) were made under the influence of alcohol, often with the patient having little to no recollection of the actual circumstances surrounding the attempt . Regarding background psychosocial stressors, surprisingly, few five (6%) reported financial issues as contributory; in contrast to this as many as 36 (45%) reported background interpersonal conflicts . All patients reviewed underwent brief individual and family counseling, with a focus on crisis intervention, support and education about suicidal risks and precautions, along with a verbal no - suicide contract and education about contact mechanisms in case of emergencies . Among the 53 (66%) patients with diagnosed psychiatric syndromes, a total of 37 (70%) patients were prescribed various psychotropic drugs . In addition, 57 (71%) patients were advised or offered regular outpatient psychotherapy . However, only 22 (28%) patients followed up in the psychiatry opd at least once after discharge from the hospital, and one patient was recorded as having died in hospital after the psychiatric consultation . Only three (4%) there was an unexpectedly high proportion of young men attempting suicide (especially associated with alcohol use); this is in contrast to most studies which suggest that females tend to attempt suicide more commonly . The rather low prevalence of psychiatric disorders in these patients, in general, is consistent with other indian studies and suggests that psychosocial, cultural, and environmental factors play at least as important a part, if not more, in causation as mental illness . It appears that the vast majority of these attempts were isolated, impulsive attempts done in anger after a specific interpersonal dispute (usually with a close family member), often under the influence of alcohol . Neither serious mental health issues such as depression and psychosis nor commonly - assumed long - term psychosocial stressors such as financial issues seem to be present in the majority of cases . Although a majority of the cases were impulsive attempts (86%), there was history of previous self - harm only in a minority of cases (14%), and only a small percentage of patients (16% of males and 14% of females) were diagnosed with personality disorders . Why impulsivity seems to be so dominant a factor in these presentations is an area for potential future research both in terms of identifying psychopathological targets for primary prevention as well as exploring the sociocultural determinants of impulsivity in this area . Surprisingly, for a rural culture where joint family systems are so central a way of life, it appears that the most common background stressors or precipitating issues were actually family interpersonal conflicts . A factor long assumed to be protective as far as suicide goes the strong joint family system actually appears to be the most common immediate trigger for suicide attempts in this population . This is in stark contrast to the standard literature on suicide, particularly from western countries where living alone is a significant risk factor . We suggest that community level family interventions that target interactions in families with high - risk patients (such as those with alcohol use disorders, depression, and past history of impulsive behavior) might help to address this problem, particularly by sensitizing family members to be vigilant for high - risk triggers (such as a major interpersonal dispute in the context of alcohol intoxication). The vast majority of the attempts were agricultural poison ingestion (pesticides, insecticides, or fertilizers) mostly because these are easily available and within anyone's reach in these rural communities . Since a considerable majority of these were impulsive attempts of low intentionality, it appears that merely limiting accessibility of these agricultural products could potentially have prevented these attempts . This represents as important area for primary prevention strategies as has already been successfully demonstrated in a rural area in tamil nadu . The strengths of this study include its rural, resource - poor setting as well as its attempts to identify locally - relevant and population - specific targets for exploring primary prevention strategies . First, the sample size is still too small for making significant analytical comparisons; consequently, the interpretations must be considered only provisional until a more substantial sample size can be recruited in the future . Second, only patients who presented to the hospital after a suicide attempt were included, and this probably represents a very selective population among suicide attempters . It may not be possible to generalize findings from the study population to other groups such as those who died before reaching the hospital or who could not come to the hospital due to financial or accessibility issues . Employing community - based surveys and research using government and legal records in addition to clinical data from the hospital records may help broaden the nature of the sample population in future studies in this area . Nevertheless, we think that simple and locally - relevant research work like this by rural and resource - poor service providers might be a step in the direction of developing adaptive solutions to the problem of suicide in the country . These preliminary findings suggest that a considerable number of suicide attempts in rural madhya pradesh perhaps even a majority are isolated impulsive attempts following triggers that are most often interpersonal disputes, many times under the influence of alcohol . Young men seem to be attempting suicide in greater numbers than would be expected, and alcohol use disorders are frequently associated with this trend . The most common psychiatric syndrome present among the attempters is alcohol use disorder, followed by depression, and personality disorders . Psychosocial stressors, especially interpersonal conflicts within the family appear to be at least as important factors as mental illnesses in contributing to suicide attempts in the area.
Laparoscopic radical prostatectomy (lrp) and retropubic radical prostatectomy (rrp) are well - established procedures for the management of localized prostate cancer . Recently, cancer treatment strategies have focused more on patient quality of life after surgery, thus requiring minimally invasive techniques . In 2002, an initial experience with extraperitoneal laparoscopic radical prostatectomy (elrp) showed similar oncological outcomes to intraperitoneal prostatectomy without intraperitoneal complications . Due to the low perioperative and postoperative mortality rate, postradical prostatectomy incontinence, which interferes with the patient's quality of life, has become an issue . One - year continence rates are excellent after rrp and lrp in larger series . However, the early return of continence remains a challenge and has a major impact on the postoperative patient's health - related quality of life . Further understanding of the anatomy of adjacent structures of the prostate, such as the bladder neck, urethra, fascia, ligaments, and neurovascular bundle (nbv), led to the development of nerve - sparing elrp (nselrp) and the most recent intrafascial nselrp, which shows a positive surgical outcome of early continence . In this study, we report our experience with 50 consecutive patients who underwent intrafascial nselrp by a single surgeon . Among 62 patients, 50 patients with clinically localized prostate cancer at the time of preoperative magnetic resonance image (mri) with psa levels less than 10 ng / ml and gleason scores less than 7 (3 + 4) were included for intrafascial nselrp from november 2011 to april 2012 . If there were any suspicious lesions with extracapsular extension of tumors or high gleason scores (7 [4 + 3] to 10), conventional radical prostatectomy and lymph node dissection were performed . A small group of patients underwent intrafascial nselrp despite their higher psa levels of more than 10 ng / ml because they had low gleason scores (6). In short, the preparation of the space of retzius began with a 2-cm sized incision in the infraumbilical crease laterally to the midline, which was then carried down to the posterior rectus sheath where a balloon trocar was inserted and the preperitoneal space was developed . Finally, the camera trocar was placed and further trocars were inserted with attention to the course of the epigastric vessels and peritoneum . Next, the 12-mm assistant port, 12-mm operator port, and 5-mm operator port were placed sequentially . The fatty tissue surrounding the prostate and pelvic space was removed to expose the landmark structures such as the pubic arch, symphysis, endopelvic fascia, bladder, and prostate . The anterior surfaces of the bladder and prostate as well as the endopelvic fascia became visible to the operator . The removal of preprostatic fibro - fatty tissues facilitates more definite differentiation between the prostate and the bladder . The endopelvic fascia and pubo - prostatic ligament were not incised and the deep dorsal venous complex was not ligated at the beginning of the procedure . A bilateral incision of the periprostatic fascia was made medial to the puboprostatic ligament (ppl) and directed to the base of the prostate . The right plane of dissection was recognized when the surface of the prostate was completely smooth . As a result, the development of a plane between the prostate and overlaying fascia was possible and the operator could detach the prostate from its enveloping fascia . All lateral periprostatic fascia, endopelvic fascia, and puboprostatic fascia remained intact (fig . Careful coagulation - free dissection is necessary during bilateral seminal vesicle dissection because the pelvic plexus and nvb run in close proximity to the tip of the seminal vesicle ., somerville, nj, usa) were used to ligate the seminal vesicular artery . When the vas deferens and seminal vesicle were both fully dissected, the denonvilliers fascia was visualized . The denonvilliers fascia was stripped down to find the correct plan for the intrafascial dissection of the prostate . After identification of the prostate capsule, the dissection was gradually directed toward the apex of the prostate in the midline to avoid injury of the nbvs . The prostate was fully detached from its surrounding fascias but was still attached by the pedicles and the apex . The assistant took the seminal vesicle and vas deferens and gently elevated them ventrally to allow clear sight of the prostatic pedicle . The prostatic pedicles were then clipped and cut in a step - by - step manner directly on the surface of the prostatic capsule without excessive traction or electrocautery . When dissecting the nbv, care must be taken to avoid damage to the nvb . After the correct plane was opened, the dissection was performed by using cold scissors in an essentially avascular plane . Meticulous dissection is needed between the lateral side of the prostate and the lateral remnant of the periprostatic fascia toward the apex of the prostate to preserve the accessory nvb (fig . The dissection was performed on the opposite side of the prostate pedicle and the nvb in the same fashion as that of the primary side . Finally, the prostate was completely detached from the surrounding fascias, bladder, prostatic pedicles, and nvbs . Sharp dissection of the prostate from the external sphincter and urethra at the site of the apex was performed . The assistant retracted the prostate to secure a clear view of the apex margin of the prostate . Dissection and division of the external urethral sphincter and prostate were carefully made (fig . Dissection of the urethra was performed proximally very close to the prostate to preserve the urethral length as much as possible . Vertical dissection was performed with cold scissors alone to achieve complete division of the prostate from the urethra . After identification of no contrast leakage at the anastomosis site, the foley catheter was removed . Postoperative evaluation of continence was performed by evaluation of the number of pads used per day . Postoperative follow - up was defined at 2 weeks, 6 weeks, and 3 months . Complete continence was defined as usage of no pads and patient's report of no urinary leakage . 18.0 (ibm co., armonk, ny, usa), with statistical significance considered at p<0.05 . Spearman's correlation test was used to evaluate the factors related to early continence recovery after nselrp . Among 62 patients, 50 patients with clinically localized prostate cancer at the time of preoperative magnetic resonance image (mri) with psa levels less than 10 ng / ml and gleason scores less than 7 (3 + 4) were included for intrafascial nselrp from november 2011 to april 2012 . If there were any suspicious lesions with extracapsular extension of tumors or high gleason scores (7 [4 + 3] to 10), conventional radical prostatectomy and lymph node dissection were performed . A small group of patients underwent intrafascial nselrp despite their higher psa levels of more than 10 ng / ml because they had low gleason scores (6). In short, the patient was placed in the supine position with mild head - down tilt . The preparation of the space of retzius began with a 2-cm sized incision in the infraumbilical crease laterally to the midline, which was then carried down to the posterior rectus sheath where a balloon trocar was inserted and the preperitoneal space was developed . Finally, the camera trocar was placed and further trocars were inserted with attention to the course of the epigastric vessels and peritoneum . Next, the 12-mm assistant port, 12-mm operator port, and 5-mm operator port were placed sequentially . The fatty tissue surrounding the prostate and pelvic space was removed to expose the landmark structures such as the pubic arch, symphysis, endopelvic fascia, bladder, and prostate . The anterior surfaces of the bladder and prostate as well as the endopelvic fascia became visible to the operator . The removal of preprostatic fibro - fatty tissues facilitates more definite differentiation between the prostate and the bladder . The endopelvic fascia and pubo - prostatic ligament were not incised and the deep dorsal venous complex was not ligated at the beginning of the procedure . A bilateral incision of the periprostatic fascia was made medial to the puboprostatic ligament (ppl) and directed to the base of the prostate . The right plane of dissection was recognized when the surface of the prostate was completely smooth . As a result, the development of a plane between the prostate and overlaying fascia was possible and the operator could detach the prostate from its enveloping fascia . All lateral periprostatic fascia, endopelvic fascia, and puboprostatic fascia remained intact (fig . Careful coagulation - free dissection is necessary during bilateral seminal vesicle dissection because the pelvic plexus and nvb run in close proximity to the tip of the seminal vesicle . Five - millimeter titanium clips (ligaclip, ethicon inc ., somerville, nj, usa) were used to ligate the seminal vesicular artery . When the vas deferens and seminal vesicle were both fully dissected, the denonvilliers fascia was visualized . The denonvilliers fascia was stripped down to find the correct plan for the intrafascial dissection of the prostate . After identification of the prostate capsule, the dissection was gradually directed toward the apex of the prostate in the midline to avoid injury of the nbvs . The prostate was fully detached from its surrounding fascias but was still attached by the pedicles and the apex . The assistant took the seminal vesicle and vas deferens and gently elevated them ventrally to allow clear sight of the prostatic pedicle . The prostatic pedicles were then clipped and cut in a step - by - step manner directly on the surface of the prostatic capsule without excessive traction or electrocautery . When dissecting the nbv, care must be taken to avoid damage to the nvb . After the correct plane was opened, the dissection was performed by using cold scissors in an essentially avascular plane . Meticulous dissection is needed between the lateral side of the prostate and the lateral remnant of the periprostatic fascia toward the apex of the prostate to preserve the accessory nvb (fig . The dissection was performed on the opposite side of the prostate pedicle and the nvb in the same fashion as that of the primary side . Finally, the prostate was completely detached from the surrounding fascias, bladder, prostatic pedicles, and nvbs . Sharp dissection of the prostate from the external sphincter and urethra at the site of the apex was performed . The assistant retracted the prostate to secure a clear view of the apex margin of the prostate . Dissection and division of the external urethral sphincter and prostate were carefully made (fig . Dissection of the urethra was performed proximally very close to the prostate to preserve the urethral length as much as possible . Vertical dissection was performed with cold scissors alone to achieve complete division of the prostate from the urethra . In short, the patient was placed in the supine position with mild head - down tilt . The preparation of the space of retzius began with a 2-cm sized incision in the infraumbilical crease laterally to the midline, which was then carried down to the posterior rectus sheath where a balloon trocar was inserted and the preperitoneal space was developed . Finally, the camera trocar was placed and further trocars were inserted with attention to the course of the epigastric vessels and peritoneum . Next, the 12-mm assistant port, 12-mm operator port, and 5-mm operator port were placed sequentially . The fatty tissue surrounding the prostate and pelvic space was removed to expose the landmark structures such as the pubic arch, symphysis, endopelvic fascia, bladder, and prostate . The anterior surfaces of the bladder and prostate as well as the endopelvic fascia became visible to the operator . The removal of preprostatic fibro - fatty tissues facilitates more definite differentiation between the prostate and the bladder . The endopelvic fascia and pubo - prostatic ligament were not incised and the deep dorsal venous complex was not ligated at the beginning of the procedure . A bilateral incision of the periprostatic fascia was made medial to the puboprostatic ligament (ppl) and directed to the base of the prostate . The right plane of dissection was recognized when the surface of the prostate was completely smooth . As a result, the development of a plane between the prostate and overlaying fascia was possible and the operator could detach the prostate from its enveloping fascia . All lateral periprostatic fascia, endopelvic fascia, and puboprostatic fascia remained intact (fig . Careful coagulation - free dissection is necessary during bilateral seminal vesicle dissection because the pelvic plexus and nvb run in close proximity to the tip of the seminal vesicle . Five - millimeter titanium clips (ligaclip, ethicon inc ., somerville, nj, usa) were used to ligate the seminal vesicular artery . When the vas deferens and seminal vesicle were both fully dissected, the denonvilliers fascia was visualized . The denonvilliers fascia was stripped down to find the correct plan for the intrafascial dissection of the prostate . After identification of the prostate capsule, the dissection was gradually directed toward the apex of the prostate in the midline to avoid injury of the nbvs . The prostate was fully detached from its surrounding fascias but was still attached by the pedicles and the apex . The assistant took the seminal vesicle and vas deferens and gently elevated them ventrally to allow clear sight of the prostatic pedicle . The prostatic pedicles were then clipped and cut in a step - by - step manner directly on the surface of the prostatic capsule without excessive traction or electrocautery . When dissecting the nbv, care must be taken to avoid damage to the nvb . After the correct plane was opened, the dissection was performed by using cold scissors in an essentially avascular plane . Meticulous dissection is needed between the lateral side of the prostate and the lateral remnant of the periprostatic fascia toward the apex of the prostate to preserve the accessory nvb (fig . The dissection was performed on the opposite side of the prostate pedicle and the nvb in the same fashion as that of the primary side . Finally, the prostate was completely detached from the surrounding fascias, bladder, prostatic pedicles, and nvbs . Sharp dissection of the prostate from the external sphincter and urethra at the site of the apex was performed . The assistant retracted the prostate to secure a clear view of the apex margin of the prostate . Dissection and division of the external urethral sphincter and prostate were carefully made (fig . Dissection of the urethra was performed proximally very close to the prostate to preserve the urethral length as much as possible . Vertical dissection was performed with cold scissors alone to achieve complete division of the prostate from the urethra . All patients underwent cystography on the 4th postoperative day . After identification of no contrast leakage at the anastomosis site, postoperative evaluation of continence was performed by evaluation of the number of pads used per day . Postoperative follow - up was defined at 2 weeks, 6 weeks, and 3 months . Complete continence was defined as usage of no pads and patient's report of no urinary leakage . Statistical analysis was performed by using pasw ver . 18.0 (ibm co., armonk, ny, usa), with statistical significance considered at p<0.05 . Spearman's correlation test was used to evaluate the factors related to early continence recovery after nselrp . The mean operation time was 149.3 minutes (range, 90 to 240 minutes). The mean hospitalization time was 6.3 days (range, 4 to 19 days), and the mean catheterization time was 5.5 days (range, 3 to 26 days). Postoperative continence results are shown in table 2 . At the 2-week follow - up after surgery, 14 patients (28.0%) achieved total continence and on average 2.3 pads were required for the other incontinent patients . A total of 35 patients (70.0%) were continent at 6 weeks after surgery, 8 patients (16.0%) had mild stress incontinence (1 to 2 pads), and 7 patients (14.0%) required> 2 pads per day (fig . 4). A total of 31 patients were available for follow - up at 3 months after surgery, and 26 patients (83.9%) were pad - free . Compared with conventional nerve - sparing lrp, intrafascial nselrp showed early continence recovery (table 2). In patients with a relatively old age of above 65 years, preoperative low prostate volume and low gleason score were related with early continence at 6 weeks after surgery (table 3). Signs of early recovery of potency (morning erection or erection sensation) were reported by 19 patients (38.0%) at 6 weeks after surgery and the rate increased to 54.8% at 3 months after surgery . But from the 30 cases, the positive surgical margin rate decreased to 9.5% (table 4). Two patients (4.0%) with anastomosis site leakage were treated with prolonged catheterization for an additional 1 week . Two patients (4.0%) with acute urinary retention after catheter removal were treated with re - catheterization for 1 week . It has been shown that the incidence of postoperative incontinence depends on the urologist's experience, patient's age (increased frequency after 70 years), and whether the operative technique includes minimal distal incision of the endopelvic fascia, preservation of the bladder neck, bilateral nerve - sparing surgery, or preservation of the ppl . In the american urological association guidelines, the reported risk of urinary incontinence ranges from 3 to 74% for radical prostatectomy . Continence mechanisms involve many structures, including the ppl, denonvilliers' fascia, levator muscle, endopelvic fascia, and internal and external sphincters . Ventrally, the proximal prostate is covered by muscle fibers originating from the outer longitudinal bladder muscle and extending over the gland . The pubovesical / ppls (pv / ppls) are paired fibrous bands originating from visceral endopelvic fascia . They insert on the distal third of the posterior surface of the pubic bone adjacent and anterior to the urethral sphincter . The visceral component of the endopelvic fascia covers the pelvic organs including the prostate, bladder, and rectum, and it is fused with the anterior fibromuscular stroma of the prostate at the upper ventral aspect of the gland [12 - 14]. Along the pelvic sidewall at the lateral aspect of the prostate and bladder, the parietal and the visceral components of the endopelvic fascia are fused . As a fascial condensation, this fusion is often recognizable as a whitish line and is named the fascial tendinous arch of the pelvis . Access to the lateral prostate may be gained by incision of the endopelvic fascia either medial or lateral to this fusion . The pv / ppls stabilize the prostate, urethra, and bladder to the pubic bone and are considered an important part of the " suspensory system " of the continence mechanism [15 - 19]. Some authors have suggested that preservation of these ligaments during radical prostatectomy may improve early recovery of urinary continence, but no definitive evidence has yet been established . Preservation of the pv / ppls is facilitated by using the perineal and laparoscopic approach, whereas during open retropubic prostatectomy, the pv / ppls are more difficult to preserve . Some authors have suggested that avoiding incision of the endopelvic fascia during radical prostatectomy, often combined with an intrafascial nerve - sparing procedure, might improve early recovery of urinary continence as well as improve postoperative erectile function, but definitive evidence has yet to be established . The incision of this fascia immediately lateral to the fascial tendinous arch incises the levator ani fascia (laf) and leaves the muscle fibers of the levator ani bare and the laf adherent to the prostate . An incision of the visceral endopelvic fascia medial to the fascial tendinous arch results in a dissection plane that leaves the levator ani muscle covered with its fascia without exposure of its fibers . The result is a prostate covered only by prostatic fascia (pf), when present, and not by a layer of laf . This fascia is not a discrete single - layered structure stretching over the lateral surface of the prostate . Laparoscopic surgery may offer an improved identification of these structures, resulting in less damage to the structures around the prostate . We could also identify these structures during the operation, which is related to recovery of postoperative urinary continence . In addition, others have showed that continence rates correlate with differences in the mean functional urethral length and the existing differences in the maximal urethral closure pressure postprostatectomy . Reported an earlier return of continence with a ppl - sparing technique versus a nonsparing technique, but the final outcomes were equivalent . We believe that during mobilization of the prostate and especially during apical dissection, the intactness of the urethral supporting structures are of paramount importance because this avoids shear stress to the urethra as well as possible denervation . In our study, we observed an earlier return to continence in patients who underwent intrafascial nselrp compared with previous conventional nerve - sparing lrp . Complete continence was achieved by 70.0% of patients who underwent nselrp at 6 weeks after surgery and by 83.9% at 3 months after surgery . In the control group, however, 55.2% of patients achieved complete continence at 6 weeks after surgery and 75.0% at 3 months after surgery . Reconstruction methods such as periurethral suspension stitch, bladder neck reconstruction, and posterior reconstruction have also been shown to provide improved early continence recovery, but the results are still controversial . Therefore, we focused on the preservation of normal structures rather than performing reconstruction after destruction of these structures . Studies have reported that older age may be the only increasing risk factor for postprostatectomy incontinence, but in our study, increased prostate volume and gleason score were related with post - prostatectomy incontinence in the older aged group (age over 65 years). We speculate that large prostate volume and old age may be related to preoperative bladder and external urethral sphincter dysfunction leading to interference with postprostatectomy recovery of continence . A preoperative urodynamic study may be helpful in proving the relationship between prostate volume and old age with postoperative incontinence . Anatomical studies have illustrated the prostatic neuroanatomy in detail, detecting additional neural tissues to the nerve bundles on the anterior midpart and posterior surface of the prostate . Costello et al . Recently showed that most of the nvb descends posteriorly to the seminal vesicle . The nerves pass anteriorly and converge at the midprostatic level, and when they approach the apex, they diverge again . The anterior and posterior nerves of the nvb are separated by 3 cm at the level of the base of the prostate . At this anatomic site, the cavernosal nerves are not easily distinguished from the surrounding tissues and care should be taken during urethra - vesical anastomosis . Walsh proposed that the nvb is enclosed within the two layers of the lateral pelvic fascia composed of the lateral layer of the levator fascia and the medial layer of the pf . Kiyoshima et al . Proved that the nvb was located on the posterolateral region of the prostate in 48% of their patients . In the remaining patients (52%), the nvb was widely distributed on the entire lateral aspect of the prostate without any specific localization . Thus, the authors proposed performing wide dissection of the lateral aspect of the prostate during radical prostatectomy to preserve the nvb . Therefore, meticulous dissection and disuse of electro - cauterization at the lateral and apex sides of the prostate are required to preserve the nvb by intrafascial nselrp . Anastasiadis et al . Reported potency rates of 30% and 41% at 12 months after lrp (n=230) and open retropubic prostatectomy (n=70), respectively . After preservation of one or both nvbs, the potency rates increased from 37 to 44% with the retropubic approach and from 46 to 53% with lrp, respectively . Patients younger than 60 years who underwent bilateral nvb preservation were reported to be potent in 72% and 81% of cases, respectively . Reported rates of erections of 96.5%, 90.7%, and 84.3% and rates of intercourse of 69.0%, 52.8%, and 37.3% at 12 months after bilateral rrp in men <55 years, 55 to 65 years, and> 65 years, respectively . In our study, the patients were generally old aged and were not sexually active; thus, we could not obtain potency recovery data . However, 19 patients (38.0%) reported subjective signs of potency recovery (morning erection or erection sensation) at 6 weeks after surgery, and at 3 months after surgery, the percentage was increased to 54.8% of patients . The oncologic data of 1,000 lrps at the montsouris institute revealed positive surgical margin rates of 6.9% for pt2a and 34% for pt3b tumors . In our study, the overall positive surgical margin rate was 34.0% . We consider this high positive margin rate as a " learning curve . " In fact, from 30 cases, positive surgical margin rates decreased compared with the first 30 cases (51.7% vs. 9.5%). The initial results are promising, providing favorable functional outcomes and oncologic results similar to other rp techniques.
Internal nasal valve incompetence (invi) is an often overlooked cause of nasal obstruction which in turn can mistakenly be attributed to other anatomical variations such as septal deviation and turbinate hypertrophy . It is characterised by the collapse during inspiration of the upper lateral cartilages; a narrowing of the angle between the dorsal septum and upper laterals can also contribute but to a lesser extent . A wide range of techniques exists in which surgeons use alar batten and/or spreader grafts, butterfly grafts, lateral crural strut grafts, alar rim grafts, and lateral suspension sutures . Alar batten grafts were first shown by toriumi et al . As an effective technique for correction of internal nasal valve collapse . Since then they have been widely used, but a reliable case series has yet to be published . When single - technique series are published, they often concentrate on the cosmetic outcomes . Recent papers have called for a single technique by a single operating surgeon to be assessed . Predictors of which patients will benefit from alar batten graft functional rhinoplasty are also not well described . Without actively examining for invi, a clinician may not identify the cause of symptoms which many patients find to have a considerable impact on their quality of life . Various scoring systems such as the nose scale do not specifically relate to the symptoms of invi . While clinicians could carry out rhinomanometry to assess the severity of invi, how this relates to symptom improvement following an invasive procedure, there is a need for a simple yet effective method for assessing patients preoperatively . We present a large case series of one specific technique, alar batten grafts, to treat invi; this includes the use of nasal strips as a positive predictor in the success of surgery . One hundred and seven consecutive patients with invi seen in the routine ear, nose, and throat clinics of the east and north hertfordshire nhs trust district were included in this prospective study over a seven - year period . All patients were assessed pre - operatively by the senior author for clinical confirmation of invi . This assessment included verification of nasal airflow restriction, worse on inspiration, in either newly presenting patients or those not previously improved by other surgical procedure(s). Patients were assessed for a positive cottle's manoeuvre for evidence of invi on inspiration . They were also assessed for airflow improvement after decongestion with xylometazoline 0.1% spray whereby those cases with nasal congestion secondary to inferior turbinate hypertrophy who improved with decongestion were excluded . Patients were asked to complete a pre - operative questionnaire detailing previous procedures, nasal trauma, and the use of nasal strips during pre - operative assessment . All patients were asked to try adhesive nasal strips prior to surgery and asked if the strips had improved nasal breathing . Visual analogue scales (vas) (0100 mm) were used to assess the degree of nasal blockage (where 0 is the least possible blockage and 100 the worst possible) and quality of life (where 0 is severe impact on quality of life and 100 excellent quality of life). This was completed at pre - operative assessment and then postoperatively at the appropriate follow - up appointments, at 1 week, 6 weeks, 6 months, and 12 months . The wilcoxon signed - rank test was used to assess the nonparametric data at the post - operative time intervals indicated above . Some patients were followed up for longer periods of time with the vas but numbers were too low to be clinically relevant . It was the senior author's practice to use batten grafts only as the single method for correction of invi . The aim of this technique was to strengthen the lateral walls, thus preventing internal nasal valve collapse, rather than increasing the cross - sectional space or reconstructing the external nasal valve . All patients were consented for functional rhinoplasty with graft harvesting from septal or auricular cartilage . Following preparation of the nose with moffat's solution, a killian's incision was made . The cartilage was assessed for size and quality intraoperatively and kept in 0.9% saline solution until required . If the amount of septal cartilage harvested was deemed insufficient in amount and/or quality, auricular cartilage was harvested instead . Conchal cartilage was obtained following marking the antihelical fold through the posterior aspect of the pinna with bonney's blue ink applied with a 16 g gauge (green) needle . An incision was made on the posterior surface of the pinna along the line of the blue marks, conchal cartilage harvested, and the incision closed with dissolvable sutures . Grafts were fashioned in strips from the harvested cartilage, measuring 1220 mm by 68 mm . This was dependent on the dimension of the lateral nasal wall as evaluated by the senior author and also partially related to the quality of quadrilateral cartilage available . To raise pockets for graft insertion, an incision was made at the dorsal edge of the septum at the junction of the upper lateral cartilages (ulcs). The caudal edges of the ulcs were dissected just to the edge of the piriform aperture and a pocket fashioned . Following this, area between the ulcs and lateral crura of the lower lateral cartilages (see figure 1). The scroll area itself is left intact with the graft laying cephalic to the s - shaped scroll . Once inserted, the grafts were secured with 5/0 ethilon to the septum to prevent migration . Eighty - five patients (79.4%) responded to post - operative questionnaires reevaluation of their symptoms using the exact vas assessment tool used pre - operatively . Post - operative followup (and repeat of vas assessment) was performed according to patient clinical need, and hence patients were assessed at different post - operative time points . For those patients who completed both pre- and post - operative questionnaires (67 patients), 91% of patients reported long - term improvement in nasal blockage on vas using the wilcoxon signed - rank test; p values for this test can be seen in table 1 . Nasal breathing: prior to surgery, the median vas of nasal blockage (0 mm = no blockage, 100 mm = full blockage) was 73.5 (range 18100). Post - operatively, this improved to 32 mm, 17 mm, 15 mm, 19 mm, and 25 mm for 1-week, 6-week, 6-month, 12-month and 18-month periods respectively (see table 1 for ranges). This is displayed in figure 2, and 3 patients felt there was a decline in nasal patency following surgery; 2 patients experienced an initial improvement then decline . Quality of life scores: similar to nasal patency scores, quality of life scores also improved post - operatively (albeit based on a slightly lower figure of 59 patients, 88%). Two patients felt there was no improvement in qol following surgery, 3 patients felt there was a decline, and 3 patients felt an initial improvement followed by decline . Of the 107 patients initially included in the study, one patient developed unsightly nostril asymmetry as a result of inadvertent hitching of the graft to the lower lateral cartilage necessitating a subsequent cosmetic surgical treatment . The graft was seen to have resorbed significantly necessitating further surgery using auricular cartilage graft replacement for the original septal batten grafts; revision grafting was successful in all occasions . One patient required removal of the graft at a second procedure as the initial procedure had failed to improve nasal airflow and the graft was deemed unsightly . Following initial examination, fifty - four patients did so and of them 46 patients found that the nasal strips improved their symptoms . Following surgery, 49 patients within this group had improvement in post - operative nasal blockage scores (odds ratio 2.152 (95% confidence interval 0.3034.001)). This was similar to quality of life scores with odds ratio of 2.580 (95% confidence interval 0.5664.594). The technique concentrates on improving the structural integrity of the internal nasal valve rather than increasing its cross - sectional area . This is thought to be the key in treating this condition and probably accounts for the improvements in nasal obstruction score experienced by patients . Symptomatic invi is due to significant collapse of the ulcs during nasal inspiration either during exercise or at rest . Alar batten grafts improve the rigidity of the ulcs preventing collapse during negative upper airway pressure during inspiration (with septoplasty where necessary, although all patients have undergone a degree of submucous resection for graft harvesting or during previous septal surgery at a separate date). As collapse of the internal nasal valve is a dynamic process, the alar batten graft is not required to improve the overall nasal patency . Our study has provided evidence that the procedure is helpful to patients not only for short - term 6 weeks but also for longer periods of time . It is difficult within a busy nhs department (from which the majority of patients were recruited) to follow up nhs patients a long term due to a variety of factors the most important of which are demanding on - clinic follow - up slots, frequent change in clinical staff and patient migration to other areas . We readily admit that longer followup would be desirable but we feel that the current data as presented is at least useful . Another area for criticism is the use of a vas to assess quality of life; while not recognised as a specific health utility to assess this aspect of surgical outcome, it does provide a useful insight into patient satisfaction in regard to their nasal surgery with minimal time input for the user . We felt that just assessing nasal blockage as the sole outcome would be too biased opinion in the technique and we wanted a more holistic view of outcomes but appreciated that patients did not always have time to complete lengthy questionnaires . Several different types of grafts have been used such as lateral crural strut grafts and spreader grafts, sometimes in combination with alar batten grafts . Graft insertion is often combined with other procedures such as inferior turbinate reduction and can concentrate on cosmetic outcome rather than functional results or the correction of external nasal valve correction . This case series adds valuable data to previously published work and is one of the only case series performed by one operating surgeon . Many patients presenting with invi have had previous nasal surgery with unsuccessful results / outcomes . We feel that this is often due to mis - diagnosis leading to unsatisfactory outcomes following routine nasal procedures . We would advise clinicians to include cottle's manoeuvre when performing routine examination of the nose for nasal obstruction although it should only be used in quiet inspiration as applicable to routine respiration . We would advise this during general inspection of the nose and prior to decongestion with xylometazoline spray . In our experience, patients with invi often find immediate relief during this manoeuvre although it can be nonspecific and should not be used as the sole basis for recommending surgery for invi . Nasal strips are a useful positive predictor of those who will benefit from alar batten grafts . By using nasal strips in pre - operative assessment, the clinician can identify those patients who will improve both in terms of nasal obstruction and in quality of life (odds ratio 2.15 and 2.58, resp . ). Ideally all patients in our study would have used the strips but as these are not provided on the nhs and come therefore at the patients' own cost, we did not insist on patients' participation . Inspiratory nasal function but to our knowledge the relationship between this and post - operative outcomes has not previously been documented . With such a simple, noninvasive test, clinicians now have an excellent diagnostic tool in the investigation of such patients . Clinicians should use cottle's manoeuvre to examine all patients with nasal obstruction prior to application of nasal decongestants but must recognise that its results are user dependent and that oversupporting the upper lateral cartilages will give a false impression of possible surgical outcomes.alar batten graft to provide structural integrity of the internal nasal valve is a reliable technique with good outcomes in both nasal patency and quality of life scores . This technique has been refined by the senior author and shows that alar batten grafts alone (rather than in combination with spreader grafts) are adequate.improvement in nasal patency with nasal strips is a good predictor of those patients who will benefit from alar batten grafting . Clinicians should use cottle's manoeuvre to examine all patients with nasal obstruction prior to application of nasal decongestants but must recognise that its results are user dependent and that oversupporting the upper lateral cartilages will give a false impression of possible surgical outcomes . Alar batten graft to provide structural integrity of the internal nasal valve is a reliable technique with good outcomes in both nasal patency and quality of life scores . This technique has been refined by the senior author and shows that alar batten grafts alone (rather than in combination with spreader grafts) are adequate . Improvement in nasal patency with nasal strips is a good predictor of those patients who will benefit from alar batten grafting.
Osteopetrosis is a rare bone disease, with an estimated prevalence of 1 case in every 20,000 people (1). This disease, caused by metabolic imbalances resulting from genetic mutations, was first reported in 1904 by the german radiologist heinrich albers - schnberg, as reviewed by bollerslev (2) and garca et al (3). Within one of a limited number of reports on this disease, garca et al (3) demonstrated osteomyelitis of the mandible in a patient with osteopetrosis . The pathophysiological mechanism of the disease involves an osteoclastic dysfunction that results in an impaired bone resorption ability; as a result, bone modeling and remodeling are inhibited . The cortical bone mass is therefore able to increase in density and develop into a hard, marble consistency when the medullary bone is remodelled . Two types of osteopetrosis exist: malignant and benign, categorized based on mortality rate, and disease severity is highly heritable (1). Malignant osteopetrosis, or autosomal recessive osteopetrosis has a poor prognosis associated with fatality in infancy . Patients with aro demonstrate a survival rate of 7%, even subsequent to curative treatment such as allogenic hemopoietic stem cell transplantation (4). Currently, it is understood that autosomal dominant osteopetrosis (ado) is a relatively benign disorder caused by a missense mutation in the clcn7 gene (5). Benign osteopetrosis typically has a late onset in children and adults, the symptoms are milder and prognosis is better than in malignant osteopetrosis . However, curative care is not available for patients with the benign form, thus only supportive care is provided . Osteopetrosis is difficult to treat and can cause complications, such as chronic osteomyelitis and bone fractures, as a result of poor blood supply and iatrogenic fractures . As there is no cure, the current study presents a case of chronic mandibular osteomyelitis and a maxillary sinusitis - generated fistula of benign osteopetrosis . The current recommended treatment options include high - dose systemic antibiotics, debridement of the necrotic region and hyperbaric oxygen (hbo) therapy (3). However, the current report describes treatment without the use of high - dose antibiotics or hbo therapy, a justified approach due to the insufficiency of the blood supply to the bone . Treatment of the debrided fistula, which included repeated flushing and cleaning using nasal endoscopy every 2 weeks in the acute infected period, was successful . Therefore, the present study investigated whether the debridement and drainage of pus from fistulas generated by maxillary sinusitis and chronic osteomyelitis is an effective treatment method . In december 2014, a 50-year - old chinese female was admitted to the xiamen chang gung hospital (xiamen, china) for the evaluation and treatment of chronic purulent nasal discharge in the left side of the nose . The patient reported that the discharge appeared 3 years previously, following a tooth extraction from the left side of the mouth . Intraoral examination revealed exposed necrotising bone in the left maxillary region, sixth, seventh and eighth tooth agenesis in the upper left side and fistula formation . The pus had accumulated in the mouth and the left side of the nose in the fistula, generated as a result of maxillary sinusitis . Previous medical history revealed that the patient was diagnosed with osteopetrosis at the age of 19 years old, with six rib, femoral and thoracic vertebra bone fractures (fig . 1). The patient did not accept any treatment for the osteopetrosis, but was fitted with a plaster cast for 6 weeks . However, the patient developed splenomegaly due to secondary bone marrow suppression, subsequent to which the spleen was resected . Examination of the patient's family history revealed that her older sibling was diagnosed with osteopetrosis at the age of 23 years old, but the patient's child had not developed the disease . Non - contrast computed tomography scans (ge healthcare life sciences, chalfont, uk) displayed evidence of low bone density in the posterior left side of the mandible with destruction of the vestibular cortex that was suggestive of a fistula, resulting from chronic osteomyelitis with maxillary sinusitis (fig . A bone marrow puncture, hematoxylin and eosin staining and brightfield imaging of these structures were performed . The medullary cavity was obliterated and the bone marrow was reported to be suppressed, determined by hematoxylin and eosin staining using an olympus bx51 brightfield microscope (fig . The sequestra were removed and debridement of the necrotic region was performed via nasal endoscopy, without antibiotics or any other form of treatment a month later, intravenous low - strength antibiotic therapy (1 g cefazolin, every 8 h; mosinter group, ltd ., ningbo, china) was administered for a week, and the fistula was flushed and drained of pus via nasal endoscopy every 2 weeks . After a week, the infection had been successfully treated without the necessity of additional antibiotic treatment, chronic osteomyelitis was resolved and the fistula was replaced with fresh granulation after 2 months (fig . Osteopetrosis is a rare hereditary disease caused by an imbalance in bone metabolism, resulting from decreased osteoclast activity (6). This leads to a lack of bone remodeling, which can result in a high mortality rate in children within the first 10 years of their life (6). Early onset osteopetrosis is known as severe infantile malignant autosomal recessive disease, or intermediate mild autosomal recessive disease (6). This condition is typically fatal as a result of anemia with congestive heart failure or sepsis due to bone overgrowing in the bone marrow space . In adults, the same disease is known as benign ado with a late onset, and has a lower mortality rate (3). At present, the curative treatment for malignant osteopetrosis is allogenic hemopoietic stem cell transplantation (4); however, only supportive care is available for benign osteopetrosis (7). Therefore, symptom management of benign osteopetrosis is important to increase the patient survival rates . The present study reported the case of a patient diagnosed with benign osteopetrosis at the age of 19 years, with fractures identified based on presentation and radiology . In benign osteopetrosis, the most common complications are chronic osteomyelitis, occurring in 10% of cases involving the mandible (3), and bone fractures, in 78% of cases (8). The patient in the current study presented with chronic purulent nasal discharge in the left side of the nose, following tooth extraction from the left side of the mouth 3 years earlier associated with chronic osteomyelitis . Since the healing process in the bone is slow, it is difficult to fight and prevent infection in osteopetrosis (1). In ado, care must be taken when removing the sequestra, since it is easy to create iatrogenic fractures due to dense bone, which are difficult to repair once the bone is fractured (9). Currently, administering a high - dose of systemic antibiotics with debridement of the necrotic region alongside hbo therapy is recommended (3). However, as the patient in the current report presented with insufficient blood supply to the bone due to osteopetrosis, the use of high - dose antibiotics was not suitable . Instead, pus from the fistula was flushed and drained via nasal endoscopy every 2 weeks for 1 month, subsequent to careful removal of the sequestra and debridement of the necrotic region . Low dose and intensity antibiotics were administered following debridement, and the infection was cleared . To the best of our knowledge, this is the first case report indicating that debridement with low doses of antibiotics may be effective in the treatment of osteopetrosis . Notably, patients with osteopetrosis should be aware of good dental care and oral hygiene . Decayed teeth should be endodontically treated and tooth extraction should be avoided, if possible, on the account of necrosis development . Care must therefore be taken when removing the necrotic bone to prevent iatrogenic fractures, which may occur due to inexperience of the surgeon (9). The findings of the present study demonstrated that debridement and drainage of pus from fistulas are important, and possibly a more effective strategy compared with high - dose antibiotic usage alone in osteopetrosis . In conclusion, the complications of osteopetrosis include bone fractures and chronic osteomyelitis, which are difficult to treat . In a patient with osteopetrosis currently, there is no cure for osteopetrosis and, therefore, symptom management is important to increase the patient survival rates . In the present study, it was demonstrated that debridement and drainage of pus from fistulas may be more effective than high - dose antibiotic usage in treating osteopetrosis.
Hepatic injury of various etiologies, such as chronic viral infections (mainly hcv and hbv), excessive alcohol consumption, metabolic disorders, or autoimmune insults, leads to the development of liver fibrosis . Fibrosis is a prolonged and exorbitant wound healing response causing the accumulation of redundant extracellular matrix (ecm). Ecm consists of a dense mesh of macromolecules, polysaccharides, and proteins, particularly -smooth muscle actin and different types of collagen, forming insoluble fibers and microfibrils . Its main function is to support the structure and functioning of the tissue during healing processes . In the physiological state, balance between ecm deposition and degradation is controlled by numerous matrix metalloproteinases (mmps), which are digesting / degrading the particular components of ecm . In the course of fibrosis, however, mmps are markedly inhibited by tissue inhibitors of mmps (timps) that are upregulated in response to the chronic liver insult . Continuous scarring may eventually lead to the development of liver cirrhosis, end - stage liver disease, or hepatocellular carcinoma [1, 2] (figure 1). These fibrogenic stimuli include reactive oxygen species (ros), hypoxia, inflammatory and immune responses, hepatocytes apoptosis, and steatosis . Response to these signals owing to persistent liver injury instigates the recruitment and transformation of the resident quiescent liver fibroblast (hepatic stellate cells, hscs) to the highly activated, proliferative, motile, and contractile myofibroblast phenotype (figure 1). . The activation process is initiated by the release of many growth factors such as platelet - derived growth factor (pdgf) and transforming growth factor (tgf-), profibrogenic cytokines and chemokines by the injured hepatocytes, and inflammatory cells particularly macrophages and other nonparenchymal cells . Deposition of the dense and complex net of scar tissue in the space of disse, where hscs reside, causes significant changes in the sinusoid architecture . Fenestrations in the structure of liver sinusoidal endothelial cells (lsecs) are gone and hepatocytes lose their microvilli . Although hscs remain the primary source of myofibroblasts, it has now become clear that other cell types can also contribute to myofibroblasts population including portal fibroblasts, bone - marrow derived cells, and possibly epithelial - mesenchymal transition (emt) and contribute to the liver scarring . First symptoms of the liver impairment in most of cases are indicating disease development into cirrhosis and this commonly occurs after 1520 years, when the prognoses of survival and recovery are dramatically reduced . A major difficulty in developing disease - specific therapy is the lack of accurate and established diagnostic techniques for long - term monitoring of disease progression and therapy responses and to optimize disease treatment strategies [3, 4]. Liver biopsy has been considered as the gold standard for the diagnosis and staging of liver fibrosis but is invasive and painful and has numerous limitations including risk of bleeding, sampling errors due to disease heterogeneity, and inter- and intraobserver variability [46]. Moreover, liver biopsies only sample 1/50,000 of the liver, and undersized or fragmented samples may therefore underestimate hepatic fibrosis [4, 5, 7]. Recently, guidelines by easl - aleh have been published summarizing and validating clinical use of noninvasive tests for evaluation of liver disease severity and prognosis . The tremendous advancement in the biomedical research over the last decade led to the development of novel, rapid blood tests for diagnosis of liver fibrosis . Several commercial biochemical and serum tests classified into class i and class ii biomarkers are developed . Class i biomarkers are associated with the mechanism of fibrogenesis, either as secreted matrix - related components or as a result of ecm synthesis or turnover, for example, hyaluronan . Class ii biomarkers are indirect methods which are grouped into panels such as (a) european liver fibrosis test (elf) (n - terminal propeptide of collagen type iii, hyaluronic acid, timp1, and age), (b) fibrotest (alpha-2-macroglobulin, haptoglobin, apolipoprotein a1, gamma - glutamyl transpeptidase [ggt], total bilirubin, and alanine transaminase), (c) fibrosis-4 index (fib-4) combining standard biochemical tests (platelets, alt, and ast) and age, (d) hepascore (age, sex, total bilirubin, gamma - glutamyl transferase, 2-macroglobulin, and hyaluronic acid), (e) aspartate and transaminase to platelet ratio (apri), and (f) forns score (platelet count, prothrombin index, ast, alpha-2-macroglobulin, ha, and blood urea) which have been developed recently [913]. However, these tests rely on indirect markers and lack specificity as these markers can be influenced by unrelated diseases . Nevertheless, recent studies indicate that the results from the serum panels might predict risk of decompensation and overall survival more accurately than biopsy [9, 10, 12]. Number of emerging technologies have recently been developed for diagnosing and staging liver fibrosis over the past years such as ultrasonography (us), computerized tomography (ct), and magnetic resonance imaging (mri). However, these imaging modalities are dependent primarily on structural and morphological alterations in the liver and these alterations are usually identified in advanced stage of fibrosis . Currently, transient elastography (te) (fibroscan, echosens, paris, france) is the most widely used method for noninvasive and rapid measurement of liver stiffness . Te uses a probe consisting of an ultrasonic transducer and a vibrator that emits low - frequency shear waves (50 hz) propagating through the liver tissue . The speed of the shear waves is directly related to liver stiffness and can be expressed in kilopascal (kpa). Several studies have evaluated te for diagnosis of hepatic fibrosis and cirrhosis with relatively high specificity and sensitivity [1518]. Point shear wave elastography (pswe) or acoustic radiation force impulse (arfi) involves mechanical excitation of tissue using short - duration acoustic pulses that produce shear waves, expressed in m / sec, which directly correlates with the extent of liver fibrosis [1925]. Another promising technique, 2-dimensional shear wave elastography (2d - swe), is based on the combination of a radiation force induced in tissues by focused ultrasonic beams and a very high frame rate ultrasound imaging sequence capable of catching in real time the transient propagation of resulting shear waves . 2d - swe expressed either in m / sec or in kpa has an advantage of being implemented on a commercially available ultrasound machine . New magnetic resonance imaging (mri) based imaging techniques have recently gained substantial interest: magnetic resonance elastography (mre), dynamic contrast - enhanced mr imaging (dce - mri), perfusion weighted imaging (pwi), and diffusion weighted imaging (dwi) [2729]. Magnetic resonance elastography (mre) is similar to ultrasound based elastography techniques and can determine liver stiffness by analysis of mechanical waves propagating through the liver [3034]. Diffusion weighted imaging (dwi) is a magnetic resonance technique that quantifies the diffusion of water molecules in tissues that can be quantified as apparent diffusion coefficient (adc). Collagen fibers in the liver would inhibit water diffusion thereby leading to a decrease in adc and therefore can be quantitatively used to assess liver fibrosis, but the technique has limitations since factors like steatosis can also affect adc . Dynamic contrast - enhanced mr imaging (dce - mri) and mr perfusion weighted imaging (mr - pwi) rely on the intravenous administration of mr contrast agents that can more precisely reveal hepatic hemodynamic changes [3638]. However, these mri - based techniques are time - consuming and cost - ineffective . Another novel and developing mr based imaging modality, molecular mr imaging, represents a unique implementation of mr modality to visualize, characterize, and measure biological processes at the cellular and molecular level with high spatial resolution . The specific contrast agents (or probes) can be endogenous and exogenous probes can be generated by encapsulating paramagnetic (gadolinium) or superparamagnetic (iron - oxide) metals in different nanoparticles . Molecular mr imaging is based on the development of mr imaging probes composed of contrast generating materials, for example, gadolinium or iron - oxide, and molecular targets, for example, ecm binding probes such as collagen i (ep-3533), fibrin - fibronectin (clt1-peptide), elastin (emsa), and v3-integrin (c(rgdyc)-uspio) [39, 40]. Overall, no single method can provide the detailed information as histological examination but using noninvasive modalities can differentiate between mild and significant fibrosis and can potentially avoid unnecessary liver biopsy in a subgroup of patients . While these methods have provided some impressive results, there remains a paucity to validate their use in disease management or assessment of potential antifibrotic therapies . Although molecular mri of liver fibrosis is currently developing, the conception of target specific molecular mri approach can open up new horizons and avenues for the diagnosis and effective management of this life - threatening disease . The term targeted therapy (tt) describes the set of treatment strategies aiming to inhibit or alter specific molecules or molecular pathways leading to certain disorders and diseases . Some of the molecularly targeted agents exert the cytotoxic or cytostatic effects on the specific target cell types, while others inhibit the activity of the particular enzymes or proteins or boost the immune system activity against pathogenic mechanism . One of the main advantages of such approach is its specificity the principle of design is to affect only the pathologically transformed cells and processes, thus minimizing the adverse effects [41, 42]. Most important part of the targeted therapy development is to determine the appropriate molecular target proteins and enzymes, hormones, peptides, genes, and specific reactions involved in the pathological processes that, upon alteration, can lead to the disease resolution / reversion . Three main types of the targeted therapy design can be distinguished: small molecule drugs: relatively small moieties which are able to target molecules and processes inside the cell [4346]monoclonal antibodies: large proteins produced by the immune cells that are able to highly specifically identify and bind with the targets on the cell surface or outside the cells [4749],targeted conjugates: delivery systems consisting of the therapeutic moiety, such as delivery vehicle or protein carrying therapeutic agent conjugated with the targeting ligands [5052]the antifibrotic therapeutic approaches are broadly classified among several categories: elimination of the primary cause of injury, for example, alcohol abstinence in alcoholic liver diseasesreduction of inflammation and immune response or inhibition of hepatocyte apoptosis / injury to avoid hsc activationresolution of fibrosis by inhibiting scar tissue formation, increasing matrix degradation, inhibiting hsc activation, or stimulating hsc apoptosisinhibition of signaling pathways (extracellular and intracellular) responsible for activation, contraction, and proliferation of hscs small molecule drugs: relatively small moieties which are able to target molecules and processes inside the cell [4346] monoclonal antibodies: large proteins produced by the immune cells that are able to highly specifically identify and bind with the targets on the cell surface or outside the cells [4749], targeted conjugates: delivery systems consisting of the therapeutic moiety, such as delivery vehicle or protein carrying therapeutic agent conjugated with the targeting ligands [5052] elimination of the primary cause of injury, for example, alcohol abstinence in alcoholic liver diseases reduction of inflammation and immune response or inhibition of hepatocyte apoptosis / injury to avoid hsc activation resolution of fibrosis by inhibiting scar tissue formation, increasing matrix degradation, inhibiting hsc activation, or stimulating hsc apoptosis inhibition of signaling pathways (extracellular and intracellular) responsible for activation, contraction, and proliferation of hscs there is an intensified focus on the development of antifibrotic therapies for chronic liver diseases in the past years . Advanced pathological and molecular understanding of the fibrosis pathogenesis has instigated identification of novel therapeutic and promising drugs in preclinical models . Furthermore public health impact of liver diseases and novel diagnostic technologies for the assessment of fibrosis has resulted in increased clinical trials in this field . In this review, clinical studies concerning targeted therapies against liver fibrosis of diverse etiology are reviewed and summarized in table 1 . In general, the biggest emphasis is on the small molecule drugs; so far these therapeutics were the most frequently investigated and the progress in this field is currently the most advanced . Reviewed studies mostly are randomized trials on the parallel two or more groups of patients (parallel assignment design); less frequently there are also single group assignments . Most of the studies are randomized and double - blinded to ensure the minimal risk of the results manipulation or bias . Clinical trials are mostly performed on patients with nash (nonalcoholic steatohepatitis), liver fibrosis, or cirrhosis with chronic hepatitis c infection and nafld (nonalcoholic fatty liver diseases) since these diseases are the most frequently occurring reasons for the development of liver fibrosis . Clinical trials in chronic liver diseases present unique challenges, because clinical events that could be used as trial primary endpoints (e.g., histological assessment of fibrosis) can vary depending on the etiology of the liver disease; therefore the study outcomes largely rely upon noninvasive surrogates . Current clinical trials are primarily based on pathological characterization of liver biopsy to assess fibrosis progression but now serum tests such as hepascore, elf, fibrotest and noninvasive imaging modalities like te or mr are characterized as surrogate endpoints . In the following list, liver histology necroinflammation: nafld activity score and knodell scorefibrosis: histopathological and immunohistochemical analysis necroinflammation: nafld activity score and knodell score fibrosis: histopathological and immunohistochemical analysis serum tests serum markers: alt, ast, alp, ggt, and albuminserum marker panels: elf test, apri, and fib-4lipidomic analysis serum markers: alt, ast, alp, ggt, and albumin serum marker panels: elf test, apri, and fib-4 liver function tests insulin sensitivityglucose toleranceindocyanine green clearance testsgalactose elimination tests indocyanine green clearance tests galactose elimination tests noninvasive tests liver stiffness measurement: transient elastography (fibroscan); shear wave elastography; magnetic resonance elastography; acoustic radiation force impulse (afri)liver fat measurement: mri and spectroscopy (mrs) liver stiffness measurement: transient elastography (fibroscan); shear wave elastography; magnetic resonance elastography; acoustic radiation force impulse (afri) liver fat measurement: mri and spectroscopy (mrs) clinical scores meld scorechild - pugh scoreishak scoremetavir score . Small molecule drugs are the group of the targeted therapeutic agents typically with molecular weight below 1000 da . They can be delivered intravenously or orally and, due to their small size, enter the target cells (cross the cell membrane); typically they are also able to penetrate the blood - brain barrier . The complex process of discovery and development of small molecule drugs mostly consists of two combined strategies: (i) knowledge - based design employing the knowledge about the structure of the target and its inhibitors / ligands and/or (ii) random high throughput screening of libraries of small molecules to search for the molecules with potential activity towards / against the target . Following extensive screening, eventually, the prospective compounds are further investigated in vitro and in vivo for the therapeutic efficacy and, if applicable, enter further the clinical development phase [54, 55]. Some of the major clinically challenged targets of the small molecule drugs are mentioned below . Activated hscs express a diverse group of nuclear receptors acting as transcription factors, for example, peroxisome proliferator - activated receptor (ppar) and farnesoid x receptor (fxr), that play an important role in hsc regulation . Ppar is highly expressed in the quiescent hscs and upon activation its expression diminishes . Following treatment with ppar ligands / agonists, ppar expression is restored, and hsc activation and collagen expression are reduced in vitro . Clinical trials using pioglitazone showed significant improvement in steatosis, inflammation, and insulin resistance in nash patients [59, 60] (table 1), while clinical trials using ppar agonists farglitazar (gi262570) [61, 62] showed no effective treatment in patients with chronic hcv infection (table 1). Fxr, another nuclear receptor, is highly expressed in the liver and small intestine . It is responsible for maintaining homeostasis of bile acids and cholesterol and regulates transcription of multiple genes involved in bile acids synthesis and transport . Fxr is also expressed in hscs and activation of fxr in hscs is associated with significant decrease in collagen production . Activation of fxr occurs via binding with bile acids such as deoxycholic or lithocholic acid, although many synthetic ligands are also known . However, most fxr ligands failed the preclinical and clinical assessment because of poor pharmacokinetics or toxicity issues . Nevertheless, synthetic fxr agonists px-102 and px-104, developed by phenex pharmaceuticals, showed promising safety and tolerability profile in healthy subjects (px-102, clinical trial nct01998659; nct01998672) and px104 is currently tested in a phase 2a study in patients with nafld (nct01999101). Int-747 (6-ethyl chenodeoxycholic acid or 6-ecdca or obeticholic acid), semisynthetic fxr agonist, showed improvement of the histological and biochemical markers, ameliorated fibrosis, inflammation, and steatosis in nash patients . Obeticholic acid is currently in clinical trials for long - term treatment of cholestatic liver diseases (table 1). Ras is an important hormonal regulatory mechanism of the blood pressure and body fluid homeostasis . The key ras protein, angiotensin ii (ang ii), is produced in the liver from its precursor angiotensin i by the proteolytic cleavage by angiotensin i converting enzyme (ace). Ang ii exerts its diverse biological effects by binding with one of its multiple receptors, particularly ang ii type 1 receptor (at1-r), overexpressed in activated hscs . Ang ii induces hsc activation, proliferation, and contraction, as well as increased tgf, timp1 expression, and collagen deposition . Therefore, ang ii and its interaction with at1-r are considered to play an important role in liver fibrogenesis and its blocking by ace inhibitors (acei) or at1-r blockers (arbs) may be an effective therapeutic option for treatment of liver fibrosis and they are already in clinical trials, for example, losartan, irbesartan, and candesartan and moexipril (table 1). Losartan, irbesartan, and candesartan share similarities in the chemical structure and they all are at1-r blockers, in contrast to moexipril, which is an ace inhibitor . Clinical trials evaluating long - term losartan effects in chronic hepatitis c patients showed decreased inflammation, reduced expression of fibrogenic mediators, and decreased ecm (collagen i) accumulation [71, 74]. Furthermore, treatment markedly decreased ang ii induced oxidative stress in hepatic fibrosis [71, 74]. Prolonged exposure to the at1-r blocking treatment in patients with chronic hcv infection was proven to be safe and well tolerated . Ras has been also shown to be associated with hypertension; therefore, candesartan (at1-r inhibitor), widely used for the therapy of hypertension and heart failure, has shown promising results in the clinical trials for alcoholic liver fibrosis in combination with ursodeoxycholic acid (udca). It was demonstrated that candesartan significantly improved the treatment outcomes in comparison to udca and reduced the fibrosis scores and -sma positive fibrotic area in biopsies . Relative expression of fibrogenic markers was downregulated and the arterial blood pressure was shown to be significantly reduced . However, long - term treatment with irbesartan (arb and antihypertensive drug) in severe fibrosis with chronic hepatitis c showed no substantial improvement in fibrosis scores, arterial pressure, and organ stiffness in the treated group, despite the fact that treatment was safe and well tolerated . In addition, ace inhibitor moexipril treatment did not show beneficial effects in primary biliary cirrhosis patients . Furthermore, in halt - c cohort study, acei / arb therapy did not retard the progression of fibrosis . Due to ambiguous results, further controlled studies are required to evaluate the long - term efficacy of arbs / acei . Endocannabinoid system plays an important role in various liver diseases including viral hepatitis, nafld, and alcoholic liver disease . Cannabinoid receptors cb1 and cb2 are upregulated in chronic liver diseases and several studies have convincingly demonstrated antagonism between cb1 and cb2; that is, cb1 promotes while cb2 suppresses liver damage [77, 78]; therefore cb1 antagonists and cb2 agonists were investigated as potential therapeutic approaches for liver diseases . Clinically, daily cannabis (cb1 and cb2 agonist) promoted fibrosis progression in chronic hepatitis c . Rimonabant cb1 antagonist was successfully tested in clinical trials in obese patients and showed reduction in body weight, improved metabolic function, and improved insulin resistance . However, depression and psychoactive side effects led to the termination of clinical rimonabant drug use . Currently, efforts are directed towards development of novel cb1 antagonist with improved specificity that lacks neuropsychiatric adverse effects . Other neurotransmitters, for example, opioids and serotonin (5ht), and their receptors are other potential therapeutic targets in liver fibrosis . Opioid antagonist naltrexone and 5ht antagonist methiothepin have shown antifibrotic activity in animal models of liver disease [81, 82], but clinical trials are needed to demonstrate their long - term tolerability and efficacy . Since inflammation promotes progression of liver fibrosis, use of anti - inflammatory drugs poses a potential and rationale therapeutic approach . Corticosteroids (e.g., prednisone, prednisolone, methyl prednisone, and triamcinolone) are used for the treatment of liver diseases, most commonly autoimmune hepatitis with improved outcome and survival . However, the adverse effects of long - term corticosteroid therapy are still the major causes of morbidity and mortality . Another anti - inflammatory approach is to inhibit release of inflammatory cytokines or to neutralize it with receptor antagonists . Upregulated tnf production is one of the initiating events in the liver injury leading to release of proinflammatory cytokines resulting in fibrosis . Pentoxifylline (ptx) is a potent phosphodiesterase inhibitor, which suppresses tumor necrosis factor (tnf) production . Ptx was also shown to be hepatoprotective since it reduces oxidative stress, which is important contributor in the hepatic pathologies and fibrogenesis . Ptx has been registered for numerous clinical trials concerning its potential therapeutic efficacy in diverse fibrotic disorders [75, 8588]. Long - term treatment with ptx in nash patients demonstrated significant improvement of both histological features and significant improvement in the liver fibrosis in comparison to placebo - treated group . Despite the fact that ptx activity is being associated with tnf inhibition, the study failed to demonstrate the tnf downregulation . Finally, it was also concluded from the study that ptx treatment was safe and well tolerated by patients and there were no severe adverse side effects . Following hepatocyte injury, hepatic macrophages secrete inflammatory chemokines or cytokines, for example, c - c chemokine ligand type 2 [ccl2 or mcp1 (monocyte chemoattractant protein-1)], driving the recruitment and migration of pro - ccr2 and ccr5 positive inflammatory monocytes to the liver . Ccr2 and/or ccr5 antagonism has been suggested as a potential approach for the treatment of inflammatory diseases and fibrosis [91, 92]. Cenicriviroc (cvc), a ccr2/ccr5 antagonist, is currently being evaluated for the treatment of nash and liver fibrosis (centaur, nct02217475, table 1). Cenicriviroc showed favorable safety profile in hiv - infected patients in a phase 2b study and in patients with hepatic impairment . Another target molecule is galectin-3 (gal-3), pleiotropic -galactoside - binding lectin, that was shown to play an important role in the liver fibrosis . Gal-3 possesses strong proinflammatory properties and is able to activate macrophages and stimulate their migration . Gr - md-02 (galactoarabino - rhamnogalacturonate) is a potent inhibitor of galectin-3 that showed remarkable therapeutic effects in thioacetamide - induced liver fibrosis in rats and was submitted for 3 clinical studies concerning liver fibrosis . Phase 1 study evaluating safety of gr - md-02 in patients with nonalcoholic steatohepatitis (nash) and advanced fibrosis is already completed [65, 98, 99]. Results showed that the drug was safe and well tolerated in nash patients with liver fibrosis and demonstrated improvement in fibrosis and inflammation [100102]. Two upcoming clinical trials will evaluate gr - md-02 efficacy for the treatment of liver fibrosis in patients with advanced fibrosis and cirrhosis originating in nash . Oxidative stress or reactive oxygen species (ros) generation also plays an important role in initiation of fibrogenesis by activation of hscs; therefore inhibition of oxidative stress or ros inhibits inflammation resulting in amelioration of liver fibrogenesis . Hence, a number of antioxidants, for example, s - adenosyl - l - methionine (same), silymarin, phosphatidylcholine, n - acetylcysteine (nac), and vitamin e, are and have been tested in clinical trials (refer to table 1) with beneficial effects . During liver fibrosis, a number of receptor tyrosine kinases, that is, pdgfr (platelet - derived growth factor receptor), vegfr (vascular endothelial growth factor receptor), fgfr (fibroblast growth factor receptor), and egfr (epidermal growth factor receptor), were significantly upregulated on activated hscs . Many fibrotic and proliferative cytokines, for example, pdgf, tgf, fgf, and vegf, signal via these receptors tyrosine kinases resulting in the activation of intracellular signaling pathways resulting in differentiation and proliferation of quiescent hscs [2, 103105]. Antagonism of these pathways via tyrosine kinase inhibitors attenuates liver fibrosis in preclinical experiments on animal models . Sorafenib, multitargeted tyrosine kinase inhibitor, was shown to attenuate liver cirrhosis, portal pressure, and angiogenesis . In a pilot clinical trial, recently, multicentered randomized clinical trial was carried out to study the effect of sorafenib on portal pressure in patients with cirrhosis (nct01714609, table 1). Erlotinib, egfr kinase inhibitor, attenuated liver fibrosis and hcc development in experimental animal models by suppression of egfr phosphorylation and inhibition of hsc activation . Currently, clinical trial is ongoing to evaluate the effects of erlotinib in inhibition of fibrogenesis and hcc prevention (nct02273362, table 1). Apoptosis signal - regulating kinase 1, ask1, a serine / threonine kinase, promotes oxidative stress responsive pathway and leads to the activation of downstream p38 mitogen - activated protein kinases (mapk) and c - jun n - terminal kinase (jnk), which stimulates inflammatory cytokines production, matrix remodeling genes expression, and abnormal cell proliferation . Ask1 and p38 have been positively correlated with the fibrosis stage in patients with nafld . Selonsertib (or gs-4997) is a highly selective and potent (ask1) inhibitor that inhibits ask1 by competitive binding to the catalytic domain of ask1 . In vivo in murine model, treatment with gs-4997 reduced fibrosis and steatosis, thus ameliorating the liver disease, thereby suggesting ask1 inhibition as the promising therapeutic approach . Pharmacokinetics of gs-4997 have been already evaluated in phase 1 clinical study in adult with normal or impaired liver function (nct02509624) and are currently registered in other clinical trials (table 1). Mammalian target of rapamycin (mtor) is a serine / threonine protein kinase that is able to regulate cell growth and proliferation by controlling the protein translation . Mtor performs its action by formation of mtor complexes 1 and 2 (mtorc1 and 2) that further transmit the signal to the downstream effector proteins, that is, ribosome kinase p70s6 and 4e - bp1, which are directly responsible for mrna translation . During fibrosis, mtor is highly dysregulated and was shown to be involved in the tgf responsiveness of the fibroblasts . Mtor inhibitors were first shown to possess immunosuppressive properties and to date they are used as the immunosuppressive drugs preventing posttransplant organ rejection as well in autoimmune diseases (e.g., rheumatoid arthritis). The foremost mtor inhibitor is rapamycin (or sirolimus); however due to its stability and solubility issues, new derivatives have been developed with improved safety and pharmacokinetics . Everolimus, one of the above - mentioned analogues, has been investigated in patients after liver transplantation [116, 117]. Monoclonal antibodies (mabs) are very relatively recent and becoming an essential element of the present pharmacotherapy [41, 47]. Utilization of mabs causes less adverse side effects and, alone or in combination with other drugs, can give remarkable results . There are many clinically approved mabs therapies for different types of diseases used either as monotherapies or as combined treatments (e.g., cetuximab, herceptin with docetaxel or paclitaxel). Monoclonal antibodies are rather new approach in the liver fibrosis treatment; therefore the development of the field is relatively in early stage . Connective tissue growth factor (ctgf) is a heparin - binding ecm - associated protein, highly upregulated during liver injury . It stimulates ecm deposition (particularly collagen i and fibronectin) and is involved in ecm remodeling, important features of liver fibrosis . Monoclonal antibody against ctgf (fg-3019) was developed by fibrogen for treatment of the fibrotic disorders . Fg-3019 was investigated in idiopathic pulmonary fibrosis (ipf) patients, and, after 2 years of the treatment, fg-3019 was proven safe and well tolerated in ipf patients . Fg-30149 is recently being tested in phase 2 trials in subjects with liver fibrosis as a result of a chronic hepatitis b infection . Vascular adhesion protein-1 (vap1) is an endothelial glycoprotein that promotes leukocytes trafficking from the blood to the site of inflammation . Upon injury and inflammation, vap1 translocates from intracellular storage to the cell surface . Soluble form of vap1 (svap1) is also able to initiate oxidative stress and secrete, via nfb, potent proinflammatory mediators . It was shown that serum levels of svap1 are markedly elevated in patients with chronic inflammatory liver diseases . Blockade of vap1 inhibits inflammatory responses by attenuating leukocyte recruitment and oxidative stress [120, 121]. Btt-1023, a human monoclonal antibody against vap1, will be assessed in the clinical study in patients with primary sclerosing cholangitis . This autoimmune liver disease is characterized by the progressive destruction of the hepatic bile ducts, which in turn leads to liver fibrosis and cholestasis . Preclinical studies showed efficient binding of the antibody with vap1 in the inflamed sites in vivo, as assessed by pet scans . Elevated levels of lysyl oxidase - like-2 (loxl2) expression were found in patient samples from liver fibrosis and primary biliary cirrhosis; moreover it was found that the upregulation of loxl2 is limited to the fibrotic areas . Loxl2 is an copper - dependent matrix metalloenzyme that enables collagen cross - linking thus creating a dense mesh of scar tissue . Loxl2 is therefore an interesting target for the hepatic fibrosis treatment and numerous approaches to inhibit loxl2 have been developed . Primarily, as it is copper - dependent enzyme, its activity can be impaired with the copper - binding ligands, such as d - penicillamine and -aminopropionitrile (bapn) [147, 148]. Nevertheless, the most promising approach is the use of monoclonal anti - loxl2 antibodies . They provide a specific allosteric inhibition of enzyme, by the binding with the scavenger receptor cysteine - rich (srcr) domains, which are the catalytic center of the molecule [146, 147]. There were at least two types of antibodies reported: ab0023, murine monoclonal antibody against loxl2, used in in vivo studies [146, 147] and simtuzumab (sim or gs-6624 and formerly ab0024), monoclonal antibody against human loxl2 . Simtuzumab is currently registered in 11 clinical trials (https://www.clinicaltrials.gov/), from which 6 are related to fibrotic liver diseases . The preliminary results of the pilot study in patients with liver fibrosis reported that sim was well tolerated at the applied doses (10 mg / kg) with no serious adverse effects and, due to its mechanism of action, provides a very promising antifibrotic therapy for patients with hepatic fibrosis . Additionally, another clinical trial has been launched recently evaluating simtuzumab in combination with gs-4997 (or selonsertib) in patients with nonalcoholic steatohepatitis (nash) and fibrosis (table 1). Targeted conjugates are the most diverse and the newest from of the described approaches; however the concept is already more than a hundred years old, as is the idea of magic bullet by paul ehrlich . The targeted conjugates are combining the features attributed to small molecule drugs and monoclonal antibodies . It can consist of the delivery vehicle, for example, protein carrier (hsa), liposome, polymeric nanoparticles, micelles, or nanoformulation, containing the active component, such as small molecule drug, small interfering rna (sirna), micro rna (mirna), cytokine, an active peptide, or a therapeutic protein . Targeting ligand is attached to guide the delivery carrier to the specific site in the body, based on the specific ligand - receptor interactions . This allows the preferential accumulation of the conjugate in specific target cells, tissues, or organs . Additional advantage of the targeted conjugates is that they provide the opportunity for theranostic approach (therapy and diagnostics). Application of the detectable moieties in the design, such as magnetic nanoparticles as the delivery vehicles or the fluorescent ligands on the surface of the conjugate, is to detect the accumulation of the particles in the target site . Moreover, magnetic nanoparticles can serve as the contrast agents in magnetic resonance imaging and nanobubbles can provide the contrast enhancement in ultrasonography imaging . There are many strategies and nanoformulations explored for the treatment of liver fibrosis in preclinical animal models . The strategies are developed to target different receptors on different liver cell types, that is, hepatocytes, hepatic stellate cells, kupffer cells (liver macrophages), and liver sinusoidal endothelial cells . The only representative of targeted conjugate in targeted therapies against liver fibrosis and a very promising drug which is currently under investigation in phase 1b/2 clinical trial is vitamin a - coupled lipid nanoparticle (liposome) containing sirna against collagen - specific chaperone heat shock protein 47 (hsp47) [3, 157, 158]. Hscs expresses retinol binding protein (rbp) receptor that regulates retinol (vitamin a) storage in hscs and is an interesting target for hsc - specific drug delivery . Hsc - targeted liposomes (nd - l02-s0201) carry sirna against hsp47, which facilitates collagen secretion by ensuring triple - helix procollagen formation, and are implicated in translational regulation of procollagen synthesis [159, 160]. Downregulation of collagen production can result in the amelioration of fibrosis and reversion of cirrhosis . Recently, lawitz et al . Presented the preliminary results from the clinical trials performed on healthy subjects as well as on the patients with advanced liver fibrosis . Nd - l02-s0201 was well tolerated in both groups of subjects without dose limiting toxicity neither in a single administration nor in multiple doses . Furthermore, in the liver fibrosis patients, 6 out of 8 patients showed at least 1-stage improvement in the liver fibrosis suggesting beneficial effects of targeted approach . As presented in this review, the development of the targeted therapies against fibrotic diseases is in relatively advanced stage . Numerous drugs are being assessed in the phase 2 clinical trials while some of them also reached phases 3 and 4 . Multiple studies are currently ongoing, and the already completed trials revealed high potential of emerging drugs in ameliorating hepatic fibrosis of various etiology . However, besides the already investigated mechanisms and drugs, there are still some target proteins and pathways that remain to be elucidated . Numerous promising molecular targets are currently under preclinical investigation and will be evaluated in the clinical trials . Nevertheless, taken all together, there is remarkable improvement in the development of targeted therapies against fibrotic diseases and, in noninvasive technologies, many drugs are already being tested but many exciting targets still remain to be explored and further investigated . It is giving hope for the patients that clinically approved efficacious treatment will emerge soon.
Two 30-year - old male patients visited the dental clinic for disabled at seoul national university dental hospital . They went through a routine dental check - up and radiographs were taken as aids to examine their oral health . On panoramic radiographs, both of the patients were presented with mesially impacted mandibular third molars, dental caries, and slight alveolar bone resorption . In addition, they both had bilateral calcified stylohyoid complex (fig . 1). Using the measurement method by jung et al5 for calcified stylohyoid complex, the length of styloid process were measured . One patient showed the severely calcified stylohyoid ligament on the right side, measuring 84.4 mm in length and 11.2 mm in width . The left stylohyoid ligament was partially calcified measuring 55.2 mm in length and 5.17 mm in width . The other patient also showed the severely calcified stylohyoid ligament on the right side measuring 72.3 mm in length and 8.3 mm in width . The left stylohyoid ligament was partially calcified measuring 67.8 mm in length and 4.3 mm in width . The pattern of calcification was similar; both sides of the calcification involved the regions of tympanohyal, stylohyal, and ceretohyal, and the calcification on the right side was thicker . They did not display such symptoms as neck pain or headache, and they had no history of trauma . No surgical treatment was performed on both patients for the calcified stylohyoid complex . After dental treatment including extraction and caries treatment with root canal treatment, their follow - up images showed no changes of the calcified stylohyoid complex (fig . Elongated styloid process, a kind of soft tissue calcification, is commonly identified on panoramic radiographs . Since it usually does not impose any discomfort to patients, it is usually not treated surgically . However, some patients complain of neck pain, sore throat, foreign body sensation, and dysphagia, which require surgical interventions in addition to complete understanding of the etiology and the possibility of its relation to any syndromes . The exact cause of the elongated styloid process due to calcified and ossified bone and ligament is unknown . It is believed that any trauma in the cervicopharyngeal region, especially after tonsillectomy, might stimulate a subsequent growth of the styloid process.7 there is a controversy over the relations between trauma history and calcified stylohyoid complex since there are many cases with no trauma history . In addition, it was suggested that local chronic irritations, surgical trauma, endocrine disorders in female at menopause, persistence of mesenchymal elements, growth of the osseous tissue, mechanical stress, or trauma during development of styloid process could result in calcified hyperplasia of the styloid process . Even the twins had no history of trauma, they had stylohyoid complex calcification in the similar area in this case . Even though there were some reports on the relationship between calcified stylohyoid complex and numerous general medical conditions,6,7 there was no such literature which mentioned its genetic factor okabe et al8 found a significant correlation between the length of the calcified stylohyoid complex and serum calcium concentration level and heel bone density . Meanwhile, macdonald - jankowski9 reported the significant differences in morphologies of the stylohyoid complex between londoners and hong kong chinese . This racial difference might indirectly indicate the genetic effects on the calcification of stylohyoid complex . However, there was no such report that suggested that stylohyoid complex calcification was caused by genome . This case suggests a possibility that the stylohyoid complex calcification might be originated from genetic factor.
Targeted drug delivery guided by molecular imaging approaches is a burgeoning area of clinical research, particularly for the treatment of cancer . This approach involves an optimized delivery of a therapeutic molecule and an imaging probe to the disease site, thereby using selective diagnosis and effective pharmacotherapy in unison for management of several diseases . Successful utilization of this strategy requires integrated knowledge and versatile approaches in multidisciplinary fields such as cell and molecular biology, chemistry, material science, and physics and has opened up vast prospects in pharmacokinetics, therapeutic target discovery, drug delivery research, and quantification of multiple biomarkers in diseases . The major goal of this approach is to use molecular imaging to maximize effective therapy in diseased tissues and to minimize systemic drug exposure in order to reduce toxicities . In the past decade, innumerable studies have been reported on the synergistic use of molecular imaging with targeted drug delivery, and this strategy has now matured with promises to fulfill the vision of personalized medical treatment in the near future . In order to minimize the effects of toxicity and improve therapeutic effects, it is essential to deliver the therapeutic drugs to the right site, in the right time, and in the right concentration . Ideally, the drug should act as a magic bullet that possesses perfect specificity to targeted lesions and has no side effect on the rest of the body . Controllable and selective delivery of drugs improves bioavailability by preventing premature degradation and enhancing uptake, maintains drug concentration within the therapeutic window by adjusting the drug release rate, and reduces side effects by targeting to disease site and target cells . The ability to deliver therapeutic drugs locally, in a minimally invasive manner, has advanced drastically with the growth of molecular imaging techniques . Molecular imaging approaches have been implemented in areas ranging from new therapeutic target discovery to effectively monitoring tumor pharmacokinetics and drug distribution to modulation of drug release at the target site . When molecular imaging probes are coadministered as part of the drug delivery system, it can help to achieve multiple goals, such as real - time and concurrent assessment of drug delivery efficiency / targeting, in vivo fate of drug and sites of localization / accumulation, modes of excretion, imaging, and monitoring the progress of drug treatment, in a single dosing . When an image - guided approach is not used, there is neither any means to track or image the in vivo fate nor the ability to measure the delivery efficiency of drugs . Also, the bioavailability, therapeutic efficacy, and dose response of drug treatment has to be estimated based on separate sets of experiments which might render the process cumbersome and cost - ineffective . However, molecular imaging of the drug delivery process involves several challenges and is affected by several factors such as target expression, type of drug, in vivo accessibility of the receptor (e.g., vascular density, vascular permeability, and interstitial pressure), enhanced permeability and retention (epr) effect, receptor internalization, tracer protein dose, and timing of imaging . Nevertheless, this approach has the potential for patient selection for targeted therapy and monitoring the therapeutic response after the drug is delivered . Currently, several noninvasive image - guided modalities are being used in biomedical and clinical settings, which include magnetic resonance imaging (mri), computed tomography (ct), positron emission tomography (pet), single photon emission computed tomography (spect), optical imaging, and ultrasonography . Among these, pet, spect, and optical imaging are regarded as quantitative or semiquantitative imaging modalities, whereas ct and mri are normally used for anatomical imaging . The relative advantages and limitations of these imaging modalities have been elaborately discussed in several review articles . In particular, pet offers picomolar sensitivity and is a fully translational noninvasive functional imaging technique with high sensitivity and accurate quantification and thus helps in measuring biological processes at the molecular and the metabolic levels in vivo . However, the limited spatial resolution of the pet images might sometimes make it difficult to accurately define the regions of interest (rois). Unnecessary radiation exposure to the nontargeted organs due to highly energetic -rays (511 kev) emitted by the pet radioisotopes is also a cause of concern . Nevertheless, one has to acknowledge that no single imaging modality can provide information on all aspects of structure and function . Therefore, investigation of a subject using multiple imaging modalities is highly desirable and is rapidly gaining popularity . In this review, we aim to provide a timely and comprehensive overview of the pet image - guided drug delivery approaches reported to date, with focus on quantitative assessments of tumor - targeted therapeutic delivery, distribution, uptake, and response . The development of various carriers for site- and event - specific targeting and controlled drug release are summarized, and the great potential and intriguing opportunities for future development which might help in bringing this exciting research avenue closer to a clinical reality are discussed . The interest in using pet as a molecular imaging modality in clinical research has steadily grown during the last 23 decades, and has now gained considerable importance in routine hospital practices because of its ability to diagnose diseases in early stages and monitor therapeutic responses . In a typical scenario of pet imaging, a suitable compound is radiolabeled with positron - emitting radionuclides such as f, cu, ga, or zr and administered to a living subject . The positron that emerges from the radionuclide decay travels a short distance before being annihilated with an electron to release two 511 kev rays, which are approximately 180 apart . The 511 kev rays can be detected by a ring of detectors configured in the coincidence mode in the pet camera . The registered events are reconstructed into a three - dimensional image which provides information on the spatial distribution of the radioactivity as a function of time in the living subject . Nowadays, pet is increasingly used in combination with ct as a hybrid imaging modality in clinical settings, to obtain higher resolution by fusing both functional and anatomical information at the same time . Pet also plays an important role in the process of drug development and evaluation, whereby understanding drug action and establishing dosage regimens and treatment strategies have been most crucial ., o can replace the stable analogues in drugs and biomolecules, and hence it is possible to synthesize pet probes with the same chemical structure as the parent unlabeled molecules without altering their biological activity . Low bioavailability, insufficient targeting, and poor localization in desired tissue / organ, adverse side effects, etc . Targeted drug delivery systems have the potential to improve these undesirable features, and when used in conjunction with pet imaging, they are effective in increasing safety to efficacy ratio and decreasing dose, which in turn reduces adverse reactions and toxicity of drugs . Pet can also provide information on the kinetics, dosimetry, and distribution of drugs in the diseased and normal tissues within the field of view as well as the clearance pattern in a biological system . Pet image - guided drug delivery is expected to play an increasingly important role in realizing the full potential of the next generation of therapeutics . For this purpose, it is essential to choose radioisotopes of appropriate half - lives to match the pharmacokinetics of the drug carriers used . Generally, for inorganic drug carriers (such as silica nanoparticles, superparamagnetic iron oxide nanoparticle, gold nanoparticles, quantum dots etc .) Which are expected to have circulation half - lives of a few hours, short - lived or intermediate - lived radioisotopes such as ga (t1/2 = 68 min), f (t1/2 = 109.8 min), sc (t1/2 = 3.9 h), ga (t1/2 = 9.7 h), cu (t1/2 = 12.7 h), etc . However, for organic drug carriers (such as carbon nanotubes, polymeric nanoparticles, micelles, liposomes, etc . ), which can circulate in vivo for more than 1 day, intermediate - lived or long - lived radioisotopes such as ga (t1/2 = 9.7 h), cu (t1/2 = 12.7 h), zr (t1/2 = 78.4 h), or i (t1/2 = 4.17 day) would be the ideal choices for pet image - guided drug delivery . The choice of suitable radioisotopes is also governed by the conjugation strategies adopted for radiolabeling the drug carrier . The radiolabeled agent must demonstrate high in vitro and well as in vivo stability for successful use in pet image - guided drug delivery . The current revolution in targeted drug delivery is fueled by the innovations in material science, organic chemistry, functional genomics, and proteomics which have created carriers that are biodegradable (which can be slowly dissolved in vivo by biological means), biocompatible (which can remain in a biological system without causing any adverse effect), targeting, and stimulus - responsive (which can control drug biodistribution in response to specific stimuli). In addition to increased selectivity against diseased cells, these delivery systems can also solve problems associated with drug instability in the biological environment as well as issues related to the modulation of drug . Two different approaches are used for drug loading and delivery for pinpoint targeted treatment of cancer cells . In the first approach, chemotherapeutic drugs are loaded onto multifunctional drug carriers such as liposomes, micelles, nanoparticles, microparticles, microbubbles, dendrimers, copolymers, intestinal pathogen, etc . Owing to the convenience in modifying the surface properties of these carrier systems, they can be conjugated with various targeting ligands such as monoclonal antibodies, antibody fragments, peptides, and other small molecules . The carriers are either directly conjugated to targeting ligands or derivatized for interactions with specific adapters that are conjugated to the targeting vectors . Streptavidin / biotin interaction is one good example used for binding various carriers to targeting proteins and antibodies . In addition to delivery of chemotherapeutic drug molecules for therapy, these carriers also carry pet radionuclides or other contrast agents for diagnosis of the diseases . Such drug delivery strategies are an important move toward achieving simultaneous diagnosis and therapy of diseases, which have recently been termed as drugs (e.g., therapeutic radionuclides) are conjugated with the targeting ligands using suitable bifunctional linkers . Unlike the first approach, here the drug and the imaging label (pet radionuclide) do not necessarily share the same delivery carrier . For diagnosis or monitoring therapeutic response, pet imaging is carried out separately in this case by conjugation of the targeting ligands with suitable pet radioisotopes . Another striking difference between the two approaches is that, in the former, the delivery of the drug to the target tissue can be achieved by both passive and active targeting, while, in the latter, the drug is delivered primarily due to active targeting . In passive targeting, the drug carriers such as nanoparticles, liposomes, micelles, etc also, therapeutic concentrations can be much lower than optimal at the tumor site by simply relying on epr - mediated accumulation, and therefore passive targeting is generally not preferred for drug delivery . More efficient and selective uptake of drug into the target cells is achieved by active targeting wherein the drug carriers are conjugated with targeting ligands, as mentioned earlier . Active targeting requires careful identification of tumor biomarkers, as well as selection of specific molecules that can bind to such markers in a selective and directed manner . Targeted drug delivery vehicles can then be internalized by tumor cells via receptor - mediated endocytosis / phagocytosis, resulting in elevated concentration of drugs in tumor tissue . Thus, the concept of theranostic agent is not just limited to chemotherapy but also has a relevant role to guide in radiation - based targeted therapies . Various drug carrier systems have been radiolabeled with different positron emitter radionuclides for image - guided drug delivery, most of which are summarized in table 1 and discussed in the following text . Albumin is an attractive macromolecular carrier that may be modified suitably for biomedical imaging applications . Such carriers have also been studied for drug and gene delivery in vitro and in vivo, through cavitation . Generally, albumin - based carriers are biodegradable, nontoxic, metabolized in vivo to produce harmless degradation products, nonimmunogenic, easy to purify, and soluble in water allowing ease of delivery by injection and thus ideal candidates for image - guided drug delivery procedures . A significant amount of drug can be incorporated in the albumin based carrier systems because of different binding sites present in the albumin molecule . Owing to the defined albumin primary structure and high content of charged amino acids (e.g., lysine) on the surface, albumin - based carriers offer the possibility of direct electrostatic adsorption of positively (e.g., ganciclovir) or negatively charged (e.g., oligonucleotide) molecules without the requirement of any other compound . In addition, these carriers can easily be prepared under mild conditions by coacervation, controlled desolvation, or emulsion formation . Commercially, albumins are obtained with significant quantities from egg white (ovalbumin), bovine serum (bovine serum albumin, bsa), and human serum (human serum albumin, hsa) and also available from soybeans, milk, and grains . The chelator - free radiolabeling of macroaggregated human serum albumin with ga (t1/2 = 68 min) for pet imaging was first described by even et al . Subsequently, this procedure was improved, and development of a kit for labeling macroaggregated human serum albumin with ga for pet imaging of liver anomalies was reported by okada et al . The kit was clinically tested and was found useful in the evaluation of the function of the reticuloendothelial system . In a similar study, maus et al . Reported the radiolabeling of different commercially available human serum albumin kits with ga.in vivo pet imaging showed that ga - labeled human serum albumin was mainly retained in the lungs . No decrease in activity or migration of particles from the lungs was observed during the first 1 h (1 half - life of ga), which demonstrated the in vivo stability of the radiolabeled albumin over that period of time . Also, no significant retention of ga - labeled human serum albumin particles in the liver was detected . The authors concluded that this approach could be used to estimate the liver - to - lung shunt and eliminate extrahepatic macroaggregate deposition in patients with primary and secondary liver malignancies, warranting y - based radioembolization therapy . In a recent development, liao et al . Prepared albumin shelled microbubbles filled with perfluorocarbon (c3f8) gas to enhance the contrast in ultrasound imaging . Additionally, the microbubbles were radiolabeled with n - succinimidyl-4-[f]fluorobenzoate (f - sfb) and also conjugated with antibodies targeting vascular endothelial growth factor receptor 2 (vegfr2) using avidin the radiolabeled microbubble shells could thus be used as dual - modality (pet and ultrasound) imaging agent . The f - labeled, albumin - shelled, vegfr2-targeted microbubbles had a lifetime of 30 min in the blood pool and demonstrated a highly specific adherence to tumor vessels in mice bearing human breast cancer . The size of the microbubbles was on the order of several micrometers and therefore should be retained in the tumor vasculature after intravenous injection . However, dynamic micropet imaging showed a relatively low tumor uptake of 1% id / g, even 1 h post injection . The low tumor uptake might be attributed to attachment of f - sfb on the surface of the microbubble, which might have influenced the targeting efficiency of the antibody . The targeted microbubbles accumulated rapidly in both the liver and lung and cleared slowly from the blood circulation . The trends found in micropet imaging were further corroborated by ex vivo biodistribution studies . The specificity of the binding of targeted microbubbles to endothelial vegfr2 was further validated by comparing the results of targeted and nontargeted contrast - enhanced ultrasound imaging . The authors concluded that the f - labeled albumin - shelled microbubbles can be used for targeted drug delivery to vegfr2 in breast cancer, guided by the dual - modality (pet / ultrasound) functional imaging approach . In all these studies, development of only imaging strategies using albumin - based platforms have been described without direct relation to drug delivery . However, there are several other reports on utility of drug - loaded albumin - based carriers and controlling drug release using ultrasound energy in such systems . Therefore, it was expected that tracking disease progression would be analogous to tracking drug delivery using albumin - based carriers . Liposomes are concentric, closed bilayer membranes of water - insoluble polar lipids that can that can be used to encapsulate biomolecules and drugs for targeted delivery while protecting their bioactivity . Soluble drugs can be loaded in the aqueous core and the hydrophobic drugs partitioned in the lipid bilayer . They are widely used not only in delivery of a variety of anticancer drugs but also in delivery of antineoplastic agents, antimicrobial compounds, immunomodulators, anti - inflammatory agents, cardiovascular drugs, etc . The widespread interest in the use of liposomal systems for drug delivery stems from their biocompatibility, biodegradability, and nontoxicity and the ease of controlling their size during the preparation process . Currently, there are several commercially available liposomal formulations for cancer therapy, including doxorubicin (doxil), daunorubicin (daunoxome), cytarabine (depocyt), myocet, and vincristine (onco - tcs). The recent advances in the use of radiolabeled liposomes for imaging as a tool in personalized medicine have been summarized in a recent review . Generally, liposomal systems are coated with poly(ethylene glycol) (peg) to increase the circulation time in blood and decrease uptake in the reticuloendothelial system (res). Radiolabeling of liposomes with pet radioisotopes generally requires the use of chelator molecules in the aqueous core or conjugation on the lipid bilayer . The radiolabeled liposomal systems employed in pet studies must be carefully designed as lower stability of radiolabeled agent might obscure image - based assessment of particle pharmacokinetics . Seo et al . Reported the development of a method for radiolabeling liposomes with cu for imaging and drug delivery monitoring using pet . Bifunctional chelators, such as, 6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane - n, n,n,n-tetraacetic acid (bat), (6-(6-(3-(2-pyridyldithio)propionamido)hexanamido)benzyl)-1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (teta - pdp), and 4-(2-(2-pyridyldithioethyl)ethanamido)-11-carboxymethyl-1,4,8,11-tetraazabicyclo(6.6.2)hexadecane (cb - te2a - pdea) were radiolabeled with cu, and the radiolabeled conjugates were attached to maleimide lipids in the liposome . The radiolabeled liposomes were found to be stable in mouse serum even after 48 h of incubation . In vivo pet studies demonstrated that liposomal activity was high in the blood pool from 0 to 6 h and slowly cleared out through the res . The presence of the peg spacer between the chelator and the lipid did not significantly alter the labeling efficiency and the clearance rate of liposomes from the blood pool . The study was further extended by the same group of authors to characterize the in vivo clearance of cu - labeled distearoyl and dipalmitoyl lipids included within pegylated liposomes.in vivo pet imaging studies established that changes in lipidacyl chain length can result in desorption of lipid from the liposomal anchorage and interaction with blood components . Therefore, this factor should be considered for liposomal pet studies as desorption can rapidly alter the apparent pharmacokinetics . In another study, peterson et al . Developed a remote loading method using 2-hydroxyquinoline ionophore, to carry cu across the membrane of preformed liposomes and deliver it to an encapsulated copper - chelator . A highly efficient loading (> 95%) and retention stability (> 99%) was obtained adopting this approach . In vivo pet imaging studies demonstrated that a maximum tumor uptake of 5% id / g with high tumor to muscle ratio could be achieved . The cu - liposomes reached a maximum level in the liver and spleen after 4 h and subsequently remained at a constant level . Also, the cu - liposomes remained in the blood pool for> 24 h. the method provided cu - labeled liposomes with excellent imaging properties due to the high concentration of cu inside the liposomes and restricted exchange of cu with the biological environment due to the protective barrier constituted by the liposomal membrane . The same group of authors investigated the suitability of cu - labeled liposomes for imaging somatostatin receptor expression in neuroendocrine tumor model . The peptide octreotate (tate) was covalently attached to the pegylated liposomes with an encapsulated positron emitter cu . This peptide is routinely used in clinic for imaging somatostatin receptor - positive tumors by scintigraphy.in vivo pet imaging and biodistribution studies revealed that the presence of tate on the liposomes resulted in a significantly faster initial blood clearance in comparison to control liposomes without tate . There was no significant difference in tumor uptake (5% id / g in both cases) on using cu - labeled pegylated liposomes with or without tate, suggesting that the uptake was mainly due to passive targeting . However, cu - loaded pegylated liposomes with tate showed significantly higher tumor - to - muscle (t / m) ratio (12.7 1.0) than the control - liposomes without tate (8.9 0.9). The tumor accumulation and t / m ratio achieved in this study suggest that lioposomal systems might be used as carriers of radionuclides for therapeutic use and also for delivery of chemotherapeutic drugs . Tumor associated macrophages (tams) have been shown to play a major role in the growth and spread of several types of cancer . Locke et al . Reported pet imaging of tams in a mouse model of pulmonary adenocarcinoma, using mannose coated liposomes radiolabeled with cu.in vivo pet imaging and biodistribution studies revealed that radiolabeled mannosylated liposome accumulated in tams and exhibited little accumulation in remote lung areas at 6 h post injection . Further, it was verified by confocal microscopy that the pet signal was due to liposome internalization by tams . Urakami et al . Developed a methodology for one - step labeling of liposomes with f. solid - phase transition method was utilized, and high labeling efficiency and visualization of liposomal trafficking in mice by real - time analysis were obtained by pet . The same group reported the development of an efficient method for preparation of f - labeled liposome - encapsulated hemoglobin . Using the radiolabeled liposome, the oxygen transfer even in an ischemic brain could be monitored by dynamic pet . In another study, radiolabeling of pegylated liposomes with [f]fluorodipalmitin ([f]fdp) was reported by marik et al . Radiolabeled diglyceride was synthesized by the incorporation of f into the lipid molecule by nucleophilic substitution of p - toluenesulfonyl moiety . While free [f]fdp was rapidly taken by the liver, spleen, and lungs, liposome incorporated [f]fdp was observed to circulate in blood vessels for nearly 90 min . Adopting the previously reported procedures, f and cu - labeled liposomes were prepared by paoli et al . The liposomes were preconjugated with suitable fluorophores (calcein or af-750), for dual - modality pet / optical imaging . A model hydrophilic drug was encapsulated in the liposomal system and administered in mice bearing bilateral met-1 tumors . Using in vivo pet imaging and ex vivo fluorescent imaging of tumors, the authors could demonstrate that the accumulation of the drug was increased by up to 177-fold by liposomal encapsulation . Recently, oku et al . Reported the radiolabeling of liposomes [modified with peg or ala - pro - arg - pro - gly (aprpg) peptide] with 1-[f]fluoro-3,6-dioxatetracosane, which enabled imaging of gliomas by pet with higher contrast than that obtained with [f]fluorodeoxyglucose ([f]fdg). The liposomes did not accumulate in the normal surrounding brain tissue due to blood brain barrier protection, and using this approach, even a very small sized (1 mm) brain tumor could be specifically imaged with the radiolabeled liposome (figure 1). Mitchell et al . Developed a series of liposomal systems with oligoethylene glycol spacers of differing lengths between the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (dota) chelator and the lipid headgroup . A suitable fluorophore, n-(fluorescein-5-thiocarbamoyl)-1,2-dihexa - decanoyl - sn - glycero-3-phosphoethanolamine triethylammonium salt, was attached to the liposome, which could also be chelated to gd (for mri), in (for spect), or cu (for pet) and used for multimodal imaging . The effective radiolabeling and noninvasive imaging strategies developed thus far might aid further research on pet image - guided drug delivery using liposomal carriers in the near future . Pet imaging of brain tumor using peg - modified liposomes (top panel) and aprpg - modified liposomes (middle panel), labeled with 1-[f]fluoro-3,6-dioxatetracosane . The other regions of the brain showed a low background . On the contrary, [f]fdg imaged the whole brain, although the accumulation was higher in the tumor region (bottom panel). Miceller structures are important carriers for drug delivery because they can form relatively small and uniform size structures, be prepared from a variety of amphiphilic materials, increase solubility of hydrophobic molecules, and incorporate multiple functionalities into a single structure . When tagged with suitable contrast agents, these systems can also be used for molecular imaging as well as image - guided drug delivery . Among the various miceller structures, the inner core is the hydrophobic part of the block copolymer, which encapsulates the water - insoluble drug . The outer shell or corona of the hydrophilic block of the copolymer is often composed of peg, and it protects the drug from the aqueous environment and also imparts particle stability and excellent dispersibility in an aqueous solution . Owing to these characteristics, polymeric micelles have several advantages as drug carriers such as enhancing the aqueous solubility of hydrophobic drugs, prolonging the circulation time of the drug in the blood, improving the in vivo stability of the drug, providing both passive and active tumor targeting abilities, and reducing nonspecific uptake by the reticuloendothelial system . In vivo tumor targeting and drug delivery properties of polydiacetylene (pda) micelles (diameter 10 nm) were investigated by mackiewicz et al . In a breast cancer model . Such small sized micelles can better diffuse through blood vessel walls and reach deeper tumor tissues due to the epr effect . The authors synthesized different micelles with coatings consisting of either nitrilotriacetic acids (nta) or peg chains of variable lengths and tested for their ability to passively target tumor . Among them, 2 kda peg - coated micelle (pda - peg2000) was identified as the most promising carrier in terms of longer blood residence time, higher tumor uptake, and better imaging contrast . Fluorescence diffuse optical tomographic imaging indicated a tumor uptake of 3% of the injected dose of pda - peg2000 . The diffusion of pda - peg2000 micelles inside the tumor was further evidenced and quantified by pet imaging using f - fdg colocalization . Drug delivery application of the cargo was also assessed using micelles loaded with paclitaxel, a hydrophobic anticancer drug, which showed good in vitro cytotoxicity and in vivo tumor growth inhibition . Thus, the potential of pda - micelles for drug delivery could be successfully demonstrated in this study . In another study, cho et al . Reported a novel drug delivery strategy using poly(ethylene glycol)-block - poly(-caprolactone) (peg - b - pcl) micelles . Three different drugs, namely, paclitaxel (cytotoxic agent), cyclopamine (hedgehog inhibitor), and gossypol (bcl-2 inhibitor), were loaded on peg - b - pcl micelles and evaluated in xenograft models of ovarian cancer . Multi - drug - loaded peg - b - pcl micelles were nanoscopic, fairly stable in aqueous solution, and capable of simultaneous as well as sustained release of each of the three drugs in vitro . In vivo studies based on bioluminescence imaging and 3-deoxy-3-f - fluorothymidine (f - flt) pet imaging revealed that multi - drug - loaded peg - b - pcl micelles had significantly less tumor burden than use of paclitaxel alone . Also, f - flt - pet images clearly showed that multi - drug - loaded peg - b - pcl micelles significantly reduced tumor volumes over paclitaxel and vehicle controls and could thus prolong the overall survival . Thus, the authors could establish that the strategy of concurrent delivery of drug combinations of cytotoxic agents and molecular targeted agents using a micellar - based drug delivery vehicle is effective for the treatment of ovarian cancer . Recently, benezra et al . Evaluated the potential of f - labeled dasatinib derivative (ski249380, a new - generation src and platelet - derived growth factor receptor (pdgfr) inhibitor) loaded on micellar and liposomal carriers for drug delivery and uptake in xenograft models of high - grade glioma.in vivo pet imaging studies demonstrated a significantly higher tumor uptake for f - ski249380-loaded micellar formulations (4.9% id / g) compared to control group (1.6% id / g). Saturation studies using excess cold dasatinib showed marked reduction of tumor uptake values to levels in normal brain (1.5% id / g), consistent with in vivo binding specificity . The improved drug solubility, delivery, and kinetic behavior conferred by the use of these micellar f - ski249380 preparations might find utility in treatment of various types of gliomas . Despite the excellent attributes of the polymeric micelles as carriers for drug delivery, such systems suffer from insufficient in vivo stability which is affected by the surrounding environment, especially the concentration of the amphiphilic block copolymers . Upon dilution in the bloodstream, multimolecular polymeric micelles disassemble, leading to a burst release of drug and loss of tumor - targeting abilities . These limitations could be circumvented with the use of suitably engineered unimolecular micelles possessing excellent in vitro and in vivo stability . The synthesis of a multifunctional unimolecular micelle made of a hyperbranched amphiphilic block copolymer, boltorn h40-poly(l - glutamate - hydrazone - doxorubicin)-b - poly(ethylene glycol), for pet image - guided drug delivery was reported by xiao et al . The copolymer was conjugated with cyclo(arg - gly - asp - d - phe - cys) peptides (crgd) for integrin v3 targeting and macrocyclic chelators (1,4,7-triazacyclononane - n, n,n-triacetic acid [nota]) for cu - labeling and pet imaging . The anticancer drug doxorubicin (dox) was covalently conjugated onto the hydrophobic segments of the amphiphilic block copolymer arms via a ph - labile hydrazone linkage to enable ph - controlled drug release . In vivo pet imaging and biodistribution studies in u87 mg tumor - bearing mice showed higher tumor uptake for crgd - conjugated unimolecular micelles (7% id / g) than nontargeted micelles (2.5% id / g) (figure 2). Additionally, crgd - conjugated unimolecular micelles exhibited a much higher cellular uptake in u87 mg human glioblastoma cells than nontargeted unimolecular micelles due to integrin v3 mediated endocytosis, thereby leading to a significantly higher cytotoxicity when the micellar systems were conjugated with dox . The same group of authors reported the synthesis of another unimolecular micelle formed by dendritic amphiphilic block copolymers poly(amidoamine)-poly(l - lactide)-b - poly(ethylene glycol) conjugated with an anti - cd105 monoclonal antibody (trc105) and nota for pet image - guided drug delivery . As observed in the previous study, cu - labeled targeted micelles exhibited a much higher level of tumor accumulation than cu - labeled nontargeted micelles, measured by serial noninvasive pet imaging and confirmed by biodistribution studies in murine breast tumor - bearing mice . Thus, these multifunctional unimolecular micelles possessing passive and active tumor - targeting abilities, ph - controlled drug release, and pet imaging capabilities are potentially important drug delivery vehicles for image - guided therapy . (a) pet imaging of u87 tumor bearing mice at different time points post injection of cu - labeled unimolecular micelle loaded with dox (h40-crgd - cu) and cu - labeled unimolecular micelle conjugated with crgd and loaded with dox (h40-dox - crgd - cu) (b) ex vivo fluorescence imaging of u87 mg tumor, with the excitation and emission set for detecting dox fluorescence, harvested from mice injected with h40-dox - cu or h40-dox - crgd - cu . Adapted with permission from ref (86). Enzyme / prodrug therapy is one of the most promising strategies where systemic toxicity can be minimized while maintaining the therapeutic efficacy . In this process, a drug - activating enzyme is targeted or expressed in cancer cells, following which a nontoxic prodrug is administered systemically . The enzyme converts the prodrug to an active anticancer drug, achieving high concentrations in the tumor and sparing the normal tissues . However, there are certain requirements for this strategy to work in clinical context . The enzyme should be non - human or expressed at very low concentrations in the normal tissue and should have high enzymatic activity . The prodrug should be a good substrate for the enzyme but should not be activated in nontumor tissues . While the prodrug should be nontoxic, the activated drug should be highly toxic and diffusible to be taken up by the adjacent cells for a bystander cell kill effect . Currently, there are three major categories of enzyme / prodrug strategies: (a) delivery of genes that encode prodrug - activating enzymes into tumor tissue (gene encoding prodrug activating enzyme therapy, gdept, and virus - directed enzyme prodrug therapy, vdept), (b) targeted delivery of active enzymes in tumor tissue where the therapeutic enzyme is conjugated with an antibody, small molecular ligand, or peptide that binds to antigens preferentially expressed on the surface of tumor cells or in the tumor vasculature or interstitium (targeting group - directed enzyme / prodrug therapy, tdept), and (c) vasculature permeability - dependent enzyme / prodrug therapy (vpdept) in which the intratumoral delivery of the enzyme is realized through the higher permeability of tumor vasculature . Pet image - guided enzyme / prodrug strategies have been extensively reviewed and hence will be discussed briefly in the following text . Most of the studies in pet guided enzyme / prodrug based cancer therapy are based on the gdept approach . Developed a strategy which combined gene therapy with aromatic l - amino acid decarboxylase (aadc) gene and a prodrug, dopamine . Using this approach, the authors could synthesize and regulate the neurotransmitters involved in parkinson s disease . In vivo pet imaging using aadc tracer, 6-[f]fluoro - l - m - tyrosine (fmt) could measure the gene expression and thus establish the potential of enzyme / prodrug approach in delivery of therapeutic agents to the central nervous system . Also, the extent of gene expression could be effectively used to predict the therapeutic response . This approach could be further validated in another study where pet imaging with i - labeled 2-fluoro-2-deoxy-1b - d - arabino - furanosyl-5-iodo - uracil (i - fiau), a specific marker substrate for expression of the herpes simplex virus type-1 thymidine kinase (hsv-1-tk) gene, was used to identify the location, magnitude, and extent of vector - mediated gene expression in a phase i / ii clinical trial of gene therapy for recurrent glioblastoma . In this study, dynamic i - fiau - pet scans were done before gene transduction to assess the basal state of fiau - accumulation and washout of the tumor, and also after vector application to investigate whether specific fiau - accumulation did occur (figure 3a). Ganciclovir treatment (5 mg per kg twice a day over 14 days) was done starting 4 days after vector infusion . Treatment responses were recorded by repeated mri as well as pet with f - fdg and c - labeled methionine (c - met). The same pet tracer (i - fiau) was used by hackman et al . To assess the potential of double prodrug activation gene therapy using the escherichia coli cytosine deaminase (cd)-hsv-1-tk fusion gene (cd / tk) for treatment of different tumors . Pet imaging was used for monitoring expression of the cd / tk fusion gene, and the different levels of cd / tk expression in tumor models could be imaged quantitatively . The results of these studies could be utilized to develop standardized gene therapy protocols adopting enzyme / prodrug strategy for human subjects . (a) pet imaging using f - fiau to identify the location, magnitude, and extent of vector - mediated gene expression in gene therapy for recurrent glioblastoma . The region of specific i - fiau retention within the tumor after hsv-1-tk - transduction (white arrow) showed the signs of necrosis (cross hairs, right column and reduced methionine uptake [met]) after ganciclovir treatment . (b) pet imaging of hsv-1-tk activity in tumors after sindbis / tk infection . Tumor - bearing mice either received no vector treatment (tumor +, sindbis / tk) or received 3 sindbis / tk treatments via intraperitoneal injection far away from sites of tumor inoculation (tumor +, sindbis / tk +). Hsv-1-tk activity was determined after intravenous administration of f - feau as tracer . Tumors on the right shoulder of scid mice are indicated by yellow arrows, and white arrows indicate activity in urinary bladder . Adapated with permission from ref (102). The synthesis of half - mustard prodrug, 4-[(2-chloroethyl)(2-ethyl) amino]-phenoxycarbonyl - l - glutamic acid, by reductive alkylation of 4-[(2-chloroethyl)amino]-phenoxycarbonyl - l - glutamic acid was reported by malik et al . The prodrug was radiolabeled with c, and its potential for imaging antibody- and gene - directed enzyme prodrug therapy with pet could be established . The use of another prodrug, 1-(2-deoxy-2-fluoro--d - arabinofuranosyl)uracil (fau), for treatment of tumors with high thymidylate synthase catalytic activity was reported by eiseman et al . Pet imaging using f - fau was used to visualize tumors that have high thymidylate synthase catalytic activity . However, the authors did not observe high localization of f - fau in tumors compared with background, which might limit further utilization of this pet probe in clinical context . In another study, selective tumor targeting and quantitative in vivo monitoring using pet of a commonly applied gdept, based on hsv-1-tk and ganciclovir (gcv), was reported by tseng et al . Sindbis virus was used to deliver the hsv-1-tk suicide gene to tumor cells for subsequent gcv activation and tumor killing . Pet imaging using f - labeled fluoro - ethyl - arabinosyluridine (f - feau) was used to monitor the hsv-1-tk activity in tumor cells after parenteral administration of sindbis virus (figure 3b). High tumor uptake of f - feau (3% id / g) proved that the sindbis vector efficiently targeted the hsv-1-tk enzyme gene into the infected tumor cells . Also, pet imaging could be used to monitor hsv-1-tk activities after systemic sindbis vector treatments for determining the levels and tissue distribution of the vector and optimizing efficient prodrug activation for more accurate treatment planning and monitoring . This study was further extended by stelter et al ., where different molecular imaging strategies, such as bioluminescence, fluorescence molecular tomography, and pet, were used to evaluate sindbis virus mediated infection of tumor cells in vitro and in vivo . The authors concluded that the sindbis virus infection rates were not solely dependent on cellular laminin receptor expression and other factors such as cellular infection and viral replication might also be responsible . In another similar study, wang et al . Evaluated the efficacy of 4 different radiotracers, i-5-iodo-29-fluoro-1-b - d - arabinofuranosyluracil (i - fiau), 5-f - fluoro-29-deoxyuridine (f - fudr), 2-f - fluoroethyl - l - tyrosine (f - fet), and f - fdg for monitoring tumor responses using spect or pet during prodrug activation gene therapy with hsv-1-tk and gcv . Based on tumor uptake of the radiotracers, f - fudr was identified as the most suitable radiotracer for assessment of responses in tumors undergoing hsv-1-tk and gcv prodrug activation gene therapy . The enzyme -glucuronidase (-gus) has recently been investigated as a target in prodrug therapy for cancer . In order to optimize -gus - based prodrug therapies, a pet tracer, f - labeled 1-o-(4-(2-fluoroethyl - carbamoyloxymethyl)-2-nitrophenyl)-o--d - glucopyronuronate (f - feanga), was evaluated for imaging of -gus in tumor (c6 gliomas) and inflammation models.in vivo pet imaging and biodistribution studies showed high uptake of the radiotracer in tumor, high target to nontarget ratio, and rapid renal clearance . In inflammation model, the uptake of the radiotracer in inflamed muscle was significantly higher than in control muscle, thereby establishing the potential of this radiotracer to detect increased activity of -gus . The extent of -gus release in small c6 glioma tumors after a single treatment of doxorubicin (dox), carmustine (bcnu), and tumor necrosis factor (tnf-) with f - feanga pet was evaluated by the same group of authors . Pet studies confirmed that -gus was released in vivo and the distribution volume of f - feanga in c6 gliomas was increased significantly . The results obtained in this study demonstrate the potential of a two - step chemotherapy prodrug approach, in which tumors are treated with a single dose of a cytostatic drug before prodrug treatment . Recently, moon et al . Reported the synthesis of f labeled 1-(3-furyl)-4-hydroxy-5-fluoro-1-pentanone (f - f-4-im), which can be metabolized by the cyp4b1 enzyme and used for pet imaging of tumors and monitoring enzyme - activating anticancer prodrugs . Biodistribution studies in normal rats showed that the uptake of f - f-4-im was high in the lung, where cyp4b1 gene is preferentially expressed . The results were further confirmed by in vitro cell assays, and the potential of f - f-4-im for imaging of cyp4b1-transfected tumor cells and monitoring cyp4b1 enzyme / prodrug interactions could be demonstrated . It is envisaged that further development of pet guided enzyme / prodrug protocols would significantly facilitate their clinical translation with high safety and reliability . In the past two decades, the applications of nanotechnology in cancer diagnostics and therapy have attracted widespread interest and a variety of functional nanoparticles have been developed and evaluated for drug delivery, diagnostic sensors, imaging agents, and labeling probes . Nanoparticles used for this purpose vary with a size from 1 nm to few hundred nanometers and surface charge varying from negative to positive and even neutral . Particularly as drug delivery vehicles and molecular imaging tools, targeted nanoparticle based systems hold significant promise by virtue of their controllable size, high surface area to volume ratio, and customized internal and external chemistries . The major advantages of using the engineered nanoparticle based systems for such applications include (a) the ease of particle functionalization for conjugation with suitable targeting vectors such as peptides or antibodies, (b) the ability to deliver a higher concentration of contrast agent for every targeted binding event to achieve higher detection sensitivity which might permit diagnosis of the disease in its very early stage, and (c) improved treatment effects when used as drug carriers by protecting entrapped drugs from degradation, enhancing tumor uptake through the enhanced permeability and retention effect as well as receptor - mediated endocytosis and thereby achieving increased exposure of the tumor to therapeutic drugs . A variety of drug delivery systems based on metallic nanoparticles, oxide nanoparticles, polymeric nanoparticles, carbon nanostructures, biodegradable nanoparticles, etc . Have been developed for molecular imaging as well as drug delivery . For a given system, multiple factors determine the stability and fate of the delivery vehicle during storage and after administration, including size, rigidity, charge, solubility, and surface modifications of the nanoparticles . Therefore, the choice of a nanoparticle based delivery system is guided by the biodistribution, types of drugs that can be delivered using that system, and the specificity and pharmacokinetics of delivery . The different nanoparticle based systems which can be radiolabeled with suitable positron emitting radioisotopes for pet image - guided drug delivery are discussed in the following text . Among the various metallic nanoparticles reported to date, gold nanoparticles are most widely used for biomedical applications, including drug delivery and novel diagnostic and therapeutic approaches, due to their biocompatibility, small size, ease of characterization, and rich surface chemistry . The utilization of f - labeled gold nanoparticles for pet imaging was first reported by guerrero et al ., in which the gold nanoparticles of 12 nm were synthesized by citrate reduction of haucl4 . The nanoparticles were functionalized with two different peptides, ck and clpffd, and f - sfb was covalently bound to the nanoparticle conjugate . After intravenous administration of the radiolabeled nanoparticles in normal rats, in vivo pet imaging showed highest uptake of the radioactivity in the bladder . The lungs, liver, and spleen were the organs with the next highest levels of radioactivity, followed by the intestine, kidneys, and blood . The pancreas and brain, however, accumulated very low concentrations of radiolabeled nanoparticles . Clearance of the nanoparticles from the biological system took place by both renal and biliary excretions . Gold nanorods with suitable aspect ratios can absorb and strongly scatter light in the near - infrared region, which can be used for enhanced optical imaging and photothermal cancer therapy . The development of a multifunctional gold nanorod - based nanoplatform for targeted anticancer drug delivery and pet imaging of tumors was reported by xiao et al . The bare gold nanorods had a length and diameter of approximately 45 and 10 nm, respectively . An anticancer drug (dox) and tumor targeting agent (crgd) were conjugated to the pegylated gold nanorods . Also, nota was attached onto the distal ends of the peg arms for complexation with cu . Based on flow cytometry analysis, crgd - conjugated gold nanorods exhibited a higher cellular uptake and cytotoxicity than nontargeted ones in vitro . However, in vivo pet imaging and biodistribution studies showed that targeted and nontargeted gold nanorods had similar distribution pattern especially in the tumor (figure 4a). Despite this limitation, this initial attempt provided a suitable nanoplatform for possible integration of multifunctionality including molecular targeting, chemotherapy, and photothermal therapy, as well as multimodality imaging, which can potentially lead to improved therapeutic efficacy and cancer monitoring . To achieve a similar goal, xie et al . Reported the preparation of cu - labeled gold nanoshells conjugated with crgd and studied the in vivo biodistribution and tumor specificity using pet . The nanoshell used in this study was composed of a silica core (120 nm in diameter) and a gold shell (810 nm) to absorb light at near - infrared wavelengths . In vivo pet imaging suggested that tumor targeting was improved by conjugation of gold nanoshells to crgd, which was advantageous over the previous study by xiao et al . Both targeted and nontargeted gold nanoshells were cleared from the circulation by the liver and spleen . In the subablative thermal therapy study, enhanced biological effectiveness of targeted gold nanoshell the promising results obtained from this study might lead to advancement of gold nanoshells as theranostic platforms for effective cancer diagnosis and therapy . (a) targeting of integrin v3 expression in u87 mg tumor bearing mice by gold nanorods (gnr) conjugated with crgd . Pet images at different time points post injection of cu - labeled gold nanorods conjugated with dox (cu - nota - gnr - dox) and cu - labeled gold nanorods conjugated with dox and crgd (cu - nota - gnr - dox - crgd). (b) targeting of cd105 expression in 4t1 tumor - bearing mice by trc105-conjugated mesoporous silica nanoparticles . Pet images at different time points post injection of cu - labeled mesoporous silica (cu - nota - msio2) and cu - labeled mesoporous silica conjugated with trc105 (cu - nota - msio2-trc105). Tumors were indicated by yellow arrowheads . Adapted with permission from ref (142). (c) targeting of lung endothelium in c57bl/6 mice by polymeric nanoparticles conjugated with anti - icam antibody . Micro - pet images of mice at different time points post injection of cu - labeled nanoparticle conjugated with anti - icam antibody (cu - dota - np - anti - icam) and cu - labeled nanoparticle conjugated with anti - icam antibody after pretreating the mice with lipopolysaccharides (cu - dota - np - anti - icam lps treated). (d) targeting of integrin v3-expression in u87 mg tumor bearing mice by crgd - functionalized single walled carbon nanotubes (swnts). Peg5400rgd observed in the u87 mg tumor (first row) and control experiment showing blocking of swnt the arrows point to the tumors . Adapted with permission from ref (165). Another category of promising nanoplatforms which has drawn substantial interest recently is the oxide nanoparticles (such as mesoporous silica and iron oxide nanoparticles) due to their nontoxic nature, easily modifiable surface, and good biocompatibility . Shell silica nanoparticles as targeted pet / optical multimodal imaging probes was reported by benezra et al . Near - infrared fluorescent, cy5 dye - encapsulated, core shell silica - based nanoparticles were prepared and coated with peg as per the method reported by burns et al . The nanoparticle was conjugated with crgd and radiolabeled with i through a tyrosine linker . In vitro cell binding assays demonstrated the specificity of the nanoplatform toward intregrin v3 expression . In vivo pet / optical imaging and biodistribution studies showed a tumor uptake of 1.5% id / g at 4 h post injection, with high tumor to background ratio and rapid renal clearance . Owing to their favorable characteristics, such as bulk renal clearance, favorable targeting kinetics, lack of acute toxicity, superior photophysical features, and multimodal (pet / optical) imaging capabilities, such nanoparticles have received the united states food and drug administration (us fda)-investigational new drug approval for a first - in - human clinical trial . The synthesis of ultrasmall, monodisperse silica nanoconjugates for targeted dual - modal imaging of lymph nodes with metastatic tumors was reported by tang et al . The nanoparticles were functionalized with an aptamer derivative having high binding affinity for nucleolin, a protein that is overexpressed in the cytoplasm and on the plasma membrane of several cancer cells . Also, a near - infrared (nir) dye and dota were conjugated on the surface of the functionalized nanoparticle . The aptamer functionalized silica nanoconjugate was radiolabeled with cu, and in vivo pet / optical imaging studies showed markedly enhanced uptake of the radiolabeled agent in lymph nodes with metastatic tumors in a murine breast tumor model . In a similar study, kim et al . Shell silica nanoprobe for multimodal (pet / optical / mri) imaging of the sentinel lymph node . Magnetic silica nanoparticles with cobalt ferrite core and silica shell were synthesized which encapsulated nir dye on the silica shell . The surface of the nanoparticle was modified with amino group and peg for conjugation with nota, which was used for chelating ga . The triple modality nanoprobe could be successfully utilized to visualize the sentinel lymph node in mice . Thus, these multimodal silica nanostructures hold great potential for improving the accuracy of clinical tumor staging by serving as probes for efficient noninvasive targeted imaging of metastatic lymph nodes . Different chemotherapeutic drugs can also be attached on the surface of the functionalized nanoconjugates for prevention of metastases . In an interesting study, di pascua et al . Utilized commercially available mesoporous silica nanoparticles as a carrier material for the therapeutic radioisotope ho (t1/2 = 26.8 h, emax = 1.84 mev). A lipophilic acetylacetonate complex of ho was incorporated in mesoporous silica nanoparticles (80100 nm in diameter), which were subsequently irradiated in a neutron flux to produce particles containing ho by (n,) reaction . These radioactive nanoparticles were utilized to deliver effective therapeutic doses for treating ovarian cancer metastases after intraperitoneal delivery in skov-3 ovarian tumor - bearing mice . In vivo spect imaging demonstrated that most of the ho - containing mesoporous silica nanoparticles administered to ovarian tumor - bearing mice were retained in the peritoneal cavity and selectively accumulated in the tumors (33% id / g after 24 h). Radiotherapeutic efficacy was monitored using pet / ct using f - fdg which showed a decrease in tumor volume, which correlated with a marked increase in survival after treatment with 4 mbq of the radioactive nanoparticles . Though the authors could not explain the reason for the high uptake of mesoporous silica nanoparticles in ovarian tumor, this strategy might find utility in incorporation with other therapeutic radionuclides in nanostructured materials for treatment of various types of cancer . In another approach, chen et al . Reported the development of biocompatible functionalized mesoporous silica nanoparticles for actively targeted pet imaging and chemotherapeutic drug delivery . Mesoporous silica nanoparticles were surface functionalized with thiol groups, pegylated, conjugated with nota chelator and trc105 antibody (specific for cd105/endoglin), and radiolabeled with cu . In vivo pet imaging and biodistribution studies in 4t1 breast tumor bearing mice showed high tumor uptake (6% id / g) at 5 h post injection (figure 4b). The tumor uptake of radiolabeled nanoparticles not conjugated with trc105 was much lower than the tumor uptake observed with trc105 conjugated nanoparticles, indicating that active targeting was responsible for the enhanced tumor uptake . The authors also demonstrated the feasibility of enhanced tumor targeted drug delivery in vivo using trc105 conjugated mesoporous silica loaded with an anticancer drug, dox . The encouraging results obtained in this study hold promise for future image - guided drug delivery and targeted cancer therapy using this class of nanomaterials . Recently, iron oxide nanoparticles have been actively investigated as nanoplatforms for multimodal molecular imaging . The conventional drug loading approach by covalent linkage on such nanoplatforms is inefficient and suboptimal for drug release . In order to circumvent this limitation, xie et al . Synthesized iron oxide nanoparticles, modified their surface using dopamine, and encapsulated them into human serum albumin matrices, which are clinically utilized as drug carriers . The human serum albumin coated iron oxide nanoparticles were dually labeled with cu - dota and cy5.5 dye, and tested in a subcutaneous u87 mg xenograft mouse model . In vivo pet / optical / mr imaging showed a high tumor uptake (5% id / g at 4 h post injection) with high tumor to background ratio . An inhomogeneous particle distribution pattern was observed with mri, but pet and optical imaging showed homogeneous intensities at the tumor area . The human serum albumin coated nanoparticles manifested a prolonged circulation half - life . Adopting this strategy, small drug molecules can be coloaded with iron oxide nanoparticles into human serum albumin to yield theranostic agents . Recently, chen et al . Reported a chelator free approach for preparation of radioarsenic labeled iron oxide nanoparticles . The radiolabeled nanoparticle was used as pet / mri agent for dual - modality imaging in vivo and lymph node mapping . This strategy can be extended for radiolabeling iron oxide nanoparticles with as for radiotherapeutic applications . In another study, yang et al . Reported the synthesis of crgd - functionalized, dox - conjugated, and cu - labeled iron oxide nanoparticles for targeted anticancer drug delivery and pet / mr imaging.in vivo pet imaging and biodistribution studies in u87 tumor bearing mice showed that crgd - conjugated iron oxide nanocarriers showed a much higher level of tumor accumulation (5% id / g) than crgd - free ones (<2% id / g). Also, crgd - conjugated nanocarriers induced a significant amount of cytotoxicity in the u87 mg tumor cells, suggesting that dox was released from the iron oxide nanocarrier and entered the cell nucleus . Thus, the potential of iron oxide nanoparticles for combined tumor - targeting drug delivery as well as multimodal imaging could be amply demonstrated . In recent times, there has been widespread interest in the use of biocompatible and biodegradable polymer nanoparticles for drug delivery . Reported the synthesis of nanoparticles using cyclodextrin - containing polycations and sirna sequence targeting luciferase mrna . A bifunctional chelator, dota, was conjugated to the 5 end of sirna and used for labeling with cu . A dual - modality (pet / optical) imaging approach was used to investigate the biodistribution and functional activity of sirna delivered by the nanoparticles . In vivo pet / ct imaging in mice bearing luciferase - expressing neuro2a tumors was used to analyze the biodistribution and tumor localization of the sirna nanoparticles . Also, bioluminescent imaging was used before and after pet imaging to enable correlation of functional efficacy with biodistribution data . It was observed that both nontargeted and transferrin - targeted sirna nanoparticles exhibited similar biodistribution and tumor localization . However, transferrin - targeted sirna nanoparticles could reduce tumor luciferase activity by 50% relative to nontargeted sirna nanoparticles, 1 d after injection . Compartmental modeling was used to demonstrate that the primary advantage of targeted nanoparticles was associated with processes involved in cellular uptake in tumor cells rather than overall tumor localization . The authors inferred that optimization of internalization might be the key factor for effective targeted therapy using this class of nanoparticles . The utilization of pet to quantify the uptake of intercellular adhesion molecule 1 (icam-1) targeted, cu - labeled polymeric nanoparticles by the pulmonary endothelium was reported by rossin et al.in vivo pet imaging and biodistribution studies showed a 3- to 4-fold higher uptake in the lungs of mice injected with icam - targeted nanoparticles compared to that of the control group (figure 4c). The lung uptake could be further enhanced by pretreating the mice with lipopolysaccharides probably due to icam-1 upregulation . However, a considerable release of small cu - radiometabolites from the nanoparticles beginning as early as 1 h after injection was observed, suggesting poor in vivo stability of the radiolabeled conjugate . An improved strategy where the radiolabeled nanoparticle remained stable in vivo was reported by simone et al . The authors developed a polymeric nanoparticle using a poly(4-vinylphenol) polymer backbone which could directly be radiolabeled with i. the polymeric nanoparticles were coated with monoclonal antibodies targeting endothelial determinants . The radiolabeled nanoparticles were used for imaging the pulmonary vasculature and also for tracking the nanoparticle pharmacokinetics . This approach might find utility in image - guided delivery of therapeutics to the pulmonary endothelium in patients with acute and chronic respiratory diseases . The synthesis and utilization of poly(n - vinylpyrrolidone)-b - poly(-caprolactone) nanoparticles (100 nm diameter) for drug delivery was reported by zhu et al . The nanoparticles were conjugated with a near - infrared fluorescent dye, nir-797, for in vivo optical imaging . An anticancer drug, paclitaxel (ptx), was loaded in the polymeric nanoparticles with high drug loading content (> 25%) and encapsulation efficiency (> 85%). The antitumor effect of ptx - loaded nanoparticles was evaluated, both in vitro on three different cancer cell lines and in vivo on a hepatic h22 tumor bearing mouse model using optical imaging . The antitumor effects of the ptx - loaded nanoparticles were further visualized using f - fdg pet scans . By combining the tumor volumes and survival rate measurements, it could be confirmed that ptx - loaded nanoparticles exhibited superior in vivo antitumor effect than taxol (commercially available formulation of paclitaxel). In a similar study, liu et al . Reported the synthesis of poly(ethylene glycol)-poly(caprolactone) nanoparticles (70 nm diameter) and evaluated their efficacy for drug delivery . As in the previous case, the polymeric nanoparticles were conjugated with nir-797 dye for investigating the biodistribution of the drug - loaded nanoparticles using in vivo optical imaging . An anticancer drug, docetaxel (doc), could be encapsulated into the polymeric nanoparticles with a high drug loading content (20%) and encapsulation efficiency (> 80%). In vitro cytotoxicity test showed that doc - loaded nanoparticles inhibited the murine hepatic carcinoma cell line h22 in a dose - dependent manner, which was similar to taxotere, the commercialized formulation of docetaxel . However, in vivo tumor evaluation using optical imaging and f - fdg pet scans demonstrated the superiority of doc - loaded polymeric nanoparticles over taxotere . Therefore, it could be envisaged that these highly efficient and biodegradable nanoparticles might find clinical utility in pet image - guided anticancer drug delivery in the near future . In a pioneering study, zhou et al . Reported the synthesis of poly(lactide - co - glycolide) nanoparticles (70 nm diameter), which could be utilized as brain penetrating nanocarriers for the treatment of glioblastoma . In order to illustrate the translational potential of brain - penetrating nanoparticles, the authors conducted a screen of 2,000 compounds that were previously approved by us fda to inhibit patient - derived brain cancer stem cells and encapsulated the best agent (dithiazanine iodide) into the nanocarrier . The radiolabeled brain - penetrating nanocarriers were administered by convection - enhanced delivery in rats bearing brain cancer stem cell derived xenografts . In vivo pet imaging demonstrated accumulation of the nanoparticles in the brain . Also, a significantly increased survival in rats bearing brain cancer xenografts was observed, which demonstrated the potential of such brain - penetrating nanoparticles for targeted image - guided drug delivery for treatment of brain tumors . With a different strategy, chen et al . Employed anionic poly(l - glutamic acid) as a carrier to covalently link with camptothecin (an anticancer drug), enabling encapsulation into supramolecular nanoparticle vectors . Approximately five camptothecin molecules were conjugated to each polymer chain by ester bond formation, which could be degraded via esterase - mediated hydrolysis to allow controlled release of camptothecin under physiological conditions . The authors synthesized nanoparticles of two different sizes (37 and 104 nm), both of which were radiolabeled with cu and administered in mice bearing lewis lung carcinoma xenografts . In vivo pet imaging and biodistribution studies revealed that the smaller sized (37 nm) nanoparticles exhibited higher tumor accumulation due to the epr effect . The superior in vivo antitumor efficacy of the 37 nm supramolecular nanoparticles was further validated by tumor reduction / inhibition studies . Despite the encouraging results, the tumor uptake of the supramolecular nanoparticles was not impressive, which might be a deterrent for their use as potential drug delivery vehicles . However, the tumor uptake might be improved on conjugation with suitable targeting ligands . Homopolymers or copolymers have long been explored as potential carriers in targeted drug delivery . The synthesis and characterization of pegylated star - shaped copolymer nanoparticles (2570 nm size) containing core shell morphology for in vivo pet imaging was reported by fukukawa et al . These copolymers possessed a hydrophilic inner shell bearing reactive functional groups, and a central hydrophobic core . Dota was conjugated to the functional groups in the inner shell for cu labeling . In vivo pet imaging and biodistribution studies in normal rats showed that copolymers with increasing peg shell thickness showed increased blood circulation and low accumulation in excretory organs . This preliminary study suggested the potential of such systems as for in vivo tumor imaging and targeted drug delivery . The synthesis of n-(2-hydroxypropyl)methacrylamide (hpma) copolymers for pet imaging and image - guided chemotherapy of prostate cancer was reported by yuan et al . The hpma copolymer was conjugated with dota for cu labeling and also with crgd peptide for targeting v3 integrin in tumor neovasculature . The tumor localization of the radiolabeled copolymer was visualized by pet in a mouse model bearing human prostate cancer xenografts . A time - dependent increase in radioactivity uptake in tumor - bearing mice injected with the hpma - crgd - dota - cu copolymers was observed, but this phenomenon was not seen in mice injected with control hpma - dota - cu copolymers . However, the tumor uptake observed for the targeted copolymer (2.75% id / g) at 3 h post injection was slightly higher than what was observed with the nontargeted copolymer (1.29% id / g), suggesting that, along with active targeting, passive epr effect also plays a partial role in tumor localization of the radiolabeled copolymer . The findings from these studies might set the stage for further optimization and evaluation of the copolymer constructs for image - guided drug delivery in various tumor models . Owing to their unique physical and chemical properties, the use of functionalized carbon - based nanomaterials is gaining popularity in many areas of biomedical research, including molecular imaging and drug delivery . Since their discovery, the carbon nanotubes have become the most widely used carbon - based nanomaterial for biomedical applications . Pet imaging using pegylated single walled carbon nanotubes (15 nm diameter, 100300 nm length) conjugated with rgd peptides was reported by liu et al . Dota was attached to the termini of the peg chains and used to conjugate cu . In vivo pet imaging and biodistribution studies showed that peg5400 modified single walled carbon nanotubes conjugated with crgd exhibited a high tumor uptake of 1015% id / g, with high target to nontarget ratio (figure 4d). High tumor uptake of the radiolabeled single walled carbon nanotubes was observed over long periods (> 24 h). In another study, mcdevitt et al . Studied the biodistribution pattern of y labeled carbon nanotubes (1 nm diameter, 50 nm length) without peg modification in normal mice . The radiolabeled agent cleared from the blood within 3 h and distributed predominantly to the kidneys, liver, spleen, and bone . Suitable peg modification of carbon nanotubes is of paramount importance in order to reduce reticuloendothelial system uptake and prolong blood circulation time of the single walled nanotubes for finding utility in drug delivery approaches . Graphene is another structurally robust, yet highly flexible, nanoplatform with potential for use as a drug delivery vehicle . Hong et al . Reported the synthesis of covalently functionalized nanographene oxide sheets (1050 nm), which were pegylated and conjugated with anti - cd105 monoclonal antibody (trc105) for imaging tumor angiogenesis . The pegylated graphene oxide was also conjugated with nota for cu labeling . In vitro studies using human umbilical vein endothelial cells (huvecs, high cd105 expression) demonstrated strong and specific cd105-binding by the trc105 conjugated nanographene . Also, in vivo pet imaging and biodistribution studies in 4t1 tumor bearing mice showed high tumor uptake (6% id / g) within 0.5 h post injection, which remained fairly stable over time . These studies were further validated by ex vivo histological analyses, and vasculature specific targeting with little extravasation of trc105 conjugated graphene oxide could be demonstrated . The same group further evaluated ga - labeled nanographene oxide conjugated with trc105, and similar results were obtained . This work was further improved by using cu - labeled reduced graphene oxide conjugated with trc105 for in vivo tumor vasculature targeting . Reduced graphene oxide has more desirable properties for photothermal therapy than the more hydrophilic graphene oxide used in the previous studies, due to its strong absorbance in the near - infrared range.in vivo pet imaging revealed rapid tumor uptake (5.5% id / g) of cu - labeled nanoplatform with excellent tumor contrast . In all these studies, trc105 conjugated nanoparticles exhibited little extravasation in the 4t1 tumor, indicating the advantages of tumor vasculature targeting using such nanoplatforms . It can be envisaged that the promising results obtained in these studies can open up new avenues for image - guided drug delivery and cancer therapy using graphene oxide based nanoplatforms . The concept of theranostics has also played a vital role in radiation - based therapies, especially, using targeted radiopharmaceuticals . In this approach, a radiation dose is specifically administered to the cancerous lesions using peptides, proteins, or antibodies radiolabeled with suitable therapeutic radionuclides such as y, i, or lu . The same targeting ligands can also be conveniently radiolabeled with suitable positron emitters such as f, ga, cu, or zr, thereby providing exciting opportunities to guide such therapies using pet . This approach plays a dominant role in diagnosis of the disease in its early stage, validation of the targeting strategy, and development of novel therapeutic radiopharmaceuticals . Thus, it facilitates better, faster, and cost - effective decision making, helping to eliminate failures in the targeted radiotherapy pathway and advance only with the promising candidates to receive such therapies . Despite the availability of a wide variety of pet radiopharmaceuticals, f - fdg is still the most widely used radiotracer in cancer management, and the pivotal role of f - fdg - pet / ct in modern nuclear medicine needs hardly to be reiterated . Excellent review articles have appeared in recent times which have summarized the clinical diagnosis and therapeutic response evaluation using f - fdg and other f - based radiotracers, and hence these are not discussed here further . Another pet radioisotope which is gaining significant clinical attention in recent times is ga (t1/2 = 68 min). The convenient availability of this radioisotope from ge / ga generators without the dependence on onsite cyclotrons makes it economical to use for a wide variety of pet scans . In particular, from the perspective of image - guided therapy, the radiolabeled peptides targeting somatostatin receptors, overexpressed in the majority of neuroendocrine malignancies, have a great potential for both imaging and therapy of tumors where other therapies fail . The development of ga - labeled somatostatin analogues such as dotanoc (dota-1-nal - octreotide), dotatoc (dota - d - phe - tyr - octreotide), or dotatate (dota - d - phe - tyr - thr - octreotide) for pet / ct imaging has significantly improved the diagnosis of neuroendocrine tumors . In a typical example, gains et al . Investigated the efficacy of pet / ct using ga - dotatate to select children with primary refractory or relapsed high - risk neuroblastoma for treatment with lu - dotatate and also evaluated whether this is a viable therapeutic option for those children . Imaging with ga - dotatate could indicate the expression of somatostatin receptors and was of paramount importance for guiding radiotherapy (figure 5a). Post - therapy of lu - dotatate, ga - dotatate scans was used to assess the response (figure 5a), and a positive therapeutic outcome with a regression in some lesions and an apparent block of the metastatic activity could be observed in many cases . In a similar study, budiawan et al . Investigated the application and role of ga - dotatate pet / ct imaging in non - radioiodine - avid refractory thyroid cancer patients who have undergone peptide receptor radionuclide therapy (prrt) with y / lu - dotatate . The authors concluded that prrt guided by pet imaging is an effective therapeutic option with minimal toxicity, good response rate, and excellent survival benefits . In a different study, wu et al . Has reported the use of ga - labeled sir spheres as pet imaging surrogate for distribution assessment and radiation dose estimation of y - sir - spheres, which are currently used in the treatment of solid liver tumors . The authors observed that ga - sir - spheres had good in vivo stability and localization of the radiolabeled microparticles in the liver could be observed using pet imaging . Similar results were obtained by avila - rodriguez et al . On using cu or y labeled sir spheres . (i) images showing ga - dotatate avid lesions in t4 vertebral body and 3 metastases in liver (arrow). Physiologic uptake is seen in pituitary, kidneys, bladder, stomach wall, liver, and spleen . (ii) images from repeated ga - dotatate pet / ct 1 y later after 3 administrations of lu - dotatate, showing metabolic partial response with reduction in suvmax of lesion in t4 and liver and no new lesions . Adapted with permission from ref (190). (i) a patient with liver and bone metastases, and (ii and iii) two patients with multiple bone metastases . A number of lesions have been specifically indicated by arrows . Adapted with permission from ref (198). Another example, close to clinical translation, is represented by pet imaging of human epidermal growth factor receptor 2 (her2) expressions . The overexpression of the her2 is observed in 15% of breast cancers and associated with poor prognosis in terms of overall survival . Generally, y or i labeled trastuzumab is used for targeted radiotherapy of primary or metastatic breast cancer, in experimental, preclinical, or clinical settings . Pet imaging of her2- positive lesions in patients with metastatic breast cancer was reported by dijkers et al . The antibody trastuzumab was radiolabeled with zr, a pet imaging surrogate for y. administration of zr - trastuzumab at appropriate doses allowed visualization and quantification of uptake in her2-positive lesions in patients with metastatic breast cancer by pet (figure 5b). The authors concluded that pet imaging of her2 expression would aid in improving diagnosis, staging of the disease, guiding trastuzumab therapy, and monitoring the therapeutic response . A large number of other pet radiopharmaceuticals for molecular imaging and personalized cancer management have also been reported, details of which can be found in recent review articles . We would apologize to those whose work could not be presented here, mainly due to the vastness of the field and availability of enormous literature which could not be summarized in a single review . In the last several decades, major milestones have been reached in every area of cancer care and a variety of anticancer drugs have been developed which are now commercially available worldwide . Though most of these anticancer agents have the potential to be effective at sufficiently high doses, they are often associated with severe systemic side effects that cannot be tolerated by patients who are already weak due to effects of cancer . In a majority of the cases, the success of cancer therapy typically hinges upon circumventing the dose - limited toxicity while administering such drugs . Additionally, many conventional therapeutic agents often fail due to their limited ability to reach the target tissue and their poor selectivity against cancerous lesions . Ideally a drug should possess perfect specificity to cancerous cells and have no effect on the rest of the body . To achieve these objectives, a variety of drug delivery systems have been engineered for the targeted delivery and controlled release of therapeutic agents to specifically kill the cancerous cells . The full potential of the drug delivery systems extends beyond treatment, and several image - guided approaches have now been implemented in areas ranging from new therapeutic target discovery to effectively monitoring tumor pharmacokinetics and drug distribution to modulation of drug release at the target site . In particular, pet image - guided drug delivery provides a means for treating a variety of diseases with minimal systemic involvement while concurrently monitoring therapeutic efficacy . This minimally invasive approach provides a comprehensive answer to many challenges with conventional therapeutic approaches and is expected to lead to a paradigm shift in cancer patient care . The major advances in targeted drug delivery have been attributed to the recent progress in nanotechnology that has resulted in the development of nanosized drug delivery platforms having distinct advantages in cancer therapy . The versatile nanoplatforms provide opportunities for multifunctionalization so that a single platform can be used to detect and treat tumors, monitor treatment response, and thus guide therapeutic regimes . The nanoformulations can be functionalized to minimize clearance by the immune system and prolong circulation times, and also for attachment of suitable vectors (peptides, proteins, antibodies, etc .) Targeting specific receptors, thereby enhancing tumor uptake through epr effect as well as receptor - mediated endocytosis . The nanomaterial based delivery systems also result in improved treatment effects by protecting entrapped drugs from degradation during their delivery . When two or more molecular imaging techniques are used in conjunction, they would provide synergistic information when compared with any single imaging modality . Additionally, multiple therapeutic agents such as chemotherapy, antiangiogenic, or gene therapy agents can be simultaneously delivered by nanocarriers to tumor sites to enhance the effectiveness of therapy . Despite these advantages, it must be admitted that the field of pet image - guided drug delivery is still in its infancy and more systematic studies to understand the mechanisms for targeting and drug delivery would be required in order to translate these novel discoveries into clinical impact . The potential challenges to clinical translation of the drug delivery systems are in vivo characterization of the drug carriers, preclinical validation of targeting and delivery, studies of biodistribution, pharmacokinetics, pharmacodynamics, and toxicity, and scale - up manufacturing of delivery systems . Another major challenge lies in overcoming the biological barriers to deliver optimum amount of therapeutics into tumors and cells . Moreover, the performance of the drug delivery carriers depends on several limiting factors which include the synthesis method adopted, their size and shape, internal structure of the drug carrier, drug loading methodology, surface functionalization and conjugation strategy adopted for attaching targeting ligands to the carrier platforms, etc . The issues related to toxicity of nanosized drug delivery platforms can be addressed by the use of biocompatible and biodegradable polymeric nanoparticles for drug delivery, which have received considerable attention in the recent times . Also, stimulus - responsive polymeric nanomaterials can be synthesized which mimic the behavior of biological molecules, where external stimuli or changes in local environment can trigger a change in property to regulate drug release . Such smart nanoparticle systems when coupled with suitable targeting ligands can probably best minimize off - target effects and maximize programmability, thereby offering the possibility of radically changing the practice of drug delivery . While numerous obstacles face all new technologies, materializing the opportunities presented by pet image - guided drug delivery requires addressing the significant interdisciplinary challenges and biological barriers . Besides these, several other complex factors, such as considerable regulatory hurdles, limited potential market, lobbying by the manufacturers of established anticancer drugs, lack of reimbursement strategies by the insurance agencies for such novel strategies, etc ., might impede the bench to bedside translation of this promising approach . The concerted efforts of all stakeholders, which include scientists from academia as well as industries, progressive and technology savvy physicians, radiologists, surgeons, program advisory boards, regulatory authorities, and grant review panels, would be required, both to create enthusiasm for developing these new concepts and also to prevent adverse messaging based on myths, conjectures, hyperbole, and bias . This in turn would provide impetus to further research which might aid in clinical translation of pet image - guided drug delivery approaches and thus achieve the ultimate goal of personalized medicine in the near future.
Dementia is a chronic condition characterized by a progressive cognitive impairment that leads to functional disability.1 in 2015, it was estimated that approximately 47 million people worldwide were affected by dementia, and this number is expected to increase, reaching 131.5 million by 2050.2 as such, it represents a veritable public health challenge . Alzheimer s disease (ad), a pathology first described by alois alzheimer in 1907,3 is the most frequent cause of dementia in elderly . Knowledge about the etiology and pathogenesis of the disease is continuously updated,4 but there are still limitations in diagnostic capability5 and in the discovery of pharmacological treatments that would be able to stop or better prevent the disease . At present medications currently used provide only a modest symptomatic relief to a subset of patients and do not treat the underlying causes of this disease . The reasons for this failure are probably due to the scant knowledge of the cellular and molecular mechanisms implicated in ad pathogenesis and of the approved therapies that coarsely affect both cholinergic and glutamatergic neurotransmission . Conversely, many of the new drugs in development aim to modify the disease process itself by impacting one or more of the many wide - ranging brain changes caused by ad . These changes offer potential targets for new drugs to stop or slow down the disease progression . It is pathologically characterized by widespread oxidative stress, mitochondrial damage, glutamate excitotoxicity, neuroinflammation, neurofibrillary tangle (nft) formation, and -amyloid (a) deposition creating senile plaques (sps).6 these latter are constituted by a peptide, and their genesis is followed by intracellular deposition of nfts,7 as a consequence of tau protein hyperphosphorylation . The results are synaptic and neuronal dysfunction and loss.8 over the years, it has been demonstrated that other factors play an important role in the pathogenesis and progression of ad . Among them, the key role of neuroinflammation has been affirmed.9 physiologically, the inflammatory process is aimed at controlling injuries through several mechanisms to repair tissues.10 however, an increasing amount of literature confirms its role in the pathogenesis and exacerbation of ad.1114 inflammation acts to remove both the initial cause of the infliction and to eliminate the destroyed tissues and dead cells resulting from the original injury . In fact, inflammation is emerging as the real cause of the associated disease, more than a mere contribution to the exacerbation of tissue damage . Indeed, some studies have revealed that the injection of lipopolysaccharide in transgenic mice induces neuroinflammation, triggering intracellular a deposit and tau phosphorylation.15,16 the molecular processes are not necessarily the primary events . The microglial priming model suggests that the presymptomatic ad pathology, characterized by low levels of proinflammatory mediators, can act on microglia for long periods of time.17 furthermore, stress, inflammation, and infection can operate as secondary triggers, causing changes in these primed cells: they reach an activated state establishing an inflammatory response contributing to ad pathogenesis.18 from an immunological point of view, the central nervous system was always seen as a highly protected tissue, exposed to inflammatory phenomena solely in cases of infection or disruption of the blood brain barrier (bbb). Nowadays, we know that there are several cells expressing pattern recognition receptors able to induce inflammatory signaling pathways.13 these pattern recognition receptors can recognize molecular signals of microbial molecules, called pathogen - associated molecular patterns, as well as endogenous damage - associated molecular patterns (damps), that typically accumulate in infected tissues . Damps are present in diseased brains as misfolded proteins (eg, sps and nfts), aggregated peptides, or nucleic acids.19 it is clear that damps can trigger neuroinflammation by deflecting proinflammatory reactions from their helpful purpose, and this is the reason why our way of viewing neurodegenerative diseases has changed over the years . The role of the neuroinflammatory process is not exclusively attributable to innate immunity (which in the brain is constituted by microglia), but it is also caused by other brain resident cells that constitute, in one word, macroglia (ie, astrocytes, ng2-positive cells, and oligodendrocytes), as well as endothelial cells and neurons.2023 hence, it is clear that there are many characters involved in this inflammatory process . Thus, a better knowledge of the mechanisms underlying the role of neuroinflammation in ad can be an excellent starting point for the development of molecules able to counteract it . Even if a deposits can alone induce an inflammatory response that subsequently leads to ad development, it is well established that the neuroinflammatory pathophysiology is more complex and driven by the activation of different brain cells . In particular, growing evidence suggests that this phenomenon is mainly supported by glial cells, which respond quickly to brain injuries, activating a series of repair mechanisms to restore brain physiology . These cells are a highly heterogeneous population, responsible for many important brain functions.24 while microglia acts as the first form of immune defense in the brain, astrocytes are an essential neurosupportive cell type . Indeed, astrocytes finely control the environment by regulating ph, ion homeostasis, oxidative stress, and blood flow.25,26 these cells together with microglia, oligodendrocytes, neurons, pericytes, and endothelial cells constitute the neurovascular unit, responsible for the proper functioning of the bbb.27 in addition, astrocytes contribute importantly to synaptogenesis and dynamically modulate information processing and signal transmission, regulate neural and synaptic plasticity, and provide trophic and metabolic support to neurons.28,29 interestingly, data from animal models and human autopsy revealed that both sps and nfts cause an immune response in the brain and colocalize close to activated glial cells . Astrocyte and microglia acquire a reactive phenotype20 and rapidly act in response to pathology undergoing important changes in their morphology and functioning.30,31 such an activation is fundamentally a protective response aimed at removing injurious stimuli . The neuroprotective action of reactive astrocytes takes place by modulating a-mediated neurotoxicity, degrading, internalizing, and removing a, thus creating a protective barrier that surrounds plaques.3234 however, uncontrolled and prolonged activation goes beyond physiological control, and detrimental effects override the beneficial ones . In this condition, glial cells foster neuroinflammatory response, accounting for the synthesis of different cytokines and proinflammatory mediators.35,36 this condition is called reactive gliosis and is a characteristic event of ad brains (figure 1). For example, activated microglia reduces a accumulation by increasing its phagocytosis, clearance, and degradation,37 as well as by secreting factors such as the glia - derived neurotrophic factor, helpful for neuronal survival.38 recently, microglia functions aimed at a clearance were attributed to the presence of triggering receptor expressed on myeloid cells 2 (trem2), a transmembrane receptor;39,40 indeed not long ago, trem2 was identified as a risk gene for ad.41,42 it is reasonable to think that this association between trem2 and ad is due to the many functions carried out through the activation of different pathways ranging from phagocytosis to encouraging survival and proliferation, and finally promoting secretion of cytokines and chemokines.4345 even astrocytes play an important role in the maintenance of the cerebral homeostasis . These cells are responsible for the proper functioning of the bbb, provide nutrients to neurons, preserve the extracellular ion balance, and remove and degrade a.46 however, glial functions are deeply altered whenever tissue physiology is not restored . In these circumstances, the inability to counteract a and nfts accumulation constantly stimulates the machinery needed to remove debris; in this way, astrocytes actively support inflammation.47,48 several studies demonstrated that their action becomes relevant from early stages of the pathogenic process, turning to a cycle independent from a presence, neural dysfunction, cell death, and disease progression.4951 the resulting chronic inflammation is due to the release of proinflammatory molecules that act not only in an autocrine manner, allowing the perpetuation of the reactive gliosis, but also in a paracrine one, the main cause of the neuronal death that increases the pathological damage.52,53 neuronal death is determined by the release of not only inflammatory mediators, but also of reactive oxygen species, nitric oxide (no), proteolytic enzymes, complement factors, and/or excitatory aminoacids.54 at the molecular level, the release of these mediators affects neuron glia crosstalk, influencing redox enzyme sensors, receptors, and transcription factors.55 in physiological conditions, microglia protects the brain from pathogens, and, together with macroglia, helps maintain homeostasis of the tissue . In ad, all these cells became more reactive and change their morphology surrounding sps.56 this is possible because of the presence of proinflammatory receptors on their surface . Microglia is able to identify and bind a oligomers and fibrils and the amyloid precursor protein (app)57 through a large number of receptors, including scavenger receptor class a type 1, marco, scavenger receptor class b member 1, cd36, and the receptor for advanced glycation end product,58,59 g protein - coupled receptors formyl peptide receptor 2 60 and chemokine - like receptor 1,61 toll - like receptors (tlrs) tlr2,62 tlr4, and the cd14 coreceptor, and 61 integrin.63 the outcome of the bond between a and these receptors is the production of inflammatory mediators such as cytokines (interleukin [il]-1, il-1, il-6, il-8, il-12, il-18, and il-23, interferon (ifn)-, tumor necrosis factor [tnf]-, and granulocyte - macrophage colony - stimulating factor [gm - csf]),64,65 chemokines (monocyte chemotactic protein 1 (mcp1), mcp-113, fractalkine),66,67 chemoattractant proteins, prostaglandins, complement factors, thromboxanes, pentraxins, no, reactive oxygen species, leukotrienes, proteases, protease inhibitors, adhesion molecules (interaction between cd40-cd40 ligand cd40l),68 coagulation factors, and c - reactive protein, most of which are detectable in ad animal and/or in the brain or cerebrospinal fluid of ad patients.25,69,70 however, glial cells are also capable of producing some regulatory cytokines, such as il-10 and transforming growth factor- (tgf-), but in ad their release is modified, exacerbating the disease.7173 among anti - inflammatory factors, we also recall the cluster of differentiation-200 (cd200) regulated by the anti - inflammatory il-4 and expressed by neurons, t- and b - cells, whose receptor is expressed by glia . Both ad patients and mouse models show an age - related or a-induced cd200 reduction.7476 upstream of cytokines production is the activation of the nuclear factor - kappa b (nf-b) pathway,77 and the subsequent activation of mitogen - activated protein kinase (mapk) pathways, whose proinflammatory gene expression is a dependent.78 extracellular signal - regulated protein kinases (erks), stress - activated protein kinases c - jun nh2-terminal kinase (jnk), and p38 constitute the set of mapks whose action is exerted both in the cytoplasm and in the nucleus, thereby phosphorylating transcription factors . For example, p38 can contribute to neuroinflammation by inducing tnf- gene transcription, which increases the activator protein-1 (ap-1) activity,79 besides being directly responsible for tau phosphorylation.80 in turn, proinflammatory mediators increase the activity and the products of amyloidogenic pathway, especially a(142). For instance, the -secretase cell - based assays showed that tnf-, il-1, and ifn- cause the initiation of app cleavage through the mapk pathway,81 and a more recent study demonstrated that nf-b signaling, activated by tnf-, results in an increased a synthesis driven by the -secretase (bace-1) transcription.82 to the vicious circle driven by cytokines and mapks,83,84 the resulting activation of the complement cascade has to be added,85 as well as the induction of proinflammatory enzymes, such as cyclooxygenase-2 (cox-2)86 and the inducible nitric oxide synthase (inos).87 induction of these enzymes may also be linked to the excessive release of s100b (form of the s100), a neurotrophin expressed by activated astrocytes,88 which is able to induce nf-b activation,89 as well as encourage tauopathy.23 two more proinflammatory proteins, implicated in the pathophysiology of ad, belong to the s100 family: s100a9 and s100a12 . These proteins, produced by activated microglia and macrophages, are increased in ad brain and are responsible for protein complex formation.90,91 s100a9 is present within sps and a deposits surrounding blood vessels, and it is also abundant in tissues neighboring a deposits, confirming that increased s100a9 levels can stimulate peptides aggregation and deposition.92 studies report that tau hyperphosphorylation is directly affected by inflammatory mediators, including the cyclin - dependent kinase 5 (cdk5):93 il-6 stimulates neuronal protein p35, which in turn is responsible for the kinase activation that can act on tau.94 cdk5 is not the only kinase related to neuroinflammation . Recently, the role of protein kinase 2 (ck2, former casein kinase ii) has been described . In fact, ck2 immunopositive astrocytes have been found to be associated with amyloid deposits in ad brains, suggesting its involvement in the neuroinflammatory response.95 inflammatory mediators, in particular cytokines, are also responsible for increased bbb permeabilization driven by chemokines, allowing leukocyte penetration in the brain.96,97 this is possible because of altering the resistance of tight junctions, upregulation of cytokines expression, and cox-2 transcription in endothelial cells.98 for example, il-6, il-10, il-13, and prostaglandins stimulated by lipopolysaccharide may increase the influx of a across the bbb, besides upregulating app processing in the brain.99,100 another mechanism underlying the pathogenic process led by neuroinflammation is the blockage of neurogenesis, which is inhibited by some proinflammatory cytokines such as il-6, tnf-, and il-18, responsible for neural progenitor cells death, and inhibition of their differentiation.101 interestingly, these cells are located in the subgranular layer of the dentate gyrus of the hippocampus, in the subventricular zone of the lateral ventricles, and amygdala areas mainly affected by ad and cognitive impairment.102 one of the still poorly explored mechanisms that might govern the relationship between ad and neuroinflammation, but definitely is in charge of the neurodegenerative processes, involves the glycogen synthase kinase-3 (constitutively active serine / threonine protein kinase) pathway.103 this idea comes from the observation of the results obtained by blocking this kinase, which causes an increase of the anti - inflammatory il-10 and a decrease of proinflammatory cytokines as a consequence of tlrs stimulation and no production.104106 the salient events reported in this paragraph are summarized in figure 2 . Because of the knowledge acquired so far and the failure of so many antiamyloid trials, scientific interest has shifted to other features of neurodegeneration including neuroinflammation.107 evidence mentioned in the pathophysiology of neuroinflammation and its role in alzheimer s disease section shows how neuroinflammation is driven by a large number of events apparently different but strongly dependent one on the other.108 for this reason, it is difficult to identify the best target upon which to act . Recently, much work has been done, but much more research still needs to be done . It is now clear that the ad neurodegenerative process is also orchestrated by proinflammatory cytokines and their receptors, which therefore become promising targets on which to focus by means of different approaches . Blocking gene expression of cytokines, releasing or binding their receptors, or better regulating the functioning of cells implicated in the neuroinflammation are definitely strategies still in exploration.109 the possibility of of reducing tau kinase activity and oligomeric and fibrillary a accumulation by neutralizing il-1 or tnf-/tnf- receptor through antibodies has been demonstrated in murine models of ad.64,110113 in this context, the role of molecules with anti - inflammatory properties (such as minocycline) that are able to decrease astrocyte release of proinflammatory cytokines and reduce both tau and amyloid pathogenesis,114 as well as improve ad behavioral symptoms, is not less important.115 interestingly, both in vitro and in vivo studies have shown that pharmacological inhibition of cox-2 and inducible no synthase has positive outcomes.38,116120 lastly, in ad models it was observed that it is possible to obtain satisfactory results by modulating kinases that are not only directly related to tau hyperphosphorylation but also to neuroinflammation . One example is the modulation of glycogen synthase kinase-3. Experimental studies have shown that it is possible to exert anti - inflammatory effect by inhibiting this enzyme, giving us another potential therapeutic target to consider.87,88,121 in the past decades, several epidemiological and clinical studies were carried out to demonstrate the neuroprotective potential of several nonsteroidal anti - inflammatory drugs.122,123 after the pioneering work with indometacin demonstrated the ability to restore cognitive functions in the enrolled subjects, many other clinical trials have shown only unsatisfactory results.124127 since the failure of trials with classical nonsteroidal anti - inflammatory drugs, scientists tested cox-2-selective compounds effects . Once again, results were disappointing.128,129 evidence from a clinical trial with naproxen suggests its ability to reduce tau and a levels in cerebrospinal fluid and plasma.130 ad is a multifactorial disease and the inflammatory outcome, driven by glial activation, depends on the context and on the stage of the pathology . For these reasons, an ideal anti - inflammatory compound should be able to control the detrimental effects and, at the same time, preserve the physiological glial activation . An alternative and recent therapeutic approach is represented by nutraceuticals (eg, curcumin, apigenin, docosahexaenoic acid, resveratrol, and n-3 fatty acids).131134 despite encouraging preclinical results, the success rate in humans has been very low.135 complex results were obtained after vaccinating ad patients against a and nfts . A large number of studies have been done in this field, and promising data were obtained in preclinical models . Unfortunately, these encouraging findings were not replicated in clinical trials, and promising vaccines were stopped because of adverse effects such as meningoencephalitis.136 some of these studies revealed that immunization halts glial activation.137 by the physiological importance of this phenomenon, this is probably why the immunization has caused severe adverse reactions . Presently, several competing hypotheses (especially related to time of intervention) may help explain the failure of translating preclinical studies into the clinical ones, but so far there is no way to confirm which of these explanations is correct . The pathogenic role of neuroinflammation in ad is now well recognized and accepted . Nevertheless, the underlying mechanisms have not been sufficiently elucidated . First of all, there is a lack of adequate preclinical models that best mimic the disease and, in particular, the processes of glial activation and neuroinflammation . Then, another important factor is the comprehension of the role of each cellular component in the inflammatory process, for example, the identification of cell - specific biomarkers . Indeed, specifically clarifying changes in both immune system and inflammatory machinery would make available different pathways for pharmacological manipulations aimed at delaying the onset and/or the progression of the disease . Finally, it is important to define the inflammatory stages to correlate each phase to ad progression and to clarify which processes are protective and which ones are detrimental . The achievement of these goals will allow scientists to practice many other experimental approaches . The hope is to get satisfactory results from clinical studies with compounds that have been successful in vitro, ex vivo, and/or in vivo experiments, such as the administration of molecules like acetylpuerarin,138 edaravone,139 palmitoylethanolamide,38 n-[2-(4-hydroxyphenyl)ethyl]-2-(2,5-dimethoxyphenyl)-3-(3-methoxy-4-hydroxyphenyl) acrylamide (compound flz),140 oleuropeinaglycone,141 oridonin,142 protocatechuic acid,143 resveratrol,110 rutin,144 or immunotherapies145,146 and vaccinations.147 growing evidence confirms that neuroinflammation, finely orchestrated by neuronal, glial, and immune components, is a contributing cause of a aggregation, tau hyperphosphorylation, and neuronal damage and death . The resulting production of cytokines and proinflammatory molecules has initially a neuroprotective role, but subsequently becomes the cause of further neurodegeneration . Unfortunately, because of the lack of appropriate animal models, we still lack a complete understanding of the relationship between inflammatory process stages and ad progression . This could explain, at least in part, the unsuccessful results of clinical trials performed with anti - inflammatory molecules whose efficacy was significantly proven in preclinical investigations . Therefore, future experimental studies must intensively investigate the intricate paths of the neuroinflammatory process and define the best time to control it . In this way, it will be possible to achieve more focused and functional therapeutic strategies in the hope of not only alleviating but also modifying ad progression.

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