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Apesar dos sistemas adesivos autocondicionantes (saa) serem indicados para aplicao no esmalte dental, preocupao tem sido relatada com relao a sua efetividade . O objetivo deste estudo foi avaliar o padro de condicionamento cido (pca) promovido por nove saa e comparar ao pca produzido pelo cido fosfrico (35% e 34% - af) no esmalte intacto (ei) ou abrasionado (ea). Vinte e dois terceiros molares humanos foram seccionados nos sentidos msio - distal e vestbulo - lingual, e quatro fragmentos dentais foram obtidos a partir de cada dente . Metade dos fragmentos tiveram o esmalte abrasionado com lixas de sic (600) e a outra metade permaneceu intacta . Os fragmentos foram divididos em 22 grupos, de acordo com a textura da superfcie do esmalte (ei e ea) e a tcnica de condicionar o esmalte (af 34%, af 35%, adhese primer; brush & bond; clearfil protect bond primer; ibond; one - up bond f; optibond solo plus primer; tyrian spe primer; unifil bond primer e xeno iii). Os agentes condicionadores foram aplicados nos ei e ea, de acordo com as instrues dos fabricantes . Espcimes tratados com af foram lavados com gua, enquanto os dentes tratados com saa foram tratados com banhos alternados de lcool e acetona . Em ambas as superfcies (ei e ea), o pca dos af (34 e 35%) foi mais profundo e homogneo, quando comparados ao pca produzido pelos saa, exceto para o adesivo tyrian spe . A maioria dos saa menos agressiva que o cido fosfrico e seus efeitos condicionadores so reduzidos em superfcies de ei . Buonocore2 (1955) reported that bonding to enamel surface could be increased by conditioning the surface with 85% phosphoric acid for 30 seconds . Since this study, a proper enamel conditioning produces a selective dissolution, resulting in one of three etching patterns . Phosphoric acid etching removes approximately 10 m of the enamel surface, creating a porous layer and increasing the surface energy and wettability25,27 . Although the adhesion to enamel produced by phosphoric acid etching has been considered stronger and more durable, self - etching adhesives are alternative methods to prepare the tooth for restorative procedures . These adhesive systems have simplified clinical use because they do not require separated phosphoric acid etching, water rinsing or superficial moist controlling steps . However, studies evaluating self - etching adhesives are in disagreement regarding the efficacy of conditioning and monomer infiltration on enamel10,13,17,18,20 . Morphological analyses of enamel surface treated with self - etching primers have shown not very demineralized surfaces and other areas that were predominantly unetched14,16,29 . In an attempt to improve the bonding of self - etching systems to enamel, surface pre - treatments, increase in acidic monomer concentration, increase of etching time as well as different application methods the purpose of this in vitro study was to compare the effects of self - etching adhesive systems and conventional phosphoric acid etchants on the morphology and the acid etching pattern of intact and ground enamel . The hypothesis was that the self - etching systems do not etch the ground and intact enamel surfaces such as phosphoric acid gels . Twenty - two extracted, caries - free erupted human third molars were used in this study according to protocols approved by the institutional review board of the piracicaba school of dentistry teeth were obtained from patients from 19 to 25 years old and stored in saline with 0.1% thymol for no longer than 3 months . Tooth roots were severed and the crowns were longitudinally sectioned (mesio - distally and buccal - lingually directions) into four quarters, using a diamond blade (isomet, buehler ltd ., lake bluff, il, usa) under water cooling . The buccal or the lingual flat enamel surface of each fragment was chosen and selected for conditioning treatments and bonding procedures . All specimens were randomly assigned to twenty - two groups (n = 4), according to surface treatment of enamel (ground and unground) and type of acid etching (self - etching and phosphoric acid). The dental fragments from the ground surface groups had their enamel surface abraded with a #600-grit sic paper on a polishing machine (apl-4, arotec s.a . Experimental groups comprised enamel treatments with two phosphoric acid concentrations: 35% (3 m espe, st . Paul, mn, usa) and 34% (dentsply caulk, milford, de, usa); five acidic primers of two - step self - priming systems (adhese - ivoclar vivadent, schaan, liechtenstein; clearfil protect bond - kuraray medical inc ., kurashiki, okayama, japan; optibond solo plus self - etch - kerr corp ., orange, ca, usa; unifil bond - gc corp .,, schaumburg, il, usa) and four one - step self - etching adhesives (brush & bond - parkell inc ., edgewood, ny, usa; i - bond - heraeus kulzer, hanau, germany; one - up bond f - tokuyama dental corp ., taitou- ku, tokyo, japan, xeno iii - dentsply detrey, konstanz, germany). Phosphoric acids were applied to the ground and intact enamel surfaces and left undisturbed for 15 seconds, rinsed with water spray for 30 seconds and dried for 30 seconds . The composition, manufacturers and directions of self - adhesive systems are described in table 1 . Procedures a: air - dry; b: apply primer; c: leave undisturbed; d: gently air dry; e: mix liquid a and b; f: apply mixture . Specimens treated with self - etching primers were thoroughly rinsed with acetone and ethanol solutions in order to dissolve and remove the self - etching primer and adhesive resins5 . All specimens were dried, dehydrated in a desiccator for 12 hours and treated enamel surfaces were sputter - coated with gold / palladium in a vacuum evaporator (scd 050, balzers, schaan, liechtenstein). The entire surface of treated enamel was examined under a scanning electron microscope (jsm 5600lv, jeol, tokyo, japan), however, only photomicrographs of representative surface areas were taken at 10,000x magnification . Phosphoric acid etchants changed the enamel morphology for both intact and ground surfaces (figures 1 and 2). Dissolution of prism cores and boundary regions can be observed, however, the conditioning of enamel surface was not uniform along the unground surfaces (figure 2b). No distinct morphological differences between the phosphoric acid concentrations (34% and 35%) were observed on the etched enamel surfaces . Figures 3 to 11 represent the morphology of enamel surface treated with self - etching systems . The etching patterns ranged from mild demineralization to an aspect of surfaces etched with phosphoric acid . Tyrian spe self - etching primer produced dissolution of enamel surface, exposing enamel crystallites, which resulted in an etching pattern similar to that created by phosphoric acid etchants (figure 9). Mild demineralization was promoted by brush & bond, one - up bond f, clearfil protect bond and unifil bond self - etching adhesives (figures 4, 5, 7 and 10), whereas adhese, ibond, optibond solo plus and xeno iii (figures 3, 6, 8 and 11) resulted in moderate mineral dissolution . Ground enamel allowed the self - etching primers to condition the subsurface enamel, exposing crystallites when the etching pattern was considered at least moderate (figures 3a, 6a, 8a, 9a, 11a). Acidic monomers have been developed containing esters from phosphoric acid, carboxylic acid or derivatives12,14,18,21 . Their etching efficacy depends on acidic monomer, ph of adhesive solution, etching time and application method3,16,18 . They are responsible for etching the dental substrates, whereas methacrylate components, such as hema, are available for monomer infiltration and polymerization of the bonding agent8,10,12,13 . As the application of self - etching adhesives comprises simplified bonding procedures, there have been concerns regarding the longevity of bonding, according to some in vitro and in vivo results4,15,24 . Studies have shown that most of the self - etching adhesives did not etch enamel as deeply as the phosphoric acid etchants did and the shallow etching pattern could compromise the bonding to enamel6,10,15,21 . Pashley and tay18 (2001) reported that the efficacy of self - etching primers in intact enamel does not depend solely upon their etching aggressiveness, but also on monomeric composition of each material . It is also possible that the low enamel bond strengths might be caused by the high amount of unpolymerized acidic monomers remaining after curing13 . Thus, no correlation among degree of primer aggressiveness, enamel etching pattern and bond strength to unground enamel has been reported for self - etching adhesives9,23 . The etching effect of phosphoric acid etchants was similar to that previously described by retief25 (1973) and silverstone, et al.27 (1975). The prism cores and boundaries were etched by 34% and 35% phosphoric acids, causing dissolution of both inter and intraprismatic areas . The predominant etching pattern was type 2, which has the peripheral region of prisms removed and prism cores relatively unaffected (figures 1a and 2a). The unground enamel treated with phosphoric acids also showed formation of a porous surface, exhibiting the exposed enamel crystallites along the entire surface (figures . Figure 2b shows the remnants of aprismatic or prismless layer that was partially dissolved by phosphoric acid etching . In this current study, as enamel surfaces were obtained from third molars, it was possible that the treated outer enamel layer would be prismless22 . Although phosphoric acid etchants present ph below 119, unground enamel surfaces were not totally attacked or conditioned by acid etching . The ph values of all self - etching systems tested were higher than that for phosphoric acid . In general, the demineralization effects of these systems were proportional to the acidity of the acidic primers or self - etching adhesive solutions . The self - etching primers were less aggressive than phosphoric acid etchants and the conditioning effects were also reduced in unground enamel surfaces, except for tyrian spe self - etching primer . This result is in agreement with other studies that demonstrated that self - etching adhesives do not form a proper and defined acid etching pattern in intact surfaces5,10,14,21,23 . Sem observations indicated that only shallow pits were produced after some self - etching treatments in intact enamel (figures 6b, 8b, 10b and 11b). The removal of superficial, aprismatic layer by wet - grinding with 600-grit sic paper improved the etching effects . The morphological structure and composition of the intact peripheral surface of enamel is different from that of the middle enamel layer22 . These differences can be favorable for etching effects in subsurface enamel . For tyrian spe primer, the etching pattern on the ground surface was similar to phosphoric acid - etched enamel (figure 9a). However, the aggressiveness of the acidic primer has not ensured that consistent bonding may be established13,18,26 . Examination of ground enamel surfaces treated with brush & bond, clearfil protect bond, one - up bond f and unifil bond self - etching systems (figures 4a, 5a, 7a and 10a) revealed that the surfaces were predominantly unetched . Adhese, ibond, optibond bond solo plus and xeno iii self - etching systems resulted in moderate and particular etching pattern that comprised demineralization of the surface with exposure of the enamel crystallites (figures 3a, 6a, 8a and 11a). The self - etching primer mechanism ofbonding to enamel is based on nanoretentive interlocking between crystallites and adhesive resin9,26 . These morphological features of the resin - enamel bonds are different from that formed with the etch&rinse adhesive systems9,11,23,26 . This thin hybridized complex of resin in enamel produced by self - etching without the usual micrometer - size resin tags can be responsible for lower bond strength and questionable effectiveness on enamel surfaces1,28,30 . Based on scientific evidence, some authors have recommended the instrumentation of enamel before bonding, in attempt to increase the bond strength6,14,21,23 . Thus, resin bond strength achieved with self - etching systems are sometimes comparable to those achieved with phosphoric acid, despite the differences between enamel etching pattems8,12,13,14,20 . However, the controversy about the effectiveness still remain, since other studies did not show the same results of bond strength1,6,11,28,30 . Regarding acid etching technique, it is well established that phosphoric acid provides good adhesion to both ground and unground enamel2,19,25,27 . Because the action of self - etching primers resulted in much less demineralization of intact enamel surfaces, enamel abrasion during cavity preparation can favor the formation of a defined etching pattern . Since in clinical situations the enamel is usually prepared with dental drills prior to application of the adhesive system however, the effects of self - etching adhesive systems must be further studied to verify the durability of bonding to enamel . The self - etching systems did not etch the ground and intact enamel surfaces as phosphoric acid etchants did . Their conditioning effects were reduced on intact enamel surfaces, except for tyrian spe system . The acid - etched enamel patterns formed by acidic monomers were observed only on ground surfaces.
In 2009, cases of influenza like illness were first reported in mexico on march 18; the outbreak was subsequently confirmed as h1n1 influenza . A novel h1n1 swine origin influenza virus has led to a worldwide pandemic 1 . In the affected patients, a novel swine origin influenza a (h1n1) virus (s - oiv) with molecular features of north american and eurasian swine, avian, and human influenza viruses were isolated 2 . In the same month, the world health organization (who) classified the global spread of this virus as a public health event of international concern . After documentation of human to human transmission of the virus in at least three countries of two who regions, the who raised the pandemic level to 6 3 . It has spread very rapidly since the first cases were diagnosed in mexico with the subsequent spread of the virus throughout europe during the winter season . The h1n1 2009 influenza pandemic (ph1n1) has resulted in over 15921 deaths worldwide more than 212 countries as of 14 february 2010 4 . Turkey reported its first laboratory - confirmed case of influenza a (h1n1) on 16 may 2009, becoming the eighteenth country in the who european region to do so, and a second case on 17 may 2009 5 . The clinical picture in severe cases of pandemic (h1n1) 2009 influenza is markedly different from the disease pattern seen during epidemics of seasonal influenza, in that many of those affected were previously healthy young people . Current predictions estimate that, during a pandemic wave, 12 - 30% of the population will develop clinical influenza (compared with 5 - 15% for seasonal influenza) with 4% of those patients requiring hospital admissions and one in five requiring critical care 6 . Pandemic influenza a (h1n1) virus infection is the first pandemic in which intensive care units (icu) play a fundamental role . During the pandemic,, we describe futures of intensive care unit admission, demographic characteristics, treatment and outcome for critically ill patients with laboratory - confirmed and suspected infection with the h1n1 virus admitted to the three different critical care departments during winter of 2009 in turkey . In response to an outbreak of influenza a virus infection in mexico, turkish ministry of health developed a case report form . The patients were admitted to hospital and critical care units according to this case report form . Data were collected retrospectively on all patients who had influenza a 2009 related critical illness from november 1 2009 to december 15 2009 . Ethical approval was provided from the ethics committee of meram medical faculty, selcuk university, konya, turkey . Influenza - like illness (ili) is defined as fever, cough, and headache, accompanied by one or more of the following signs or symptoms: rhinorrhea, coryza, arthralgia, myalgia, prostration, odynophagia, chest pain, abdominal pain, and nasal congestion . Data were reported by the attending physicians reviewing medical charts, radiologic and laboratory records . The following information was recorded; demographic data, comorbidities, time from illness onset to hospital admission, time to first dose of antiviral delivery, microbiologic findings, and chest radiologic findings at icu admission . Intubation and mechanical ventilation requirements, adverse events during icu stay and laboratory findings at icu admission were also recorded . We classified patients according to case definitions (confirmed, probable, or suspected) developed by the world health organization and centers for disease control and prevention . A confirmed case of novel influenza a (h1n1) virus infection is defined as a person with an ili with laboratory confirmed novel influenza a (h1n1) virus infection by real time rt - pcr 8, 9 . We defined critically ill patients as those admitted to an adult intensive care unit (icu); requiring mechanical ventilation or receiving intravenous infusion of inotropic or vasopressors during the hospitalization . Severity of illness was assessed in adults using the acute physiology and chronic health evaluation (apache) ii within 24 hours of icu admission . We recorded co - morbidities and prior defined major co - morbidities as the presence of one or more of the following chronic medical conditions: asthma, chronic obstructive pulmonary diseases (copd), congestive heart failure, malignancy, neuromuscular disorders, cerebral palsy, diabetes mellitus, coronary artery diseases, heart diseases, chemotherapy, malnutrition, immunosuppressive status or renal failure . Nasopharyngeal - swab specimens were collected at admission, and bronchial - aspirate samples were obtained after tracheal intubation . Specimens were placed in transport medium and kept at a temperature from 2 to 4c . Centers for disease control and prevention (cdc) 10 . Seasonal vaccination history and radiographic findings specimens (bronchoalveolar lavage and blood) for culture sent to microbiology laboratory for detection of bacterial infection in invasive and noninvasive mechanically ventilated patients . The body - mass index (bmi, weight in kilograms divided by the square of the height in meters) was calculated . Statistical analysis; descriptive analysis included frequency (%) and mean standard deviation (sd). During the study period which is november 1 2009 to december 15 2009, 61 critically ill patients were admitted to three different critical care units in turkey due to confirmed or suspected influenza a 2009 (h1n1) infection were assessed . In 45 patients, diagnosis was confirmed by real - time pcr for pandemic h1n1 virus . In 16 patients, diagnosis was suspected according to cdc and who criteria 8, 9 . Average age was 41.52 15.7 years and, 54% were female (female: 33, male: 28). Mortality rate in males was 64.3% and in females 39.4% (p> 0.05) clinical characteristics of patients with influenza a virus infection were showed in table 1, table 2 and table 3 . In table 4 comparison between survivors and nonsurvivors were shown . Symptoms at presentation included fever (88.5%), cough (83.6%), sputum (79%) and dyspnea (96.7%). Diarrhea, nausea, and vomiting were reported in 24.6%, 39.3%, and 45.9%, respectively . The mean time from the onset of illness to critical care admission was 7.56 4.1 days (range, 2 to 22). Obesity (27.9%) and copd (14.7%) were the most common conditions in patients . There was no significant difference according to underlying medical condition in between nonsurvivor and survivor groups . A total of 3 patients (4.9%) were pregnant, of whom 2 had another underlying medical condition (asthma and heart disease). Of the 4 pregnant patients, 1 was in the first trimester, 1 was in the second trimester, 1 was in the third trimester, and 1 was in the postpartum period . At the time of icu admission, all patients had elevated lactate dehydrogenase levels (604.8 316.9 u / l), 25 (40.9%) above 500 u / l, and 7 (11.4%) above 1000 u / l . Thirty - three patients (54%) had elevated aspartate aminotransferase (144.5 178.07 u / l). U / l). Sixteen patients (26%) had increased creatinin kinase levels (mean 418.7 529.1 u / l) (range, 6 to 2573 u / l). C - reactive protein was measured in 48 patients (78.7%) with a mean of 95.1 49.5 mg / dl . Eighteen patients (24.6%) had elevated creatinine levels (> 1.2 mg / dl) at hospital admission . On admission, 11 of 61 (18%) patients who were tested had leukopenia, 27 of 61 (42.2%) had anemia, and 18 of 61 (29.5%) had thrombocytopenia . The mean time from the onset of illness to the initiation of antiviral therapy was 7.44.17 days (range, 1 to 22 days); 2 of the patients received antiviral therapy within 48 hours after the onset of symptoms . Antiviral therapy was started before admission in 4 patients, on admission in 55 patients, within 48 hours after admission in 2 patients . There was significant difference according to the time from the onset of illness to the initiation of antiviral therapy between nonsurvivors and survivors (p<0.05). Antibiotic therapy was started before admission in 32 patients and on admission in 29 patients . Patients received a mean of two different antibiotics (range, one to five); 81% of the patients received more than one antibiotic . Commonly used antibiotics included moxifloxacin (in 19 patients), linezolid (in 14 patients), ampicilline - sulbactam (in 13 patients), clarithromycin (in 13 patients), piperacillin - tazobactam (in 12 patients), imipenem (in 11 patients), third generation cephalosporin (in 9 patients), vancomycin (in 2 patients), teicoplanin (in 4 patients), and tigecycline (in 8 patients). Of 61 patients for whom data were available regarding the use of corticosteroids, 20 (32.8%) received intravenous steroids . Of the patients who received corticosteroids, 85% had an underlying medical condition; the most common conditions were copd and asthma (70%). Patients with viral primary pneumonia had bilateral patchy alveolar opacities, affecting two or five quadrants in 51 patients . All patients had a mean oxygen saturation of 65% (range, 45 to 80) in the absence of supplementary oxygen . After supplementary oxygen, all patients had a mean oxygen saturation of 83.7% (range, 49 to 98). Mean apache ii score was 18.7 6.3 (range, 6 to 37). Ards was diagnosed in 48 patients (78.6%) and ali in 4 (6.5%) of the patients . Clinical evidence of bacterial infection on icu admission was present in 7 patients (11.4%). Data on the use of mechanical ventilation in the icu were available for all patients . Fifteen of these patients were endotracheally intubated after a mean of 3.4 1.7 days . Apache ii score was higher in nonsurvivors (20.9 6.7) than survivors (16.5 5.4) (p<0.01). There were 8 obese patients in nonsurvivor group and in 7 obese patients in survival group (p>0.05). In 3th days, mean level of urea, creatinine, international normalized ratio (inr) and heart rate were higher nonsurvivors than survivors (p<0.05, p<0.05, p<0.05, and p<0.01). Pao2/fio2 ratio was lower in nonsurvivors than survivors in third icu day (p<0.05). Of all patients, 56 (91%) were mechanically ventilated on the first day of icu admission; 14 (23%) patients received invasive and 42 (68.8%) noninvasive mechanical ventilation . Apache ii score, pco2, white blood cell count and neutrophil account were higher in invasive mechanical ventilation group than nimv group . Duration of nimv and imv were 5.28 3.4 days (range, 2 to 14) and 6.92 5.8 days (range, 1 to 19) respectively . In survivors, the length of invasive mechanical ventilation ranged from 1 to 19 days (6.2 5.5days). The length of nimv ranged from 1 to 14 days (4.25 3.8 days). There were no significant differences in tidal volume or ventilation strategies between survivors and nonsurvivors . Patients who survived were more likely to have nimv use at the time of admission to the icu . Patients who died were more likely to have imv use at the time of admission to icu . Our data of critically ill patients with influenza a 2009 (h1n1) reveals that relatively younger patients are affected by the disease . There was a relatively long period of illness prior to presentation to the hospital, followed by a short period of acute and severe respiratory deterioration . These patients had severe hypoxia requiring high fio2, peep, and ventilator pressures . Within 30 days, previously published reports have highlighted cases of severe viral pneumonia affecting patients younger than the expected age of patients affected during a normal influenza season 11 . The low mean age is different from seasonal influenza, in which older patients appear more susceptible to severe diseases 12 . Importantly, severity of illness and mortality in our cohort are similar to that demonstrated previously with novel h1n1 . The first data from mexico showed that most of the patients were previously healthy 1 . In our study, the most of critically ill patients had comorbidities and there was no difference according to comorbidities between survived and died patients . A history of lung diseases, obesity, diabetes, hypertension, neurological diseases, malignancy, and heart diseases were the most common comorbidities in our study (83.6%). Among critically ill patients, obesity has been shown to be a risk factor for increased morbidity, but not consistently with mortality 15 . In our study, there was no statistically significant difference due to obesity between survivors and nonsurvivors . An early 2009 meta - analysis indicated that obesity was not associated with increased icu mortality 16 . A recent, large cohort study by gong et al . 17 prior to 2009 novel h1n1 infection, noted an association of obesity with ards but not with mortality . The canadian novel h1n1 experience likewise suggests that bmi did not differ between survivors and non - survivors 18 . Patients with h1n1 infection - related critical illness experienced symptoms for an average of 6 days prior to hospital presentation, but rapidly worsened and required care in the icu within 1 to 2 days 1 . In our study, this duration was higher than other studies 1, 18, 19 . The tendency of females to develop severe 2009 influenza a (h1n1) infection in this series is striking . A general female susceptibility has been observed in other influenza case series of variable severity including reports of h1n1 infections 18, 19 . In this report, the explanation for increased risk of death among males in this report may be due to existence of more frequent comorbidities in man . In most of infectious diseases and related conditions such as sepsis and septic shock, males represent a larger proportion of cases and have a higher mortality 20, 21 . Importantly, we found in this cohort that apache ii score may help to identify patients at high risk of death . Rarely, we used vasopressor support on day 1 following icu admission (3.2%). Chest radiographs demonstrating bilateral mixed interstitial or alveolar infiltrates were found in 90% of patients . In our study, 92% of patients required ventilator support for profound hypoxemic respiratory failure, requiring high levels of inspired oxygen and peep . However, survival rate was higher in nimv than invasive ventilation . Noninvasive ventilation has been used an alternative therapy for patients with acute respiratory failure with hopes of obviating intubation and mechanical ventilation . The results of nimv in hypoxemic respiratory failure have been conflicting, and the etiology of hypoxemia appears to be an important determinant of its success . 22 compared nimv to conventional venture oxygen delivery in patients with severe hypoxemic respiratory failure and found that nimv decreased the need for intubation . This benefit was observed in the subgroup of patients with pneumonia, but not in those with ards, in which the intubation rates were high in both groups . A meta - analysis suggests that nimv does not decrease the need for intubation, so there is not enough evidence to support its use in ards 23 . Of all patients, 56 (91%) were mechanically ventilated on the first day of icu admission; 14 (23%) invasively and 42 (68.8%) noninvasively . 24 reported that invasively ventilation was used in 82.7% of patients . In kumar's study 18, invasive ventilation was used in 81% of patients with swine flu associated respiratory failure . In our study, we used noninvasive ventilation in 68.8% of critically patients with 2009 influenza a (h1n1) on admission icu . In critically ill patients with 2009 influenza a (h1n1) infection, high levels of peep were often used to achieve adequate oxygenation . In our study, patients with ards were often had peep refractory hypoxemia . It was also noted that once patients improved and the weaning process was started, oxygenation was sensitive to small decrements in peep . Use of noninvasive mechanical ventilation has some significant problems when there is risk of transmitting infectious diseases . Use of noninvasive ventilation was identified as risk factor for transmitting infection due to exposure to aerosols during sars epidemics 25 . These were expert opinions but in an experimental model, it was claimed that noninvasive ventilatory support may increase occupational risk 26 . However it was shown multiple times that noninvasive ventilatory support may decrease mortality with avoiding from endotracheal intubation . It is difficult to identify immediately if patients are infected or not during epidemic so noninvasive ventilation can be initial chose of ventilatory support in those patients . There is always a potential harm from a withholding a procedure while there is epidemics . Even if there is risk to use noninvasive ventilation for h1n1 patients since it may save the lives, we decided to use it under strict isolation including negative pressure isolation rooms . In conclusion, we have demonstrated that 2009 influenza a (h1n1) infection - related critical illness predominantly affects young patients with little major comorbidity and is associated with severe hypoxemic respiratory failure, often requiring prolonged mechanical ventilation . Among patients admitted to icu, older age, and a requirement for invasive ventilation were associated with increased risk of death, but because there were greater numbers of younger patients in our cohort, the majority of deaths occurred in younger patients . Alternatively, nimv could be used in 2009 influenza a (h1n1) infection - related hypoxemic respiratory failure.
Patients with end - stage kidney disease (eskd) are exposed to multiple physical and psychological stressors as a result of their illness . Treatment of eskd in the form of dialysis imposes considerable stress, including potential changes in family relations, social interactions, and occupational demands . Impact of eskd has been proposed to account for its poorer quality of life (qol) compared to patients with other chronic diseases [1, 2]. Furthermore, survey data has shown significant correlation between poorer qol and higher morbidity and mortality in eskd . As opposed to the mostly invariant biological risk factors, modifiable psychosocial factors may provide avenues for successful intervention and improved clinical outcomes in this population . The renal social worker is the patients' advocate, serving as a bridge in communicating individual's needs to the medical and allied health team . Commented that a multidisciplinary team approach is critical to the overall care and qol of patients with eskd . Social workers play a central role in the care of these patients, which may be further enhanced by engaging them in the measurement and monitoring of qol . There is a paucity of data pertaining to the psychosocial factors affecting patients with eskd in australia . This study seeks to identify these issues using the renal social work database of a tertiary hospital . We identify various reasons for initial referral, subsequent consultations, and interventions performed by the renal social worker over a three - year period . Ultimately, the study would further explore the significance of social work involvement in the care of patients with eskd and lead to future improvements in service delivery . We conducted a single centre retrospective audit of the patients with eskd (chronic kidney disease stage - v (ckd - v)) who were referred to one, full - time renal social worker from january 2012 to december 2014 at royal brisbane & women's hospital, queensland, australia . Referrals for social work input were made either by healthcare staff (medical and allied health) or directly by patients and/or family members . The reason for referral varied and many individuals were referred for multiple reasons over the study period . All patients underwent an initial psychosocial assessment by the social worker, typically a 60-minute consultation where issues at index were identified . Each issue was analysed separately in the case where an individual patient had many issues identified over the study period . Instrumental support included patient advocacy; assistance with paper work / forms; referrals to relevant government agencies (e.g., department of housing, welfare services); and allied health services (e.g., psychologist, dietician, and occupational therapist). Informational support included provision of helpful resources across various domains (e.g., predialysis education, treatment adherence, aged care services, management of finances, and employment prospects). Emotional support was provided through counselling sessions and organising family meetings (e.g., for those with adjustment issues, caregiver stress, and palliative care discussions). All clinical reviews, documentation, and data collation were performed by the same social worker over the study period . Deidentified data was transposed into a spreadsheet, including patient demographics, social history, eskd management modality, summary of social worker encounters, and the respective interventions carried out . This study only included participants with eskd defined by estimated glomerular filtration rate (egfr) <15 ml / min/1.73 m or those with a functioning renal transplant (ckd - vt). Patients were subclassified into predialysis, maintenance haemodialysis (hd), peritoneal dialysis (pd), renal transplant, or palliative . Adherence is defined as the extent to which a patient complies with the prescribed treatment under limited supervision . Limited supervision involves monitoring a patient in the community, for example, review at an outpatient appointment or during outpatient dialysis . Adherence also can be defined as the extent to which a person's behaviour (taking medication, following a diet, and/or executing lifestyle changes) corresponds with agreed recommendations from a healthcare provider . Adjustment is described in relation to how the patient adapts to the multitude of stressors posed by the routine and restrictions of treatment . Referrals for treatment nonadherence and adjustment were made at the discretion of the treating team, typically when this had serious consequences on the patient's health outcome, for example, hospitalisation due to nonadherence or difficulty coping with life changes associated with eskd . Domestic assistance is a service aimed at helping people to remain independent in their home, by helping with the essential light house work tasks necessary to maintain hygiene and safety standards in the home . We used cross tabs with chi - square test to assess association of demographic variables with socioeconomic issues that were identified . We used univariate and multivariate logistic regression to determine predictors of adjustment issues and nonadherence . Age, gender, country of origin, financial status, employment status, reimbursement plan, referral before or after starting renal replacement therapy (rrt), and marital status of the patients were considered for inclusion in multivariate model . Since financial status and employment status were highly correlated, separate multivariate models were constructed for each of them to avoid colinearity . A p value <0.05 was considered as statistically significant and odds ratios with 95% confidence interval were calculated . The study included 244 patients (148 men) with mean age 62.4 (16.9) years . The majority (61.6%) of referrals to social worker were made after dialysis commencement or transplantation . Need for transportation assistance was most prevalent for those patients on hd (46/126 [36.5%]), followed by pd (15/60 [25%]) and transplant patients (7/32 [21.8%]), a significant difference between modality of rrt (p = 0.015) and a more commonly identified issue in patients referred prior to starting rrt (p = 0.004). Child protection was needed significantly more often if the country of birth was other than australia (5/92 [5.4%] versus 1/152 [0.66%]; p = 0.03). Patients referred prior to starting rrt were more likely to have adjustment problems than those referred after commencement of rrt (72.3% versus 21.3%; p <0.001, univariate analysis). In multivariate logistic regression, age, referral prior to commencement of rrt, and financial and employment status independently predicted the odds of having adjustment issues (tables 3(a) and 3(b)). Separate models were created for financial status and employment status to avoid colinearity since the two were highly correlated . Increasing age was associated with a significantly decreased risk of having adjustment issues . Compared to aged pension, patients with financial stability (salary or savings) were significantly less likely to have adjustment issues . Compared to employed patients, unemployed patients were significantly more likely to have adjustment issues (odds ratio 3.34, 95% confidence interval 1.229.13, and p = 0.018). Issues related to adherence to treatment were also common and were seen in 21.3% patients . Age and financial / employment status were significant independent predictors of nonadherence in multivariate logistic regression model (tables 4(a) and 4(b)). Increasing age was associated with a significantly lower risk of nonadherence . Compared to aged pension, disability pension was associated with a significantly greater risk of nonadherence (odds ratio 3.11, 95% confidence interval 1.10 to 8.84, and p = 0.033). Compared to employed patients, unemployed patients were significantly more likely to have treatment nonadherence (odds ratio 4.19, 95% confidence interval, 1.46 to 12.01, and p = 0.008). This study sought to assess the psychosocial challenges faced by patients with eskd in an australian population . Among the patients referred to social work, the majority were> 60 years of age, male, born in australia, on hd, unemployed, and reliant on government assistance . The most common social work consults related to patients with difficulties with adjustment, treatment nonadherence, management of finances, and domestic assistance . We found that age, timing of referral (before versus after starting rrt), financial status, and employment status were independent predictors of adjustment issues . Age, financial status, and employment status also were independent predictors of treatment nonadherence . As defined by beder, adjustment to dialysis is described in relation to how the patient adapts to the multitude of stressors posed by the routine and restrictions of treatment . Social work intervention aims to stabilise the individual with a view towards maintenance of functionality and return to work after initiation of treatment . Early intervention, especially in at - risk patient groups, has been shown to significantly decrease the degree of psychosocial maladjustment in new - start dialysis patients . Our current study suggests that patients dependent on government assistance are most at risk of maladjustment in the australian setting . These patients may therefore also be most likely to benefit from social work intervention . The kha - cari guidelines on ckd management suggest the involvement of the social worker in early stages of ckd . Of note the majority (> 60%) of patients in this study were referred after commencement of dialysis, whereas a substantial number of patients with adjustment issue were referred prior to rrt . Maladjustment has been associated with loss of employment, which is not uncommon among dialysis patients [10, 13]. Earlier referral to social worker may therefore provide an opportunity to more effectively address adjustment concerns . Importantly, earlier referral may see more new - start dialysis patients maintaining or returning to employment . Treatment nonadherence in eskd has been widely researched and remains a challenge for the care of these patients globally . Studies in dialysis patients have shown the association between decreased adherence and increased rates of depression, hospitalisation, morbidity, and overall mortality [1417]. Rates of nonadherence, risk factors, clinical implications, and appropriate interventions have been variably described in the literature, largely owing to the heterogeneity of trials . We found that younger age, patients on disability pension, and unemployed patients were at a significantly higher independent risk of treatment nonadherence . Showed that cognitive behavioural therapy improved depressive symptoms, qol, and treatment compliance in hd patients . A systematic review by chan et al . Showed the association between psychosocial variables and qol in dialysis patients concluding that targeted interventions to treat psychosocial factors may improve quality of life, morbidity, and ultimately mortality in this population . Firstly the psychosocial factors identified in this population may represent surrogate markers of patient qol; correlation of the findings with validated qol scores would be of interest . Secondly, qol scores may be an appropriate way to assess the outcome of social work interventions over time . Limitations of our study were that data were collected retrospectively from a single tertiary centre social work database . The findings may therefore underrepresent the true extent and nature of psychosocial issues faced by eskd patients . Also the number of encounters per patient was not tracked over the study's duration . This could have identified specific risk factors and/or groups that required more intensive social work follow - up . In conclusion, this study observed the demographics of eskd patients referred to social work at a tertiary hospital and found the major issues to be related to adjustment, financial difficulty, and domestic assistance . Adjustment issues were common and were more likely to be present in patients with younger age and referred before start of rrt . Patients with financial stability were less likely to have adjustment issues compared to patients on aged pension . This highlights the need for further financial assistance / support from government and nongovernment agencies . Furthermore, we propose the need for earlier and more comprehensive social work support as patients approach eskd which may lead to improvements in qol, morbidity, and mortality.
A 78-year - old person, retired as school teacher with no history of any substance abuse or medical history . He had been suffering from bilateral knee pain diagnosed as osteoarthritis for the last 20 years and was not on any steroids . He presented with 1-month duration of altered behavior characterized by excessive psychomotor activity, emotional lability, increased self - confidence, excessive talkativeness, decreased need for sleep, and increased sense of energy level . There were a few occasions when he misrecognised his daughter as his wife who had passed away 6 months ago . On examination, he was an elderly obese person weighing 92 kg with bmi> 27 . Mental status examination revealed increased irritability, increased psychomotor activity, and increased self - confidence and authoritativeness . He had similar behavior 3 years ago, diagnosed and treated as mania in the same hospital . There was positive family history in the form of his mother having had symptoms suggestive of postpartum psychosis . Investigations including hiv, vdrl, serum vitamin b12 level, tft, eeg, routine hemogram, serum electrolytes, blood urea, and serum creatinine were normal . Ecg revealed no abnormalities, except for a prolonged corrected qt interval (qtc) of 486 msec . His ct brain [figure 1] showed cortical atrophy, especially pronounced in the temporal and frontal areas . The periventricular regions in the frontal lobe showed mild hypodensity suggestive of small vessel ischemic disease . He didnt fulfill international consensus criteria for behavioral variant of frontotemporal dementia (ftd). Ct brain shows frontal cortical atrophy (top left), with frontal subcortical white matter hypodensities (top right). Widened sylvian fissures suggestive of temporal pole atrophy and basal ganglia calcifications are also seen (bottom left). Widening of temporal horns of lateral ventricles (bottom right) is further evidence of temporal lobe atrophy he was initiated with aripiprazole 5 mg / day because of his qtc being 486 msec . Given his age, we chose aripiprazole as it is less sedating and has lower chances of producing postural hypotension . He also required intravenous lorazepam 2 mg sos for his agitated behavior for the first 2 days . Aripiprazole was gradually increased to 15 mg, and by the end of 3 weeks he showed complete remission of his manic symptoms . At the time of admission in psychiatry ward, he scored 27 on mania severity on young's mania rating scale (ymrs) and it came down to 4 at the end of 3 weeks . Occurrence of first episode of mania in the elderly (after 60 years age) warrants a high index of suspicion for organic causes ., we did not find any obvious etiology for his mental illness, except for frontal and temporal atrophy and possible frontal sub - cortical white matter involvement . One possibility considered was that of ftd, but this was ruled out as he did not fulfill criteria for dementia and behavioral component of ftd . It is possible that the manic episodes were the initial manifestations of ftd in this patient and this will be tested only in the follow - up . There was family history of postpartum psychosis in his mother, which is associated with a family history of bipolar disorder and the index case had been diagnosed as mania 3 years ago with complete remission in the interval period . These two points and absence of any abnormalities in laboratory and clinical examination favored diagnosis of mania . Aripiprazole has relatively high affinity for both 5-ht2a and d2 receptors similar to other atypical antipsychotics but differs from them due to its partial agonist activity at d2 and 5-ht1a receptors . It has a more favorable side - effect profile compared to typical and even atypical antipsychotics . Aripiprazole has lesser propensity to cause extra - pyramidal symptoms, an important factor in the elderly . Other desirable properties are lesser chance for causing side - effects like dyslipidemia, glucose intolerance, hyperprolactinemia, postural hypotension, sedation, qt prolongation, and weight gain . Its efficacy has been proven as monotherapy in treating acute mania and in prophylaxis of bipolar disorder . Hence, we chose aripiprazole for our patient in view of his already prolonged qtc interval and age of 78 years . This case is unusual in that age of onset of mania is very late (75 years) and during the current second episode we could not find any organic etiology . Hence, aripiprazole may be a safe and effective antipsychotic medication for the control of mania in the elderly.
A notification was given by the emergency medical service (ems) to the ed in our hospital regarding a 40-year - old male who developed tonic when ems arrived at the scene, a ventricular fibrillation rhythm was recorded and electrical cardioversion was delivered seven times followed by the administration of intravenous amiodarone . Advanced cardiac life support (acls) was initiated, which lasted about 40 min . On arrival to ed, blood pressure was not able to be registered, but his heart rate was 104 beats / min with regular rhythm . His oxygen saturation by pulse oximetry was 87% despite receiving a fraction of inspired oxygen (fio2) of 100% . Remarkable laboratory findings included a white blood cell count of 18,800/mm (4.810.8), a lactic acid level of 15.3 mmol / l (0.52.2), a blood glucose level of 433 mg / dl (65115), and a bicarbonate level of 14 mmol / l (2431). Arterial blood gas (abg), while on mechanical ventilation and receiving an fio2 of 100%, showed a ph of 7.02 (7.357.45), a pco2 level of 66 mmhg (3545), and a pao2 level of 100 (80100). Troponin i and creatine kinase levels were elevated at 8.32 ng / ml (<0.1) and 2,799 u / l (25215), respectively . Urine toxicology screen was positive for thc but negative for cocaine, amphetamines, barbiturates, benzodiazepines, methadone, or opioids . A qualitative urine toxicology assay using liquid chromatography tandem mass spectrometry (quest diagnostics nichols institute chantilly) was negative for synthetic cannabinoids . Liver function, creatinine, lipid panel, and coagulation profiles were within normal limits . 1). A 12-lead electrocardiogram (ecg) demonstrated sinus tachycardia, with st - segment elevation in leads ii iii and avf as well as v1v5 (fig . 2). Transthoracic echocardiogram displayed severely depressed left ventricular ejection fraction of 20% (5565), global hypokinesis with apical septal akinesia, and a 3.14 cm non - mobile, calcified apical thrombus (fig . Thrombolytic therapy was not considered because of the prolonged cardiopulmonary resuscitation (cpr) time . He was admitted to the intensive care unit (icu) with the presumptive diagnosis of cardiogenic shock secondary to st - segment elevation mi . According to the hospital policy, the presence of severe persistent hypoxemia and refractory hypotension despite the use of two vasopressors was considered as the exclusion criterion to initiate therapeutic hypothermia protocol . Within 48 h, hospital course was complicated by worsening kidney function, and severely depressed glasgow coma scale despite being off sedation . Neurological examination disclosed findings compatible with possible diagnosis of brain death: dilated and fixed pupils, absence of corneal and gag reflexes, negative response to deep painful stimuli, no spontaneous breathing, and negative eye - caloric test . Head computed tomography showed diffuse loss of gray - white matter differentiation and ventricular effacement, compatible with global anoxic encephalopathy . No ischemic infarcts or hemorrhage was identified . Given the possibility that the initial neurological examination may had been confounded by the presence of central nervous system depressant drugs, a second neurological assessment was performed in the setting of a negative urine toxicology screen . Autopsy report revealed an acute anterior lateral, septal, and posterior wall mi with a mural thrombus in the left ventricle as well as hypertensive and atherosclerotic heart disease as the main causes of death . A review of the literature was undertaken to compare our case with other reported occurrences of cannabis - related mi . A medline search using the words marijuana and myocardial infarction yielded 36 cases of acute coronary events likely triggered by the consumption of marijuana . The majority of these patients were male (33, 92%) with a mean age of 30.9 years (median 30, range 1751), and eight patients (22%) died . A similar search using the words cardiac arrest and sudden cardiac death and similarly, most of these patients were male (28, 93%) with a mean age of 34.6 years (median 31, range 1561). In eight patients (27%), other recreational drugs were also found in toxicology screen (in five of these patients (63%), serum alcohol levels were positive). Summary of cases of cardiac arrest and sudden cardiac death related to marijuana consumption is shown in table 1 . The patient described in this report paired the characteristics of those previously reported events: male gender, young adult, positive serum alcohol levels, and had an unfavorable outcome . Unfortunately, our patient was not able to be transferred to another institution for coronary angiography and possible percutaneous angioplasty because of his hemodynamic instability and elevated fio2 requirements . As previously outlined by elmer et al ., patients with recreational drug - related out - of - hospital cardiac arrests are less likely to undergo cardiac catheterization (21). It is possible that our patient might have had a non - diagnosed underlying coronary artery disease that may have been aggravated by the consumption of marijuana . This may be supported by the presence of a calcified apical thrombus, which suggests chronicity . It has been suggested that consumption of thc may worsen coronary ischemia in patients with baseline coronary artery disease, potentially triggering an mi (8). Summary of cases of cardiac arrest and sudden cardiac death associated with consumption of marijuana the pathophysiological effects of marijuana in the cardiovascular system are well described, and they seem to be mediated by stimulation of the sympathetic nervous system through release of norepinephrine as well as by parasympathetic nervous system blockade (26). However, the mechanism underlying the association between marijuana use and mi has not been well established . It has been demonstrated that consumption of marijuana increases oxygen demands on the myocardium, leading to an increase in carboxyhemoglobin levels resulting in decreased oxygen - carrying capacity (27, 28). The rise in norepinephrine levels resulting from sympathetic stimulation reduces the left ventricular ejection time, thereby lowering the threshold for angina . Interference with the integrity of peripheral vascular reflex responses and vascular inflammation with the subsequent increase in platelet activation have been also proposed as mechanisms responsible for cardiac events among cannabis users (29, 30). Reversible vasospasm seems to be the most commonly suggested etiology for cannabis - associated acute coronary syndromes . One study showed that cardiovascular complications resulting from the consumption of marijuana have a mortality rate of about 25% (31). In a large epidemiological study, thc and its derivatives were reported to increase the risk of mi by 4.8 times in the first hour after use (32). Another cohort showed that in marijuana consumers, the population attributable fraction for mi was 0.8% (33). Although cannabis is not commonly associated with seizures and may actually have medical use benefits for the treatment of epilepsy (34), there have been reports of seizures resulting from acute thc intoxication (2, 3). Even though prolonged cardiac arrest leads to hypoxia, which may result in seizures, we do not consider hypoxemia to be the leading cause of seizures in this case given the initial cascade of events, since our patient was initially presented with tonic it is possible that our subject developed seizures because of the synergistic effect of marijuana and alcohol consumption (35). It is also feasible that the patient described in this report might have been under the effects of other recreational drugs unable to be identified by using conventional toxicology screens . Seizures have been reported to be a frequent feature of synthetic cannabinoids intoxication (36, 37). Unfortunately, we were not able to collect any analytical data on synthetic cannabinoids from our patients serum except from a negative urine toxicology assay . Our subject's urine toxicology screen failed to detect the presence of synthetic cannabinoids, making the role of these substances unlikely in our patient's clinical presentation . We hypothesized that the development of cardiac arrest in our patient was the result of an extensive st - segment elevation mi, in conjunction with the ventricular fibrillation likely triggered by myocardial ischemia . It is possible that the onset of ventricular fibrillation may have been provoked by tonic clonic seizures, as previously reported in few other cases (38, 39). As a consequence of the prolonged cpr time and hypoxemia, reported an 18-year - old male who succumbed to an asystole cardiac arrest while smoking marijuana and suffered hypoxic brain injury related to prolonged cardiac arrest (2). Described a 23-year - old male who collapsed with ventricular fibrillation and died after 40 min of unsuccessful cpr (17). In a large cohort of patients with recreational drug overdose - related out - of - hospital cardiac arrests, ventricular fibrillation has been sporadically reported as a result of marijuana consumption (2, 16). A possible explanation for the onset of ventricular dysrhythmias is that cannabis may increase the activity of the purkinje fibers system (6, 40). The combination of alcohol with marijuana could potentially increase the cardiac toxicity and arrhythmogenic effects of thc by alteration of atrial refractoriness and conduction velocity . Our report has two potential limitations: first, the initial ecg strip recorded by ems showing ventricular fibrillation was not able to be recovered, so it is difficult to comment on the rhythm obtained after electrical cardioversion was delivered; second, no blood levels of thc were obtained on admission and, therefore, it may be difficult to attribute the etiology of this acute coronary event directly to the consumption of marijuana . Postmortem toxicological tests were not performed . A positive urine toxicology screen for thc may be insufficient evidence in the linkage of cardiac arrest and marijuana . Thc analysis in blood samples must be considered an essential requirement to estimate and correlate the time of last intake with the occurrence of acute cardiovascular events . Unfortunately, the association of marijuana consumption and the occurrence of cardiovascular events is rarely supported by an exhaustive toxicological examination and, when such investigation is performed, it is usually limited to a positive urine toxicological examination that is not reliable for the estimation of the time of last marijuana intake . In this case, as in majority of the previously reported events, the cannabis causality was assumed based on the result of a positive urine toxicology screen for thc and the absence of other substances known to be linked to acute coronary syndrome . Despite the widespread use of marijuana, public awareness of the risk for potential cardiovascular complications remains low . Further investigations of clinical, toxicological, and epidemiological aspects are needed to enlighten causality between consumption of thc and acute cardiovascular events . Clinicians should be aware of these potential life - threatening complications resulting from the consumption of cannabis.
Reactive aldehydes, such as acrolein and malondialdehyde (mda), react with dna and form exocyclic adducts . Acrolein, an,-unsaturated aldehyde commonly found in tobacco smoke and other exogenous sources (petroleum industry waste, automobile exhaust, and overcooked food) is a mutagenic agent that has been implicated in the etiology of lung cancer . Acrolein is also formed endogenously as a byproduct of lipid peroxidation, alongside structurally related molecules such as mda . As a reactive aldehyde, acrolein condenses with deoxyguanosine (dg) through a michael addition and subsequent cyclization to form two exocyclic adducts: -oh - pdg (3-(2-deoxy--d - erythro - pentofuranosyl)-5,6,7,8-tetrahydro-6-hydroxypyrimido[1,2-]purin-10(3h)-one) and -oh - pdg (3-(2-deoxy--d - erythro - pentofuranosyl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-]purin-10(3h)-one) (figure 1). Reactions of the environmental pollutant acrolein and of the lipid peroxidation byproduct malondialdehyde with dna guanine bases generate the structurally similar exocyclic deoxyguanosine lesions -oh - pdg, -oh - pdg, and m1dg . Dna adducts have deleterious biological consequences and are believed to underlie the mutagenic effects of acrolein and its ability to promote carcinogenesis . -oh - pdg can block dna replication in human cells and can cause g to a and g to t mutations . By contrast, -oh - pdg is efficiently bypassed by certain polymerases in both bacterial and mammalian cells; however, -oh - pdg can also form inter- and intrastrand cross - links (via an open - ring aldehydic form), which are difficult to bypass and can cause mutations . Mda is an important biomarker of lipid peroxidation, a deleterious reaction between cellular lipids and reactive oxygen species produced by inflammatory processes . Similar to acrolein, mda can also form an exocyclic dg adduct, m1dg (3-(2-deoxy--d - erythro - pentofuranosyl)pyrimido[1,2-]purin-10(3h)-one). This adduct is both a strong block to replication and mutagenic, properties that may contribute to inflammation - associated human malignancies, including aging and cancer . Given the toxicity and mutagenicity of the acrolein- and mda - derived exocyclic deoxyguanosine adducts, it is important to investigate if these adducts are substrates for dna repair pathways . While the nucleotide excision repair (ner) pathway can remove these lesions from dna, acrolein, the agent responsible for generating the exocyclic dg lesions, inhibits ner . Little is known about the contribution of a direct reversal repair pathway for removal of acrolein- and mda - derived exocyclic guanine dna adducts . Alkb, the escherichia coli direct reversal dna repair enzyme, can efficiently repair a wide range of dna and rna alkyl lesions . As an fe(ii)- and -ketoglutarate - dependent dioxygenase, alkb uses molecular oxygen to oxidize and remove simple alkyl dna lesions (such as 3-methylcytosine, 1-methyladenine, 3-methylthymine, 6-methyladenine, 1- methylguanine (m1 g), 2-methylguanine(m2 g), and 2-ethylguanine(e2 g)) and exocyclic bridged lesions (n1,n - ethenoadenine (a), n1,n - ethanoadenine (ea), 3,n - ethenocytosine (c), hydroxy-3,n - ethanocytosine, and hydroxy-3,n - propanocytosine). Given the ability of alkb to remove alkyl groups from both n1 and n positions of guanine, we suspected that the exocyclic guanine adducts -oh - pdg, -oh - pdg, and m1dg could also be potential substrates for alkb . This study investigated the ability of alkb to repair the acrolein- and mda - derived exocyclic dg lesions in single - straned (ssdna) or double - stranded dna (dsdna). We established that all three exocyclic dg lesions can exist in open - ring forms in the sequence context studied, and we also investigated whether these open - ring forms are substrates for alkb repair . Using high - resolution quadrupole time - of - flight (q - tof) ms, we found that alkb can oxidize all three exocyclic dg adducts, in both the open- and closed - ring forms . The alkb activity on the lesions was more efficient in ssdna than in dsdna, with the base opposite the lesion dictating the repair efficiency . Among the three lesions studied, -oh - pdg was most efficiently repaired, followed by m1dg and -oh - pdg, respectively . By identifying and characterizing the alkb reaction intermediates and products using ms, ms / ms, and biochemical trapping experiments, a model for the chemical mechanism of repair of exocyclic dg lesions by alkb is proposed . The observation that these important lesions are oxidized by alkb provides evidence that direct reversal enzymes, such as alkb, may play important roles as modulators of the biological consequences of acrolein and mda . Oligonucleotide 16-mers of sequence 5-gaagacctxggcgtcc-3 (x = adduct) bearing exocyclic guanine adducts, -oh - pdg, -oh - pdg, and m1dg (figure 1) were prepared using the solid - phase methods and were deprotected, purified, and characterized as described previously . Table 1 shows the calculated mws of all the oligonucleotides used in this study and their observed intermediates . To determine the oligonucleotides concentrations, the extinction coefficients () of normal dna bases at 260 nm alkb incubations were performed with the alkbn11 protein, a version of alkb lacking the first 11 amino acids . This truncated protein has been previously shown to have activity similar to that of wild - type alkb . The alkbn11 protein was expressed in bl21(de3) cells and purified as described previously . The alkb incubations were performed at 37 c for 2 h in a reaction buffer containing 45 mm hepes (ph 8.0), 0.9 mm -ketoglutarate, 67 m fe(nh4)2(so4)26h2o, and 1.8 mm ascorbate, followed by hplc / q - tof ms analysis . Typically, 100 pmol dna was reacted with 5 m alkb (or enzyme diluent for control reactions) in the presence of all cofactors in a 20 l reaction volume . The alkb incubations with double - stranded dna were performed under similar conditions except that prior to alkb addition 1.2 equiv of complementary oligonucleotides were annealed by heating the mixture at 65 c for 5 min . The mixtures were cooled at a rate of 0.1 c / s to 4 c and then used in alkb reactions as described above . The alkb repair efficiencies were estimated from the fraction of starting material converted into intermediates and products (table 2). These values were determined by integrating the extracted ion chromatogram (eic) peaks corresponding to each of the species observed in the alkb reactions (starting material, intermediates, and products) and calculating the percentage of each species formed after alkb incubation relative to the total amount of material (table 3). In each reaction, the total amount of material was calculated by summing the areas corresponding to the starting material and all intermediates and products derived from the starting material . All of the values were background - corrected for the amount of species (intermediate or product) that may have been present in the alkb untreated control reactions . Trapping reactions of oligonucleotides with pfbha were performed in water at room temperature for 1 h using 5 m dna and 500 m pfbha in a 10 l incubation volume, followed by hplc / q - tof ms analysis, as described previously . The calculated mws of the trapped intermediates melting temperatures were measured on the dsdna obtained by mixing the 16-mer oligonucleotides containing lesions with equimolar amounts of the complementary oligonucleotides containing each of the four possible natural bases (c, t, g, and a) opposite the lesion site . The oligonucleotides (4 m in a total volume of 25 l) were mixed in alkb reaction buffer (45 mm hepes, ph 8, 0.9 mm -ketoglutarate, 1.8 mm ascorbate, 67 m fe(ii)) supplemented with the high - resolution melting dye lcgreen plus (idaho technologies inc ., salt lake city, ut) to a final concentration of 1. annealing was done by heating at 75 c for 5 min, followed by a slow cooling (0.1 c / s) to 4 c . Melting temperature, tm, was measured using a roche lightcycler 480 spectrophotometer, by heating the samples from 37 to 95 c at a rate of 0.1 c / s and recording fluorescence five times every degree celsius . Data analysis was performed using lightcycler480 software . For each oligonucleotide and complement pair, reduction of exocylic dg lesions (-oh - pdg, -oh - pdg, and m1dg) was performed by incubating 1 nmol of 16-mer oligonucleotide in 100 mm potassium phosphate, ph 8.0, with 1 m nabh4 for 1 h at 37 c in 10 l reaction volume . To prevent excess pressure from hydrogen gas accumulation from the nabh4 hydrolysis, subsequently, the excess nabh4 and salts were removed by passing the reaction mixtures through homemade dry - spun sephadex g50 fine (amersham biosciences) columns . After concentration by centrifugation under vacuum, the purified reduced oligonucleotides were used in alkb reactions using the conditions described previously . All samples (untreated, nabh4 treated, and nabh4 then alkb treated) were analyzed using hplc - esi - tof mass spectrometry . Oligonucleotide analyses were performed using agilent q - tof 6510 mass spectrometer (palo alto, ca) setup as follows: needle voltage, 3.5 kv; nitrogen drying gas, 8 l / min; heated capillary, 340 c; and nebulizer, 30 psig . The reaction mixtures were separated by hplc on a zorbax sb - aq column (2.1 150 mm; 3.5 m; agilent technologies, palo alto, ca) at a 0.2 ml / min flow rate at room temperature . Solvents were 10 mm ammonium acetate in water (a) and 100% acetonitrile (b). Lc ms / ms analyses were performed on the agilent 6510 q - tof instrument, operated in the negative ion mode with the following parameters: gas temperature, 340 c; esi capillary voltage, 3.5 kv; nebulizer pressure, 30 psi; drying nitrogen gas, 8 l / min; and fragmentation energy, 3035 v. the theoretical fragmentation pattern of a 16-mer oligonucleotide is shown in figure 2a; the expected monoisotopic masses of the fragments were calculated using mongo oligo mass calculator, version 2.06 (http://mods.rna.albany.edu/masspec-toolbox). A typical fragmentation spectrum (e.g., intermediate 3a/3b) is shown in figure 2b . The comparison between the calculated and observed m / z values for the ms / ms spectrum of 3a/3b is shown in figure 2c . Ms / ms analyses of the observable reactants, intermediates, and products for all of the alkb reactions with 16-mer oligonucleotides containing -oh - pdg, -oh - pdg, and m1dg are included in the supporting information (figures s10s17 and tables s2s9). Ms / ms analysis of prototypic 16-mer oligonucleotide containing the alkb - oxidized form of -oh - pdg . (top) predicted collision - induced dissociation (cid) fragmentation pattern of the 16-mer oligonucleotide . (bottom) ms / ms fragmentation spectrum of 16-mer containing the alkb - oxidized form of -oh - pdg . A comparison of predicted and observed m / z values for the ms / ms fragmentation pattern shown at the bottom of the figure is included in the supporting information (table s6). The ability of alkb to repair acrolein- and mda - derived exocyclic dg adducts was measured by incubating site - specifically modified 16-mer oligonucleotides with purified alkb protein . Following a 2 h incubation at 37 c, the reaction mixtures were analyzed using high - resolution ms . For each lesion, experiments were conducted in both the presence and absence of the alkb protein, with all of the necessary cofactors . Figure 3 shows representative ms spectra corresponding to each oligonucleotide containing an exocyclic dg lesion before and after alkb treatment . The molecular weight (mw) of each of the 16-mer oligonucleotides employed was calculated, from which the 4 charge monoisotopic mass (all c, n, etc .) Was determined (table 1). For example, the mw of the 16-mer containing the -oh - pdg lesion is 4960.88 (da); therefore, its monoisotopic 4 charge state has a theoretical m / z of 1239.21 . In this case, an m / z of 1239.20 was observed experimentally (figure 3a), which correlated well with the theoretical value (table 1). Because the ms conditions used throughout this study produced robust 4 charge states for all the oligonucleotides analyzed, all of the m / z numbers discussed below refer to 4 charge states, unless otherwise specified . The chemical structures corresponding to the peaks labeled in figure 3 as well as their proposed alkb - catalyzed transformations are shown in figure 4 . Q - tof mass spectrometry analysis of reactants and products of the oligonucleotides containing exocyclic guanine lesions incubated with alkb for 2 h. data represent the 4 charge envelopes; multiple ion mass peaks associated with each envelope reflect mostly the number of c atoms in each 4 charge packet . (a) -oh - pdg; (b) -oh - pdg + alkb; (c) -oh - pdg; (d) -oh - pdg + alkb; (e) m1dg; and (f) m1dg + alkb . Chemical structures and proposed pathways for alkb - mediated exocyclic guanine lesions transformation: (a) -oh - pdg (dotted border) and -oh - pdg (solid border); (b) m1dg (dashed border). Interestingly, the mass spectrum of -oh - pdg in the absence of alkb showed two peaks: a major peak at m / z 1239.20, which corresponds to the 16-mer containing the -oh - pdg lesion (structure 1a, calculated m / z 1239.21), and a minor peak at m / z 1234.70, which corresponds to a dehydrated form of -oh - pdg (structure 2, calculated m / z 1234.71) (figures 3a and 4a). The chemical structure of 2 was confirmed by ms / ms fragmentation analysis (figures s10s11 and tables s2s3). Upon addition of alkb protein, two new products were observed with m / z 1238.68 and 1243.18 (figures 3b, s1a, and s2). The product with mass 1243.18 was assigned to the oxidized form of the parent -oh - pdg lesion (structure 3a, calculated m / z 1243.21), consistent with the 4 m / z units increase, which in the 4 charge state corresponds to 16 da, the mass of an oxygen atom . The product with mass 1238.68 is similarly 4 m / z units higher than the dehydrated form at 1234.70, suggesting that it could be an oxidized form of 2 (structure 4, calculated m / z 1238.71) (figures s1 and s2). However, structure 4 could also arise from dehydration of the oxidized intermediate (structure 3a), being 4.5 m / z units lower than the 1243.18 species (which, in the 4 charge state, corresponds to the loss of a water molecule, 18 da). These data suggest that, under these reaction conditions, alkb can oxidize the -oh - pdg lesion (and its dehydrated form), transforming it into the diol 3a that could spontaneously lose the exocyclic bridge to restore the parental deoxyguanosine (figure 4a). However, the fully repaired deoxyguanosine product was not observed, perhaps because the amount of oxidized intermediate 3a formed was relatively small, with only 5.5% conversion (table 3a). The oligonucleotide containing -oh - pdg had an experimental m / z of 1239.23 (structure 5a, calculated m / z 1239.21). Incubation with alkb resulted in the conversion of about 60% of the -oh - pdg starting material into four new species (figure 3d and table 2) with m / z values of 1225.17 (dg, structure 6), 1243.17 (diol intermediate 3a), 1238.67 (single - dehydration product 4), and 1234.17 relative mass differences between these peaks (figure s1b) were used to propose chemical structures for all of the intermediates (figure 4a), which were further confirmed by an ms / ms fragmentation analysis (figures s12s14 and tables s4s6). The appearance of these intermediates and products suggests the following reaction course for direct reversal of -oh - pdg damage: alkb oxidizes the methylene group adjacent to the n - position of -oh - pdg to generate,-dihydroxy - pdg (3a), which can release mda to form undamaged guanine (6). The final step of mda release from 3a is analogous to the release of glyoxal from the a glycol intermediate that forms when alkb repairs the a lesion . However, intermediate 3a can also dehydrate to form 4 and, upon loss of another water molecule, intermediate 7, which is the mda adduct m1dg (figure 4a). As will be shown later, however, given the small amounts m1dg formed in the reaction of -oh - pdg with alkb, the intermediates of the m1dg reaction with alkb likely were below the limit of detection and therefore were not observed . When the oligonucleotide containing m1dg (starting material m / z of 1234.19) was incubated with alkb under similar conditions, four new mass envelopes were observed in the mass spectrum (figures 3f and s1c), besides the unreacted starting material . These peaks suggested a reaction course for direct reversal of m1dg damage by alkb (figure 4b). Specifically, the mass envelope at m / z 1238.19 was consistent with the alkb - catalyzed formation of an epoxide for m1dg (structure 8 in figure 4b, calculated m / z 1238.20, table 1). Hydrolysis of the epoxide would generate glycol 9, observed at m / z 1242.69, consistent with the calculated m / z 1242.71 . The additional species observed at m / z 1247.19 indicated the presence of the trihydroxylated intermediate 10 (calculated m / z 1247.21), which would be the hydration product of glycol 9 . The spontaneous loss of 2-hydroxy - mda from the trihydroxylated intermediate 10 would generate the undamaged guanine base, observed at m / z 1225.19 (calculated m / z 1225.21) (figures 3f and 4b). The assignment of intermediates was confirmed by ms / ms fragmentation analysis (figures s15s17 and tables s7s9). Alkb can repair alkylated nucleic acid bases in both ssdna and dsdna . While repair in the single - stranded context is typically more efficient, there are examples of lesions that are repaired by alkb equally well in ssdna and dsdna . Therefore, it was of interest to investigate whether the acrolein- and mda - derived exocyclic dg lesions were also substrates for alkb in dsdna . Alkb repair reactions were carried out with each of the three exocyclic dg adducts opposite each of the four canonical bases (t, c, g, and a) (figures s3s5). The amount of the starting material converted by alkb (table 2) was estimated by integrating the extracted ion chromatograms (eic) of each peak in the mass spectra (see experimental procedures for details). Likewise, the relative amounts of the intermediates and repair products formed in each case were also estimated (table 3a c). The data show that the efficiency of alkb - mediated oxidation of exocyclic dg adducts depended not only on the type of lesion (as seen in ssdna) but also on the nature of the base placed opposite the lesion . In general, the oxidation was less efficient in dsdna compared with that in ssdna, with m1dg being a notable exception, for which the maximum conversion of starting material was observed in dsdna when placed opposite t (table 2). As a general trend, lesions were more efficiently converted when placed opposite t than opposite any other base . The smallest conversion the quantification of intermediates and fully repaired product for each of the alkb reactions above provided some additional insights . For -oh - pdg, the alkb conversion was at least 3-fold lower in dsdna compared with that in ssdna, and the amount of diol intermediate (structure 3a) formed in the dsdna reaction was reduced by a similar amount . However, the amount of fully repaired product, dg, was disproportionally reduced (by at least 10-fold with respect to ssdna, table 3b), indicating that the final step of mda release from 3a is impeded when alkb repair occurs in dsdna . For m1dg opposite t, however, the amount of fully repaired product was maximal in ssdna (8.1%) compared with that in dsdna (1.34.6%) (table 3c), suggesting, as before, that the final step of the release of the oxidized exocyclic bridge from intermediate 10 was impeded in dsdna . For -oh - pdg in dsdna, alkb oxidation was even less efficient than that in ssdna, with the oxidized intermediates detected at correspondingly lower levels (table 3a). Just as in ssdna, no fully repaired dg product was observed in the alkb reaction of -oh - pdg in dsdna . However, it appears that the equilibrium between the hydrated (structure 1a) and dehydrated (structure 2) forms of -oh - pdg shifted toward the structure 1a in dsdna . The reactions of alkb with dsdna consistently showed that when the acrolein- and mda - derived exocyclic dg adducts were placed opposite c the efficiency of repair was significantly diminished (table 2). We hypothesize that this effect was due to the ability of exocyclic dg lesions to exist in open - ring structures . In the case of -oh - pdg and m1dg, the presence of the cognate base pairing partner (dc) in the opposite strand has been shown to induce the opening of the exocyclic ring to expose the watson crick base - pairing side of the damaged guanine and allow it to form hydrogen bonds . To test this hypothesis in our sequence context, the thermal melting temperatures (tm) of the oligonucleotides containing exocyclic dg adducts opposite all four canonical bases were measured (table 4). Consistent with this hypothesis and previous observations, the melting temperatures containing -oh - pdg and m1dg opposite c were the highest in each case (as compared to other complementary bases) and approached the tm of a duplex containing a canonical g: c base pair at the lesion site . When compared with a normal guanine, the presence of m1dg increased the melting temperature of the duplex when opposite t, g, or a. this effect was likely due to the extended aromatic ring system of m1dg, which allowed for additional favorable stacking interactions . By contrast with the other two lesions, the oligonucleotide containing -oh - pdg was not stabilized opposite any of the four canonical bases . This observation was consistent with the fact that the open - ring structure of -oh - pdg would allow only the n position of guanine to interact with the opposite base, while the n1 position would be still blocked . The -oh - pdg and m1dg lesions have been reported to equilibrate between ring - closed and ring - open forms . In dsdna, -oh - pdg predominantly exists in open - ring form when placed opposite dc, whereas in ssdna, it exists mostly in the closed - ring form . In both cases, -oh - pdg could be trapped by peptides, with the conjugation being more effective in dsdna than in ssdna . The reversible, hydrolytic ring - opening of m1dg has also been well - studied, with the ring - open form of m1dg predominating in dsdna when m1dg is placed opposite dc . The presence of open - ring forms of the three exocyclic dg lesions was studied in our sequence context by incubating the site - specifically modified oligonucleotides used in the alkb repair studies with o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (pfbha), a nucleophilic alkoxyamine that forms stable oxime linkages with free aldehydes . After 1 h at room temperature, the reaction mixtures were analyzed using high - resolution ms (figure 5). The calculated and experimentally observed m / z values for the oligonucleotides trapped with pfbha are listed in table s1 . Q - tof mass spectrometry analysis of the pfbha trapping reactions of oligonucleotides containing exocyclic guanine lesions . (a) -oh - pdg; (b) -oh - pdg + pfbha; (c) -oh - pdg; (d) -oh - pdg + pfbha; (e) m1dg; and (f) m1dg + pfbha . All three oligonucleotides containing the exocyclic dg lesions were trapped by pfbha, indicating that they can potentially exist in open - ring forms containing free aldehyde groups (figure 5). In the case of -oh - pdg, pfbha trapping provides strong evidence that the exocyclic adduct (5a) exists in equilibrium with an open - ring aldehyde form (5b). For m1dg (7), however, the complete trapping with pfbha does not unequivocally demonstrate the existence of the ring - open aldehyde forms (11b or 12b), because pfbha can react directly with the closed - ring form of m1dg and generate the oxime product without the formation of a free aldehyde . This alternative course of reaction is based on the observation that the rate of ring opening of m1dg at neutral ph in water is slower than the rate of trapping of m1dg with hydroxylamine . The presence of pfbha completely trapped the oligonucleotide containing -oh - pdg and m1dg . The parent peak of -oh - pdg at m / z 1239.21 (calculated m / z 1239.21) shifted quantitatively to a pfbha - trapped species at m / z 1287.97 (calculated m / z 1287.97). Similarly, the parent peak of m1dg at m / z 1234.20 (calculated m / z 1234.21) was completely converted to a pfbha - trapped species at m / z 1287.47 (calculated m / z 1287.46). In the case of -oh - pdg, 1a (observed m / z 1239.20, calculated m / z 1239.21) can form an open - ring structure (1b) that can be trapped with pfbha . However, as described previously, 1a is also in equilibrium with the imine 2, which could also react with pfbha to give a trapped product . Only a partial trapping of the -oh - pdg was observed (pfbha - trapped species at m / z 1287.98, calculated m / z 1287.97), with the relative amounts of untrapped oligonucleotides suggesting that pfbha reacted primarily with 1b and not with 2 . Assuming pfbha trapping of 1b is fast, these data also suggest that the formation of the open - ring form of -oh - pdg is slower than that for -oh - pdg . Given the presence of the open - ring forms of the three exocyclic dg lesions, it was of interest to investigate whether alkb could also act on the open - ring forms . Sodium borohydride (nabh4) buffered with sodium phosphate was used to lock the open - ring forms by reducing the open - ring aldehydes to alcohols, which were detected using high - resolution ms (figure 6). For -oh - pdg, upon treatment with nabh4, the parent peak at m / z 1239.23 was quantitatively converted to a new peak at m / z 1239.71, consistent with the addition of two hydrogens . Because there are no functional groups reducible by nabh4 in the closed - ring structure of -oh - pdg, or on any other normal base, the peak at m / z 1239.71 must correspond to the open - ring alcohol of -oh - pdg, structure 16 (calculated m / z 1239.72) (figures 6b and s7). Similarly, when the oligonucleotide containing -oh - pdg was treated with nabh4, the parent peak at m / z 1239.16 shifted to m / z 1239.67 (figures 6a and s6). Similar to the argument made for -oh - pdg, the only functional group that could be reduced by nabh4 is the aldehyde of the open - ring form of -oh - pdg (structure 1b, figure 4a) to generate an open - ring alcohol (structure 19, calculated m / z 1239.67). The dehydrated form of -oh - pdg (structure 2), a cyclic imine, was also reduced by nabh4, as suggested by the change in the isotopic distribution pattern and the expansion of the peak envelope toward higher masses (figure 6a). These changes are consistent with the appearance of a peak at m / z 1235.16 (figures 6a and s6), which presumably corresponded to a fully saturated 1,n - propano - dg (structure 15, calculated m / z 1235.21) (figure 6a). Both reduction products (19 and 15) are consistent with the reported nabh4 reduction of the -oh - pdg free nucleoside . Additional evidence for the formation of 15 was obtained by running the nabh4 reaction at a lower ph (5.8 instead of 7.0), which improved the yield of reduction of 2 (figure s9). However, even at low ph, reduction of 2 was not quantitative, perhaps due to the lesser reactivity of the aromatically stabilized cyclic imine toward nabh4 compared to that of an aldehyde . Q - tof mass spectrometry analysis of the reactions of oligonucleotides containing exocyclic guanine lesions with nabh4 and the subsequent incubation of the reduced forms with alkb for 2 h. each peak is labeled with its monoisotopic m / z value, in the 4 charge state . The corresponding chemical structures and (a) (top) -oh - pdg, (middle) -oh - pdg + nabh4, (bottom) -oh - pdg + nabh4 + alkb . (b) (top) -oh - pdg, (middle) -oh - pdg + nabh4, (bottom) -oh - pdg + nabh4 + alkb . (c) (top) m1dg, (middle) m1dg + nabh4, (bottom) m1dg + nabh4 + alkb . When exposed to nabh4, the m1dg - containing oligonucleotide (observed m / z 1234.19, calculated m / z 1234.21) was converted to a reduced species detected at m / z 1234.70 (figures 6c and s8). The reduced ring - open form of m1dg requires the initial addition of a water molecule to produce 11a or 12a (calculated m / z 1238.71) (figure 4b), which could subsequently be reduced with nabh4 . However, the open - ring form of m1dg is known to enolize readily and thus is not very reactive toward nabh4 . Consistent with this fact, a product with an m / z of 1239.21 corresponding to a reduced version of 11a or 12a could not be detected (figures 6c and s8); therefore, under the reaction conditions, m1dg could not be trapped as an open - ring species by nabh4 . Nevertheless, the reaction of alkb with the reduced form of m1dg provided additional insights into the regioselectivity of the alkb repair reactions on exocyclic dg structures . After nabh4 treatment, the oligonucleotides were first purified using sephadex g-50 columns to remove excess nabh4 and other salts and were then allowed to react with alkb under the same conditions as those previously described . Alkb produced more fully repaired product (dg) when incubated with nabh4-treated lesions as compared to the incubations with untreated lesions . For -oh - pdg, these reaction conditions produced detectable levels of fully repaired dg (6). The amount of the reduced open - ring alcohol 19 did not change upon treatment with alkb, whereas the amount of 15 was diminished in the alkb reaction (figure 6a). These observations indicated that the fully repaired dg was not generated from the reduced open - ring form 19, but rather from the 1,n - propano - dg reduced intermediate (15) via two successive oxidations by alkb, which would generate 3a (figure 6a). For the reduced ring - open form of -oh - pdg (16), the alkb reaction intermediates were consistent with oxidation at the carbon attached to the n position of guanine with subsequent release of a three - carbon aldehyde to generate fully repaired dg (figure 6b). This reaction was very efficient, with most of the starting material being consumed along with a high yield (50%) of dg . For nabh4-treated m1dg, the alkb reaction generated several intermediates (figure 6c), consistent with oxidation of 18 at the carbon attached to the n position of guanine to generate 12a, which upon dehydration yielded the original m1dg lesion (structure 7, observed m / z 1234.23). Subsequently, m1dg (7) reacted with alkb, as shown before (figures 3f and 4b), to generate epoxide 8, triol 10, and, upon spontaneous loss of 2-hydroxy - mda, the fully repaired product, dg . In an alternative reaction course, 18 could react with alkb to generate epoxide 20 and upon hydration, diol 21 (figure 6c). Subsequently, 21 can be further oxidized by alkb to generate triol 10, leading to additional repair product dg . These additional reaction pathways could explain the improved yield of fully repaired product, dg, when nabh4-treated m1dg was incubated with alkb . The three exocyclic dg lesions investigated (i.e., -oh - pdg, -oh - pdg, and m1dg) are important biomarkers of exposure to ubiquitous environmental chemicals and inflammation - induced lipid peroxidation byproducts, acrolein and mda . The present study demonstrates that the exocyclic dg lesions are processed by alkb by a complex but chemically understandable biochemical pathway . Among the three lesions studied in ssdna, -oh - pdg was repaired most efficiently, followed by m1dg and -oh - pdg (table 2 and table 3a c). The overall alkb efficiency at repairing the lesions was generally lower in dsdna than that in ssdna, as evidenced by the lower amounts of fully repaired product formed in each case (table 3a c). However, in the one case, when m1dg was opposite t, the total amount of starting material consumed was higher than in the incubation of alkb with m1dg in ssdna . The base placed opposite the lesion also influenced the repair reactions . Generally, when the lesions were placed opposite t, the repair was most efficient, with c being the opposite base that gave the lowest conversions (tables 2 and 3a c). These observations can be rationalized by the base - flipping mechanism proposed for alkb, in which a mispaired base (i.e., lesion opposite t) is more likely to splay out into the enzyme active site to be repaired . By contrast, when the exocyclic dg lesions are placed opposite c, a correct base pair is formed (in the case of -oh - pdg and m1dg, via their open - ring forms), which hinders base - flipping, effectively hiding the lesion from alkb and reducing the repair efficiency . This mechanism is consistent with our previous study of alkb activity on n - dg alkyl lesions, such as m2 g, e2 g, and the bulkier n - furfuryl - dg and n - tetrahydrofurfuryl - dg, which are good alkb substrates in ssdna but are poorly repaired when placed in dsdna opposite dc . Furthermore, the preference of alkb for repairing exocyclic dg lesions in ssdna suggests that transcriptionally active regions of the genome are likely to be most protected by alkb activity, which complements other repair pathways that target exocyclic dg lesions only in dsdna contexts (i.e., ner). All three exocyclic dg lesions studied have been proposed to exist in equilibrium with open - ring forms . The trapping of the -oh - pdg and -oh - pdg with pfbha (figure 5) suggests that there is a low but kinetically useful concentration of the ring - opened aldehyde form in equilibrium with the closed - ring form . Subsequently, by using nabh4 to reduce the aldehydes and trap the open - ring forms of -oh - pdg and -oh - pdg, the activity of alkb on the open - ring forms was also investigated . N-(3-hydroxypropyl)-g, the nabh4-reduced open - ring form of -oh - pdg, was shown to be a good substrate for alkb repair, even better than the closed - ring -oh - pdg substrate (figure 6b). The reduced open - ring form of -oh - pdg, n1-(3-hydroxypropyl)-g, was not significantly oxidized by alkb; instead, the reduced version of the dehydrated -oh - pdg, 1,n - propano - dg (structure 15), reacted with alkb (figure 6a). For m1dg, however, the pfbha trapping does not necessarily support the existence of the ring - opened aldehyde form, because the trapped product can also be formed by a direct reaction of pfbha amine at the carbon adjacent to n1 of m1dg . Consistent with this argument, m1dg incubation with nabh4 did not generate a measurable amount of trapped open - ring forms; the reduction yielded the closed - ring product 18, which was also a good substrates for alkb, on par with the parent species m1dg . Taken together, the trapping experiments demonstrated that alkb can process both open - ring species (such as the open - ring of -oh - pdg) and closed - ring species (such as the ones derived from m1dg). However, due to equilibria between open- and closed - ring forms, the alkb repair reactions of exocyclic dg lesions are complex, involving overlapping pathways with multiple convergence points (figure 4). Given the complexity of the repair pathways (figure 4), it is challenging to establish unambiguously which reaction pathway (i.e., oxidation of open- or closed - ring intermediates) is predominant . Specifically, is alkb processing the exocyclic dg lesions by directly oxidizing the closed - ring forms or is alkb reacting primarily with the open - ring forms, essentially simpler n1- and n - alkyl guanines that are known to be alkb substrates? For -oh - pdg, the data were more consistent with the alkb oxidation on the closed - ring form . When this substrate was reduced to the open - ring form with nabh4, no corresponding alkb oxidation intermediate was detected; the alkb reaction presumably occurred only with the reduced dehydrated version of -oh - pdg (structure 2), which is a closed - ring form (figure 6a). A possible explanation for this preference may be that the open - ring form of -oh - pdg generated a bulky alkyl group on the n1 position of guanine, which did not fit in the active site of alkb, unlike a smaller n1-methyl - dg substituent, which is repaired by alkb . For -oh - pdg, the data suggest that both the closed- and open - ring forms could be substrates for alkb . We demonstrated that alkb more efficiently repaired the reduced, open - ring form of -oh - pdg than the unreduced, closed - ring -oh - pdg lesion . This observation is consistent with previous work, which demonstrated that alkb can repair guanines substituted at n positions with alkyl groups of varying sizes, including bulky alkyl substitutents . It is noted, however, that the observed repair of the open - ring form does not rule out the possibility that alkb can additionally oxidize the closed - ring form of -oh - pdg (i.e., the 5a to 3a step of figure 4a). The data on the alkb repair of m1dg lesion are most consistent with the formation of an epoxide intermediate (8) of the closed - ring form, which subsequently gets hydrated twice to form triol 10 . Intermediate 10 can also be obtained from the oxidation and hydration of intermediates 11a or 12a, which are hydrated forms of m1dg . However, the presence of these hydrated forms was not detectable in the starting material, and their reduced forms were not observed in the nabh4 reaction (figure 6c). Therefore, the alternative pathways involving 11a and 12a are expected to play a minor role, if any, in the alkb reaction (figure 4b). The data on m1dg repair by alkb suggest that the enzyme, similar to its mechanism on a, primarily acts on the closed - ring form of the substrate . The alkb repair data on exocyclic dg lesions also provided some insight on the alkb selectivity between oxidation at n1- or n - attached carbons, in the context of an exocyclic propano adduct . By placing the three - carbon exocycle into a published alkb structure, it was determined that the exocyclic carbon atoms adjacent to the n1 and n positions are situated at similar distances relative to the iron center of alkb (figure s19). This suggests that alkb, in principle, should oxidize an exocyclic propano adduct equally well at either the n1 or n position . However, in the present study, alkb repaired the two oh - pdg lesions with very different efficiencies (table 2), with -oh - pdg being repaired more efficiently than -oh - pdg . One explanation for the different alkb reactivity toward these isomeric lesions may be the propensity to form open - ring species . The open - ring form of -oh - pdg shifts the entire alkyl substituent to the n1 position, which likely cannot be accommodated in the alkb active site; by contrast, the open - ring form of -oh - pdg contains the alkyl substituent at n, which can swivel away from the protein to potentially reduce steric clashes . On the basis of these considerations, we speculate that the most likely reaction course of the fully reduced exocyclic propano - dg (16) with alkb involves oxidations first at the n1 carbon (-position) and subsequently at the n carbon (-position) to generate the fully repaired dg . Compared with other known exocyclic alkb substrates, such as a and ea, the acrolein- and mda - derived dg lesions are repaired less efficiently under similar conditions . One possible explanation is that three - carbon bridges are bulkier than two - carbon bridges and some of them (i.e., -oh - pdg, -oh - pdg) can form puckered structures that are harder to accommodate in the active site of the enzyme . Furthermore, the position of a three - carbon exocycle between n1 and n of guanine could be suboptimal in the alkb active site compared with the two - carbon bridge between n and n1 of adenine . Finally, alkb has been demonstrated to prefer alkylated adenines compared to alkylated guanines; specifically, n1-methyl - adenine is repaired significantly more efficiently than n1-methyl - guanine . This difference in reactivity is believed to be due to the fact that n1-methyl - adenine has a positive charge on n1, whereas n1-methyl - guanine is a neutral species . Proposed that, in general, the preference of alkb toward positively charged lesions is likely due to the presence of negatively charged aspartate residue (asp-135) in the active site . Therefore, it is not surprising that this trend extends to exocyclic alkyl lesions, where the exocyclic guanines in the present study are repaired less efficiently by alkb than exocyclic adenines, in part because they are neutral species . Furthermore, the higher repair efficiency of alkb when acting on positively charged lesions could also be explained by the propensity of the alkb oxidation intermediates of such lesions to decompose and generate the fully repaired base . Consequently, it is anticipated that alkb oxidation intermediates with pka s of the base nitrogens altered in a direction that favors protonation would convert more efficiently to the fully repaired product . However, in the case of the exocyclic dg lesions, the alkb oxidation intermediates are generally more abundant than fully repaired dg, suggesting that the pka s of the n1 or n nitrogen of the intermediates are not altered significantly during the alkb reactions . The present work established the chemical competence of the e. coli dioxygenase alkb to oxidize and repair acrolein- and mda - derived exocyclic guanine lesions, suggesting that one role of alkb may be to alleviate the mutagenic and toxic consequences of reactive aldehydes derived from these primary toxicants . Additionally, the ability of alkb to repair the open - ring forms of exocyclic guanine lesions suggests that alkb may also modulate the toxicity and mutagenicity associated with the formation of inter- and intrastrand cross - links generated by the open - ring forms of the acrolein- and mda - derived exocyclic guanine lesions . Given that alkb has nine mammalian homologues (abh18, fto), it is anticipated that these repair pathways may also be operating in mammalian cells; however, the extent to which these pathways play a role in modulating the mutagenic and carcinogenic risk associated with exposure to acrolein and inflammation - derived mda is not known and remains to be established.
The incidence of the diverticulum of the small bowel varies from 0.2 - 1.3% in autopsy studies to 2.3% when assessed on enteroclysis . It may be congenital or acquired and its prevalence increases with the age, occurring mostly in the 6 decade . These diverticula occur most commonly in jejunum in around 80%, followed by 15% in the ileum and 5% in both . Small bowel motility disorders leading to raised intraluminal pressure are the established factors for their development . Most of the patients are asymptomatic, but about 10 - 30% of patients tend to develop severe complications like diverticulitis, stone formation, perforation, haemorrhage and intestinal obstruction . Variable presentation of diverticuli leads to delayed diagnosis or misdiagnosis which results in associated morbidity and mortality . In our case, the patient was presented with severe pain in the periumbilical region with vomiting, constipation and with a history of similar episodes of pain since 20 years . A 62-year - old male patient presented to surgery emergency with the complaint of severe pain in the periumbilical region, non - radiating to back since two days with 5 - 6 episodes of vomiting associated with constipation since one day . The patient had a history of abdominal pain and vomiting episodes followed by diarrhoea dating back to 20 years . Another episode, one and four years later, used to occur after regular meals with increased pain intensity . His blood pressure was 110/78 mmhg, pulse 86 per minute . On abdominal examination, his hb was 13.8 gm / dl, total leukocyte count 18,600/cmm, blood urea 32 mg / dl, serum creatinine 1.2 mg / dl, blood sugar 108 gm%, pti 83.33%, s. bilirubin 1.2 mg, sgot 22, sgpt 32, and s. amylase 75 iu . Usg abdomen revealed mild free fluid in the lower abdomen with thickened twisted mesentery along with thrombosed mesenteric vein s / o mesenteric ischaemia . A cect abdomen revealed a homogeneous circumferential wall thickening of the small bowel loops with the luminal compromise of the bowel, visualized twisting of the mesenteric vessels, and small bowel (jejunum) around the mesentery with mesenteric vessels creating the whirl sign suggestive of volvulus (figures 1 and 2) with few enlarged mesenteric lymph nodes . After initial fluid resuscitation and antibiotics, the patient was taken up for exploratory laparotomy with informed consent . On opening the abdomen, about 200 ml of fluid was aspirated and the abdomen inspected . It revealed multiple diverticula along mesenteric border of the jejunum with two clockwise complete rotation of the small bowel segment containing diverticuli around its mesenteric axis causing volvulus . Proximal jejunal loops were dilated, edematous and congested with collapsed distal gut loops with the normal anatomical position of caecum and duodeno - jejunal junction . After untwisting the volvulus segment, resection of about 1.5 feet of the affected part of the jejunum with diverticuli (figure 3) with end - to - end jejuno - jejunal anastomosis was done . After proper peritoneal toileting, the abdomen was closed in layers over a single drain placed in the pelvis . Postoperative period was uneventful and the patient was discharged in satisfactory condition on the 12 postoperative day . Diverticuli is a false pulsion diverticuli, characterized by herniation of mucosa and submucosa through the muscular layer in places of minor resistance to the intraluminal pressure typically at the mesenteric side where blood vessels penetrate the intestinal wall . Diverticula occur more commonly in jejunum and in the terminal ileum, probably due to the larger size of the vasa recta . They tend to be larger and more in number in the proximal jejunum but smaller and fewer distally . Isolated jejunal diverticulosis coexists with diverticuli of the oesophagus (2%), of the duodenum (26%) and of the colon (35%). The etiology of diverticulosis is believed to develop as a result of abnormalities in peristalsis, intestinal dyskinesis probably due to motor dysfunction of the smooth muscle or myenteric plexus in the small bowel leading to high segmental intraluminal pressures and penetration of mucosa and submucosa through weak mesenteric sites . It is commonly seen with visceral neuropathies, visceral myopathy and connective tissue disorders like ehlers - danlos syndrome, progressive systemic sclerosis, and sle . Intestinal diverticulae incidence increases with age and about 80% occurs in the 7 decade of life with slight male predominance . Majority of jejunal diverticuli are an incidental finding with most having chronic clinical manifestations which may be misdiagnosed as dyspepsia and irritable bowel syndrome . Patient presents with chronic postprandial pain, nausea, vomiting, borborgymi, alternating diarrhoea and constipation, weight loss, anaemia, steatorrhea, tenderness and fever, when symptomatic . Flatulent dyspepsia: epigastric pain, abdominal discomfort, flatulence one or two hours after meals . Characteristically, the intermittent, variable periodicity and severity of symptoms extends over many years . Complications, namely inflammation, obstruction (dynamic / adynamic), perforation, hemorrhage, and malabsorption occur in 10 - 30% of cases, which may be managed conservatively . . Mechanical intestinal obstruction can be due to enteroliths, intussusception, adhesive band formation as a result of previous diverticulitis, and volvulus . In volvulus, the diverticulum acts as a pivot, commonly around the adhesive band or the fluid filled involved heavier segment might be responsible for initiating the volvulus . In literature, cases of bowel obstruction indiverticulosis due to distended diverticula, inflammatory mass associated with diverticulitis, stricture or adhesions from recent or past diverticulitis, and intussusception at the site of the diverticulum have been reported . Lobo et al . Reported that dynamic intestinal obstruction is the most frequent complication of jejunal diverticulosis, which needs surgical intervention . Majority of jejuna diverticulosis, being asymptomatic or with mild symptoms, suspicion is difficult and the diagnosis is often missed or delayed . The enteroclysis and enteroscopy are the best diagnostic imaging, however, in emergency situations like in the present case due to time constraints its utility is limited . When complications arise, computed tomography is the best investigation . The whirl sign on abdominal ct shows spiral shape of the mesenteric vessels, accompanying the intestinal loops and their feeding vessels . On angiogram,barber pole sign most of acute complications of jejunoileal diverticulum need surgical intervention . As in our patient, conclusive diagnosis was made by diagnostic laparotomy, resulting in appropriate surgical treatment such as intestinal resection with end - to - end anastomosis . A simple diverticulectomy, as advised earlier, is no longer recommended as it causes increased incidence of postoperative leakage, sepsis, and death . The purpose of presenting this case is that the patient in our case had previous multiple episodes of abdominal pain with vomiting and diarrhoea after food intake for past 20 years . These were suggestive of subclinical and clinical diverticulitis and misdiagnosed for such a long period of time . Thus, it stresses the need for a diagnostic consideration in cases of chronic abdominal pain, diarrhoea, and discomfort in uncomplicated cases to prevent diagnostic delay and the associated mortality . Although this phenomenon is rare, we should keep in mind that intestinal diverticulosis may induce intestinal obstructions of different kinds . Thus, repeat physical examinations and x - ray films are needed and enteroclysis studies or ct scan is helpful in diagnosis.
Hiv treatment guidelines from the us department of health and human services (dhhs) have endorsed regimen simplification or switching in treatmentexperienced patients with suppressed viraemia . One simplification strategy is the discontinuation of lowdose ritonavir once viral suppression has been achieved, which has been shown in some studies to improve tolerability and reduce toxicities [2, 3, 4, 5, 6, 7, 8, 9]. Depending on the other regimen components, ritonavir discontinuation may also eliminate concern about cytochrome p4503a (cyp3a)mediated drug interactions with other drugs [2, 3, 4, 5, 6, 7, 8, 9, 10]. While atazanavir is a generally welltolerated protease inhibitor (pi) whose plasma concentration and genetic barrier to resistance are improved by coadministration with ritonavir, the combination is also associated with an increased risk of indirect hyperbilirubinaemia [11, 12, 13], which, when accompanied by scleral icterus, can prompt treatment discontinuation . This study, assure (a simplification study of unboosted reyataz with epzicom), investigated the efficacy and safety of discontinuing ritonavir in virologically suppressed participants receiving a regimen of tenofovir / emtricitabine + atazanavir / ritonavir (tdf / ftc + atv / r), one of the recommended firstline regimens in the dhhs guidelines . As tdf / ftc is not recommended for use with unboosted atv because of a drug drug interaction that results in decreased concentrations of atv, participants in this trial who were randomized to discontinue ritonavir simultaneously switched to a nucleoside reverse transcriptase inhibitor (nrti) backbone of abacavir / lamivudine (abc/3tc). This paper presents the 48week results of this study, including virological efficacy, immunological response, safety, tolerability, and hiv1 genotypic and phenotypic resistance patterns in participants who experienced confirmed virological failure . Changes from baseline in fasting lipids and in biomarkers associated with cardiovascular, bone and renal health were also evaluated . The prospective, randomized, multicentre, openlabel, phase iv assure study (epz113734; nct01102972) enrolled hiv1infected, antiretroviral (art)experienced adults (18 years of age) who were receiving a oncedaily regimen of tdf / ftc (300 mg/200 mg) + atv / r (300 mg/100 mg) for at least 6 months prior to the first day of screening . Eligible participants were virologically suppressed (defined as hiv1 rna 75 hiv1 rna copies / ml) at two consecutive timepoints (including the screening visit and one additional visit at least 28 days prior to screening). Tdf / ftc + atv / r could be a participant's initial regimen or first or second switch regimen (defined as any change in art components) as long as there was no documented history of virological failure . All previous art regimens consisted of two nrtis (either tdf / ftc or zidovudine / lamivudine) plus either a food and drug administration (fda)licensed nonnucleoside reverse transcriptase inhibitor (nnrti) or a ritonavirboosted pi, although alternative prior regimens were allowed on a casebycase basis . Participants were excluded if they were human leucocyte antigen (hla)b*5701positive, or had prior abacavir exposure, active centers for disease control and prevention (cdc) clinical category c disease, ongoing clinically relevant hepatitis and/or chronic hepatitis b virus (hbv) infection [hbv surface antigen positive (hbsag+)] or a creatinine clearanc <50 ml / min via the cockroft gault method . While a baseline genotype was not required, participants were deemed ineligible if they had known hiv genotyping results that indicated the presence of any nrti or pi mutation associated with resistance to any study drug . Women of childbearing potential were eligible if they had negative pregnancy tests at screening and baseline and agreed to use protocolprescribed contraception methods throughout the study . Additional information on inclusion / exclusion criteria, including a link to the study protocol, is available in a previous publication . All participants provided written informed consent to participate in the study, and the protocol was approved by the institutional review board for each study site . After stratification by prior art experience (tdf / ftc + atv / r as initial regimen or as first / second switch regimen), eligible participants were randomized 2:1 to simplify their regimen to oncedaily abc/3tc 600 mg/300 mg (viiv healthcare, research triangle park, nc) plus 400 mg atv once daily (two 200 mg tablets; bristolmyers squibb, princeton, nj) or to remain on oncedaily tdf / ftc (gilead sciences, foster city, ca) plus one 300 mg atv tablet boosted with a 100 mg ritonavir tablet (abbvie, chicago, il). Randomization and study drug provisioning were performed by glaxosmithkline's randomization and medication ordering system . Participants were evaluated at screening, baseline, and weeks 2, 4, 12, 24, 36 and 48; visits included routine chemistry, haematology, hiv1 rna and immunology assessments . The concentrations of specific biomarkers [bone alkaline phosphatase (bap), parathyroid hormone (pth), cterminal telopeptide (ctelopeptide), osteocalcin, urine 2 microglobulin (2m):creatinine ratio, highsensitivity creactive protein (hscrp), interleukin6 and ddimer] were evaluated at baseline, week 24 and week 48 (as were vitamin d levels). Hiv1 rna concentrations were measured using the realtime hiv1 assay (abbott molecular, inc ., des plaines, il). Adverse events and laboratory toxicities were graded using the 2004 division of aids toxicity grading scale . Glomerular filtration rates (gfrs) were estimated using the modification of diet in renal disease (mdrd) formula . Smoking status and presence of diabetes were assessed at baseline, week 24 and week 48 for framingham risk score calculation; treatmentemergent changes in fasting total, highdensity lipoprotein (hdl) and lowdensity lipoprotein (ldl) cholesterol and triglycerides were assessed using the division of aids lipid toxicity grading system . Viral genotypes and phenotypes were determined by monogram biosciences (south san francisco, ca); mutations were defined according to international aids society usa guidelines . All other laboratory tests were performed centrally by quest diagnostics (van nuys, ca). The intenttotreat (itt) population consisted of all enrolled participants randomized in the study . The primary population for efficacy analyses included all participants in the itt population who received at least one dose of study drug [ittexposed (itte)]. The virological failure population included all participants who met the protocoldefined criteria for confirmed virological failure, defined as plasma hiv1 rna 400 copies / ml on two consecutive occasions . The observed data set contained all data collected while participants were in the study; no missing values were imputed . The primary efficacy endpoint was the proportion of participants with hiv1 rna <50 copies / ml at week 24 by the time to loss of virological response (tlovr) algorithm . The proportion of participants with hiv1 rna <50 copies / ml at week 48 was a secondary endpoint . Other secondary endpoints included the proportion of participants with hiv1 rna <400 copies / ml, the change from baseline in cd4 cell count, time to virological failure, detection of genotypic and phenotypic resistance at the time of virological failure, change from baseline in fasting lipid profiles (total, ldl and hdl cholesterol and triglycerides), grade 24 aes and all saes . There were two exploratory endpoints: (1) change from baseline in neurocognition scores, to be reported in a separate paper; and (2) change from baseline in eight cardiovascular / inflammation, bone and renal biomarkers, included in this publication . Per protocol, virological response rates at week 48 were compared using a cochran mantelhaenszel test stratified by initial art regimen (tdf / ftc + atv / r as initial regimen or as first / second switch regimen). Continuous variables were assessed using the wilcoxon ranksum test, and binary responses were compared using fisher's exact test . Biomarker data were logtransformed prior to analysis, and the change from baseline was assessed using geometric mean ratios with 95% confidence intervals . The statistical analyses were performed using sas version 9.1 (sas institute inc ., cary, nc). After stratification by prior art experience (tdf / ftc + atv / r as initial regimen or as first / second switch regimen), eligible participants were randomized 2:1 to simplify their regimen to oncedaily abc/3tc 600 mg/300 mg (viiv healthcare, research triangle park, nc) plus 400 mg atv once daily (two 200 mg tablets; bristolmyers squibb, princeton, nj) or to remain on oncedaily tdf / ftc (gilead sciences, foster city, ca) plus one 300 mg atv tablet boosted with a 100 mg ritonavir tablet (abbvie, chicago, il). Randomization and study drug provisioning were performed by glaxosmithkline's randomization and medication ordering system . Participants were evaluated at screening, baseline, and weeks 2, 4, 12, 24, 36 and 48; visits included routine chemistry, haematology, hiv1 rna and immunology assessments . The concentrations of specific biomarkers [bone alkaline phosphatase (bap), parathyroid hormone (pth), cterminal telopeptide (ctelopeptide), osteocalcin, urine 2 microglobulin (2m):creatinine ratio, highsensitivity creactive protein (hscrp), interleukin6 and ddimer] were evaluated at baseline, week 24 and week 48 (as were vitamin d levels). Hiv1 rna concentrations were measured using the realtime hiv1 assay (abbott molecular, inc ., des plaines, il). Adverse events and laboratory toxicities were graded using the 2004 division of aids toxicity grading scale . Glomerular filtration rates (gfrs) were estimated using the modification of diet in renal disease (mdrd) formula . Smoking status and presence of diabetes were assessed at baseline, week 24 and week 48 for framingham risk score calculation; treatmentemergent changes in fasting total, highdensity lipoprotein (hdl) and lowdensity lipoprotein (ldl) cholesterol and triglycerides were assessed using the division of aids lipid toxicity grading system . Viral genotypes and phenotypes were determined by monogram biosciences (south san francisco, ca); mutations were defined according to international aids society usa guidelines . All other laboratory tests were performed centrally by quest diagnostics (van nuys, ca). The intenttotreat (itt) population consisted of all enrolled participants randomized in the study . The primary population for efficacy analyses included all participants in the itt population who received at least one dose of study drug [ittexposed (itte)]. The virological failure population included all participants who met the protocoldefined criteria for confirmed virological failure, defined as plasma hiv1 rna 400 copies / ml on two consecutive occasions . The observed data set contained all data collected while participants were in the study; no missing values were imputed . The primary efficacy endpoint was the proportion of participants with hiv1 rna <50 copies / ml at week 24 by the time to loss of virological response (tlovr) algorithm . The proportion of participants with hiv1 rna <50 copies / ml at week 48 was a secondary endpoint . Other secondary endpoints included the proportion of participants with hiv1 rna <400 copies / ml, the change from baseline in cd4 cell count, time to virological failure, detection of genotypic and phenotypic resistance at the time of virological failure, change from baseline in fasting lipid profiles (total, ldl and hdl cholesterol and triglycerides), grade 24 aes and all saes . There were two exploratory endpoints: (1) change from baseline in neurocognition scores, to be reported in a separate paper; and (2) change from baseline in eight cardiovascular / inflammation, bone and renal biomarkers, included in this publication . Per protocol, virological response rates at week 48 were compared using a cochran mantelhaenszel test stratified by initial art regimen (tdf / ftc + atv / r as initial regimen or as first / second switch regimen). Continuous variables were assessed using the wilcoxon ranksum test, and binary responses were compared using fisher's exact test . Biomarker data were logtransformed prior to analysis, and the change from baseline was assessed using geometric mean ratios with 95% confidence intervals . The statistical analyses were performed using sas version 9.1 (sas institute inc ., cary, nc). This study enrolled 297 participants from 43 clinical sites in the usa and puerto rico . One participant randomized to tdf / ftc + atv / r was withdrawn because of a protocol violation prior to receiving any study medication, so the itte population included 199 participants simplifying to abc/3tc + atv and 97 continuing on tdf / ftc + atv / r . Participants were recruited between april 2010 and december 2011, and the last 48week analysis visit occurred in december 2012 . Most participants (79%) were male (table 1), 34% were of africanamerican / african heritage, and 26% selfidentified as hispanic / latino . Median time on art prior to enrolment was 978 days for the abc/3tc + atv group and 1106 days for the tdf / ftc + atv / r group . Baseline participant demographics and characteristics abc/3tc, abacavir / lamivudine; art, antiretroviral therapy; atv, atazanavir; atv / r, atazanavir / ritonavir; cdc, centers for disease control and prevention; tdf / ftc, tenofovir / emtricitabine . Eightyfive per cent (253) of participants in the itte population completed 48 weeks on study (fig . 1). The most common reasons for study withdrawal were lost to followup (5%; 15 participants), consent withdrawn (4%; 12 participants), and aes (3%; 10 participants). All adverse events leading to study discontinuation were grade 1 or 2, with the exception of a grade 3 lipase increase in one participant continuing on tenofovir / emtricitabine + atazanavir / ritonavir (tdf / ftc + atv / r). Abc/3tc, abacavir / lamivudine; atv, atazanavir; atv / r, atazanavir / ritonavir; tdf / ftc, tenofovir / emtricitabine . As previously reported, 86.9% (173 of 199) of participants in the abc/3tc + atv treatment group successfully maintained hiv1 rna <50 copies / ml by the tlovr analysis at week 24 compared with 86.6% (84 of 97) in the tdf / ftc + atv / r group . The adjusted treatment difference was 0.33%, and the twosided 95% confidence interval (ci) (7.97 to 8.64%) stratified by prior art regimen excluded the predefined noninferiority margin of 12%, demonstrating the noninferiority of the abc/3tc + atv simplification regimen to the tdf / ftc + atv / r continuation regimen . At week 48, the efficacy results were also similar between treatment groups (p = 0.564), with 76% (152 of 199) of participants taking abc/3tc + atv and 79% (77 of 97) taking tdf / ftc + atv / r having hiv1 rna <50 copies / ml by tlovr analysis . In the observed analysis, there was no significant difference (p = 0.134) in hiv1 rna <50 copies / ml at week 48 between treatment groups, with response rates of 91% (154 of 169) in the abc/3tc + atvtreated group versus 96% (79 of 82) in the tdf / ftc + atv / rtreated group . There was no significant difference between treatment groups by tlovr analysis in the hiv1 rna <50 copies / ml response rate when adjusted by gender (p = 0.672), race (p = 0.645) or age (p = 0.652). At week 48, median cd4 cell counts were 603 and 590 cells/l for abc/3tc + atv and tdf / ftc + atv / r, respectively . However, the median increase in cd4 cell count from baseline to week 48 was significantly larger (p = 0.026) in the abc/3tc + atv group (+ 90 cells/l) compared with the tdf / ftc + atv / r group (+ 47 cells/l). Confirmed virological failure occurred in four of 199 (2%) participants switched to abc/3tc + atv and one of 97 (1%) participants continuing on tdf / ftc + atv / r . At virological failure, hiv1 from three participants (two receiving abc/3tc + atv; one receiving tdf / ftc + atv / r) was fully susceptible to all drugs, with no reverse transcriptase (rt) mutations and a few minor pi mutations (including l10i, g16g / e, d60e, i62v, v77i and i93l) detected, and confirmed virological failure was associated with site reports of noncompliance and/or therapy interruption . One abc/3tc + atvreceiving participant experienced initial rebound at day 14, concomitant with sitereported treatment compliance issues . No prior hiv1 genotypes or comprehensive treatment records were available, but the participant reported receiving nevirapine plus tdf / ftc for> 8 years before tdf / ftc + atv / r . Numerous hiv1 rt mutations (m41l, l74v, k103n, m184v, l210w and t215y) were detected, including thymidine analogue mutations (tams), despite no prior reported zidovudine or stavudinecontaining treatment . Multiple pi mutations (l10f, k20i, m46i, i54v, i62v, l63p, a71 t, g73 t, i84v and l90 m) were detected . The participant's hiv1 had reduced phenotypic susceptibility to multiple drugs, including abc, 3tc and atv . Several years of prior treatment with tdf / ftc and efavirenz before switching to a tdf / ftc + atv / r regimen were reported . The m41l and t215y tams and the m184v mutation were detected at failure, despite no prior reported zidovudine or stavudine treatment, as were numerous pi mutations (l10i, k20r, l24i, m36i, k43 t, m46l, i62v, v82a and l89 m); the virus from this participant remained susceptible to both abc and tdf but had reduced susceptibility to atv and 3tc / ftc . Rates of aes of moderate or greater severity (grade 24) were similar between the two groups (45% for both groups; table 2). There were few grade 24 treatmentrelated aes in either group (9% for abc/3tc + atv and 6% for tdf / ftc + atv / r). Five participants experienced grade 24 cardiac disorders during the course of the study; none were considered to be drug related . In the tdf / ftc + atv / r group, one participant had grade 2 palpitations . In the abc/3tc + atv group, two participants were diagnosed with grade 2 cardiomyopathy; the third had a prior history of congestive heart failure and developed grade 2 coronary artery disease, and the fourth participant, a former smoker with diabetes, hyperlipidaemia, and a family history of cardiovascular disease, had a grade 4 acute inferior myocardial infarction . Adverse events and laboratory abnormalities listing includes all events occurring in more than one participant . Abc/3tc, abacavir / lamivudine; ae, adverse event; atv, atazanavir; atv / r, atazanavir / ritonavir; ldl, lowdensity lipoprotein; tdf / ftc, tenofovir / emtricitabine . Adverse events leading to study withdrawal affected 4% of participants receiving abc/3tc + atv and 2% of participants continuing on tdf / ftc + atv / r; of these, only nausea, vomiting and rash were reported in more than one participant . Suspected abc hypersensitivity was reported for one participant during the course of the study; the participant was not withdrawn but restarted his original tdf / ftc + atv / r regimen with resolution of signs and symptoms . The rate of treatmentemergent or worsening grade 24 (p = 0.0026) and grade 34 (p = 0.0014) laboratory abnormalities was significantly higher in the tdf / ftc + atv / r group compared with the abc/3tc + atv group . Other treatmentemergent grade 34 laboratory abnormalities were infrequent and similar between groups (table 2). The fasting lipid levels for participants with paired baseline and week 48 results (fig . 2) were similar between treatment groups and varied little between baseline and week 48, except for a small but statistically significant increase in median hdl cholesterol levels (3.0 mg / dl; p = 0.013) for the abc/3tc + atv group . Median fasting lipid parameters with guideline cutoffs from the national cholesterol education program (ncep) for participants with paired baseline and week 48 values . A significant increase (p <0.0001) in median highdensity lipoprotein (hdl) cholesterol levels for the change from baseline (cfb) to week 48 was seen for the abacavir / lamivudine + atazanavir (abc/3tc + atv) group with no significant increase in the tenofovir / emtricitabine + atazanavir / ritonavir (tdf / ftc + atv / r) treatment group (p = 0.8994); a significant difference (p = 0.013) between treatment groups was also observed . Abc/3tc, abacavir / lamivudine; atv, atazanavir; atv / r, atazanavir / ritonavir; ldl, lowdensity lipoprotein; tdf / ftc, tenofovir / emtricitabine . At baseline, the median estimated glomerular filtration rate by the mdrd equation was 93 ml / min/1.73 m for both treatment groups . At week 48, there was a small median increase of 0.8 ml / min/1.73 m for the abc/3tc + atv group and a small median decrease of 1.3 ml / min/1.73 m in the tdf / ftc + atv / r group, but this effect was not statistically significant . The median 10year risk of coronary heart disease as measured by the framingham equation at baseline and week 48 was 2.0% and 2.0%, respectively, for the abc/3tc + atv group and 1.5% and 2.0% for the tdf / ftc + atv / r group, respectively . Between baseline and week 48, there was a significant (p <0.001) decrease resulting in improvement for the measured bone biomarkers bap, pth, ctelopeptide and osteocalcin in the abc/3tc + atv group (fig . 3a d); these biomarkers remained relatively unchanged in the tdf / ftc + atv / r group over the same time period . Comparing the two treatment groups, the decrease from baseline to week 48 in all four of these bone biomarkers was significantly larger (p <0.001) for the abc/3tc + atv group than for the tdf / ftc + atv / r group . Serum calcium levels remained similar within and between groups, with baseline mean values of 9.33 and 9.35 mg / dl for the abc/3tc + atv and tdf / ftc + atv / r groups, respectively, and mean changes of 0.05 and 0.02 mg / dl, respectively, at week 48 . Vitamin d (25 oh) geometric mean values were in the low but normal range at baseline (28.3 ng / ml in the abc/3tc + atv group and 26.4 ng / ml in the tdf / ftc + atv / r group). At week 48, these values were 24.6 ng / ml in the abc/3tc + atv group and 29.3 ng / ml in the tdf / ftc + atv / r group, representing a decline from baseline for the abc/3tc + atv group (p <0.001) and an increase for the tdf / ftc + atv / r group (p = 0.005). Biomarkers . Geometric means and 95% confidence intervals for the bone biomarkers (a) bone alkaline phosphatase, (b) parathyroid hormone, (c) cterminal telopeptide and (d) osteocalcin; (e) the renal biomarker urine 2 microglobulin: creatinine ratio; and the inflammatory biomarkers (f) highsensitivity creactive protein (hscrp), (g) interleukin6 and (h) ddimer in participants from both treatment groups with data at baseline and week 48 . Intraarm pvalues were calculated using a paired ttest on the ratio of the geometric mean concentration at baseline and the geometric mean concentration at week 48 . Interarm pvalues were calculated using a twosample ttest on the ratio of the geometric mean change from baseline to week 48 in the abacavir / lamivudine + atazanavir (abc/3tc + atv) versus tenofovir / emtricitabine + atazanavir / ritonavir (tdf / ftc + atv / r) groups . Abc/3tc, abacavir / lamivudine; atv, atazanavir; atv / r, atazanavir / ritonavir; tdf / ftc, tenofovir / emtricitabine . The renal biomarker 2m: creatinine ratio declined by 56% in the abc/3tc + atv group (p <0.001) but increased by 14% in the tdf / ftc + atv / r group (fig . 3e); the change from baseline to week 48 was significantly different (p <conversely, there were no significant changes from baseline to week 48 in any of the biomarkers associated with cardiovascular disease, inflammation or thrombogenesis that were evaluated (hscrp, interleukin6 and ddimer) either within or between treatment groups (fig . This study enrolled 297 participants from 43 clinical sites in the usa and puerto rico . One participant randomized to tdf / ftc + atv / r was withdrawn because of a protocol violation prior to receiving any study medication, so the itte population included 199 participants simplifying to abc/3tc + atv and 97 continuing on tdf / ftc + atv / r . Participants were recruited between april 2010 and december 2011, and the last 48week analysis visit occurred in december 2012 . Most participants (79%) were male (table 1), 34% were of africanamerican / african heritage, and 26% selfidentified as hispanic / latino . Median time on art prior to enrolment was 978 days for the abc/3tc + atv group and 1106 days for the tdf / ftc + atv / r group . Baseline participant demographics and characteristics abc/3tc, abacavir / lamivudine; art, antiretroviral therapy; atv, atazanavir; atv / r, atazanavir / ritonavir; cdc, centers for disease control and prevention; tdf / ftc, tenofovir / emtricitabine . Eightyfive per cent (253) of participants in the itte population completed 48 weeks on study (fig . 1). The most common reasons for study withdrawal were lost to followup (5%; 15 participants), consent withdrawn (4%; 12 participants), and aes (3%; 10 participants). All adverse events leading to study discontinuation were grade 1 or 2, with the exception of a grade 3 lipase increase in one participant continuing on tenofovir / emtricitabine + atazanavir / ritonavir (tdf / ftc + atv / r). Abc/3tc, abacavir / lamivudine; atv, atazanavir; atv / r, atazanavir / ritonavir; tdf / ftc, tenofovir / emtricitabine . As previously reported, 86.9% (173 of 199) of participants in the abc/3tc + atv treatment group successfully maintained hiv1 rna <50 copies / ml by the tlovr analysis at week 24 compared with 86.6% (84 of 97) in the tdf / ftc + atv / r group . The adjusted treatment difference was 0.33%, and the twosided 95% confidence interval (ci) (7.97 to 8.64%) stratified by prior art regimen excluded the predefined noninferiority margin of 12%, demonstrating the noninferiority of the abc/3tc + atv simplification regimen to the tdf / ftc + atv / r continuation regimen . At week 48, the efficacy results were also similar between treatment groups (p = 0.564), with 76% (152 of 199) of participants taking abc/3tc + atv and 79% (77 of 97) taking tdf / ftc + atv / r having hiv1 rna <50 copies / ml by tlovr analysis . In the observed analysis, there was no significant difference (p = 0.134) in hiv1 rna <50 copies / ml at week 48 between treatment groups, with response rates of 91% (154 of 169) in the abc/3tc + atvtreated group versus 96% (79 of 82) in the tdf / ftc + atv / rtreated group . There was no significant difference between treatment groups by tlovr analysis in the hiv1 rna <50 copies / ml response rate when adjusted by gender (p = 0.672), race (p = 0.645) or age (p = 0.652). At week 48, median cd4 cell counts were 603 and 590 cells/l for abc/3tc + atv and tdf / ftc + atv / r, respectively . However, the median increase in cd4 cell count from baseline to week 48 was significantly larger (p = 0.026) in the abc/3tc + atv group (+ 90 cells/l) compared with the tdf / ftc + atv / r group (+ 47 cells/l). Confirmed virological failure occurred in four of 199 (2%) participants switched to abc/3tc + atv and one of 97 (1%) participants continuing on tdf / ftc + atv / r . At virological failure, hiv1 from three participants (two receiving abc/3tc + atv; one receiving tdf / ftc + atv / r) was fully susceptible to all drugs, with no reverse transcriptase (rt) mutations and a few minor pi mutations (including l10i, g16g / e, d60e, i62v, v77i and i93l) detected, and confirmed virological failure was associated with site reports of noncompliance and/or therapy interruption . One abc/3tc + atvreceiving participant experienced initial rebound at day 14, concomitant with sitereported treatment compliance issues . No prior hiv1 genotypes or comprehensive treatment records were available, but the participant reported receiving nevirapine plus tdf / ftc for> 8 years before tdf / ftc + atv / r . Numerous hiv1 rt mutations (m41l, l74v, k103n, m184v, l210w and t215y) were detected, including thymidine analogue mutations (tams), despite no prior reported zidovudine or stavudinecontaining treatment . Multiple pi mutations (l10f, k20i, m46i, i54v, i62v, l63p, a71 t, g73 t, i84v and l90 m) were detected . The participant's hiv1 had reduced phenotypic susceptibility to multiple drugs, including abc, 3tc and atv . Several years of prior treatment with tdf / ftc and efavirenz before switching to a tdf / ftc + atv / r regimen were reported . The m41l and t215y tams and the m184v mutation were detected at failure, despite no prior reported zidovudine or stavudine treatment, as were numerous pi mutations (l10i, k20r, l24i, m36i, k43 t, m46l, i62v, v82a and l89 m); the virus from this participant remained susceptible to both abc and tdf but had reduced susceptibility to atv and 3tc / ftc . Rates of aes of moderate or greater severity (grade 24) were similar between the two groups (45% for both groups; table 2). There were few grade 24 treatmentrelated aes in either group (9% for abc/3tc + atv and 6% for tdf / ftc + atv / r). Five participants experienced grade 24 cardiac disorders during the course of the study; none were considered to be drug related . In the tdf / ftc + atv / r group, one participant had grade 2 palpitations . In the abc/3tc + atv group, two participants were diagnosed with grade 2 cardiomyopathy; the third had a prior history of congestive heart failure and developed grade 2 coronary artery disease, and the fourth participant, a former smoker with diabetes, hyperlipidaemia, and a family history of cardiovascular disease, had a grade 4 acute inferior myocardial infarction . Adverse events and laboratory abnormalities listing includes all events occurring in more than one participant . Abc/3tc, abacavir / lamivudine; ae, adverse event; atv, atazanavir; atv / r, atazanavir / ritonavir; ldl, lowdensity lipoprotein; tdf / ftc, tenofovir / emtricitabine . Adverse events leading to study withdrawal affected 4% of participants receiving abc/3tc + atv and 2% of participants continuing on tdf / ftc + atv / r; of these, only nausea, vomiting and rash were reported in more than one participant . Suspected abc hypersensitivity was reported for one participant during the course of the study; the participant was not withdrawn but restarted his original tdf / ftc + atv / r regimen with resolution of signs and symptoms . The rate of treatmentemergent or worsening grade 24 (p = 0.0026) and grade 34 (p = 0.0014) laboratory abnormalities was significantly higher in the tdf / ftc + atv / r group compared with the abc/3tc + atv group . Other treatmentemergent grade 34 laboratory abnormalities were infrequent and similar between groups (table 2). The fasting lipid levels for participants with paired baseline and week 48 results (fig . 2) were similar between treatment groups and varied little between baseline and week 48, except for a small but statistically significant increase in median hdl cholesterol levels (3.0 mg / dl; p = 0.013) for the abc/3tc + atv group . Median fasting lipid parameters with guideline cutoffs from the national cholesterol education program (ncep) for participants with paired baseline and week 48 values . A significant increase (p <0.0001) in median highdensity lipoprotein (hdl) cholesterol levels for the change from baseline (cfb) to week 48 was seen for the abacavir / lamivudine + atazanavir (abc/3tc + atv) group with no significant increase in the tenofovir / emtricitabine + atazanavir / ritonavir (tdf / ftc + atv / r) treatment group (p = 0.8994); a significant difference (p = 0.013) between treatment groups was also observed . Abc/3tc, abacavir / lamivudine; atv, atazanavir; atv / r, atazanavir / ritonavir; ldl, lowdensity lipoprotein; tdf / ftc, tenofovir / emtricitabine . At baseline, the median estimated glomerular filtration rate by the mdrd equation was 93 ml / min/1.73 m for both treatment groups . At week 48, there was a small median increase of 0.8 ml / min/1.73 m for the abc/3tc + atv group and a small median decrease of 1.3 ml / min/1.73 m in the tdf / ftc + atv / r group, but this effect was not statistically significant . The median 10year risk of coronary heart disease as measured by the framingham equation at baseline and week 48 was 2.0% and 2.0%, respectively, for the abc/3tc + atv group and 1.5% and 2.0% for the tdf / ftc + atv / r group, respectively . Between baseline and week 48, there was a significant (p <0.001) decrease resulting in improvement for the measured bone biomarkers bap, pth, ctelopeptide and osteocalcin in the abc/3tc + atv group (fig . 3a d); these biomarkers remained relatively unchanged in the tdf / ftc + atv / r group over the same time period . Comparing the two treatment groups, the decrease from baseline to week 48 in all four of these bone biomarkers was significantly larger (p <0.001) for the abc/3tc + atv group than for the tdf / ftc + atv / r group . Serum calcium levels remained similar within and between groups, with baseline mean values of 9.33 and 9.35 mg / dl for the abc/3tc + atv and tdf / ftc + atv / r groups, respectively, and mean changes of 0.05 and 0.02 mg / dl, respectively, at week 48 . Vitamin d (25 oh) geometric mean values were in the low but normal range at baseline (28.3 ng / ml in the abc/3tc + atv group and 26.4 ng / ml in the tdf / ftc + atv / r group). At week 48, these values were 24.6 ng / ml in the abc/3tc + atv group and 29.3 ng / ml in the tdf / ftc + atv / r group, representing a decline from baseline for the abc/3tc + atv group (p <0.001) and an increase for the tdf / ftc + atv / r group (p = 0.005). Biomarkers . Geometric means and 95% confidence intervals for the bone biomarkers (a) bone alkaline phosphatase, (b) parathyroid hormone, (c) cterminal telopeptide and (d) osteocalcin; (e) the renal biomarker urine 2 microglobulin: creatinine ratio; and the inflammatory biomarkers (f) highsensitivity creactive protein (hscrp), (g) interleukin6 and (h) ddimer in participants from both treatment groups with data at baseline and week 48 . Intraarm pvalues were calculated using a paired ttest on the ratio of the geometric mean concentration at baseline and the geometric mean concentration at week 48 . Interarm pvalues were calculated using a twosample ttest on the ratio of the geometric mean change from baseline to week 48 in the abacavir / lamivudine + atazanavir (abc/3tc + atv) versus tenofovir / emtricitabine + atazanavir / ritonavir (tdf / ftc + atv / r) groups . Abc/3tc, abacavir / lamivudine; atv, atazanavir; atv / r, atazanavir / ritonavir; tdf / ftc, tenofovir / emtricitabine . The renal biomarker 2m: creatinine ratio declined by 56% in the abc/3tc + atv group (p <0.001) but increased by 14% in the tdf / ftc + atv / r group (fig . 3e); the change from baseline to week 48 was significantly different (p <conversely, there were no significant changes from baseline to week 48 in any of the biomarkers associated with cardiovascular disease, inflammation or thrombogenesis that were evaluated (hscrp, interleukin6 and ddimer) either within or between treatment groups (fig . In this randomized, multicentre assure study, the proportion of participants maintaining suppression of hiv1 rna <50 copies / ml by tlovr at week 48 was similar between participants switching to abc/3tc + atv (76%) and those maintaining on tdf / ftc + atv / r (79%). The efficacy and tolerability of abc/3tc + atv demonstrated in this study complement those observed in the aries trial, where artnave patients initiated treatment with abc/3 tc + atv / r, and after 36 weeks the virologically suppressed population was randomized to continue on the same regimen or switch to a regimen of abc/3tc + atv . Viral suppression was maintained 48 weeks postrandomization in 86% of patients in the abc/3tc + atvtreated group and in 81% of those taking abc/3tc + atv / r, accompanied by lower rates of treatmentrelated grade 2 4 aes and a decrease in hyperbilirubinaemia from 14% to 4% for the abc/3tc + atvtreated group . In assure, switching to abc/3tc + atv was also accompanied by fewer treatmentemergent or worsening aes, driven largely by a lower rate of moderate to severe hyperbilirubinaemia and by a modest but significant increase in median cd4 cell count . Similarly, in the induma study, previously antiretroviralnave patients received two nrtis plus atv / r for 26 to 30 weeks, and virologically suppressed patients were randomized to unboosted atv or maintained atv / r . After 48 weeks there was no significant difference in virological suppression between the groups (78% for atv and 75% for atv / r), while hyperbilirubinaemia decreased by 50% (from 32 to 16%) in the unboosted atv group . In the assure study, significant improvements in the bone turnover markers pth, bap, ctelopeptide and osteocalcin were observed following the switch to abc/3tc + atv, with little change over time in the tdf / ftc + atv / r group . Initiation of art is often associated with decreases in bone mineral density that slow or stabilize over 24 to 48 weeks of therapy; these losses tend to be larger with tdfcontaining regimens compared with nontdfcontaining regimens [20, 21, 22, 23]. Bone biomarkers have been studied in treatmentnave patients initiating therapy [24, 25, 26, 27], but few randomized trials have included treatmentexperienced adults [28, 29, 30]; results from these treatmentexperienced studies were generally consistent with our findings that bone biomarkers were more favourable in patients taking abc/3tc compared with tdf / ftc and probably reflect clinically relevant differences between these regimens in bone turnover . The improvement in bone markers observed after switching from tdf in our study was not explained by changes in levels of calcium or vitamin d. vitamin d levels at baseline and at week 48 remained within the lower end of the normal range for both treatment groups, and a small decline was noted for these levels in the abc/3tc + atv treatment group . Switching to abc/3tc + atv also resulted in improvement in the 2m: creatinine ratio, a validated marker of combined glomerular and tubular health . While the association between tdf and renal impairment is well studied (for example [31, 32, 33, 34],), only one randomized trial, assert, which compared abc/3tc with tdf / ftc both in combination with efavirenz in treatmentnave patients, has included the 2m: creatinine ratio . After 48 weeks on therapy, the 2m: creatinine ratio increased by 24% in the tdf / ftc group and decreased by 47% in the abc/3tc group (p <0.0001), a result consistent with the results observed in assure (albeit in treatmentnave patients). In this study, switching to abc/3tc + atv was not associated with significant changes in the markers of inflammation and coagulation, interleukin6, hscrp and ddimer . Several studies found no increase in markers of cardiovascular disease with initiation of abc compared with other nrtis [28, 29, 34, 35, 36, 37, 38, 39], while study a5202 reported a significant increase from baseline in hscrp after 96 weeks in treatmentnave patients randomized to abc/3tc, with no significant change in patients randomized to tdf / ftc, although interleukin6 levels decreased significantly in both the abc/3tc and tdf / ftctreated groups . Four other studies have examined cardiovascular markers in treatmentexperienced patients: steal, swap, bicombo, and swift . As in our study, none of these trials reported a difference between abc/3tc and tdf / ftc in hscrp, interleukin6 or ddimer over 48 weeks of postrandomization treatment . Wildtype virus at virological failure was detected in three of five participants with confirmed viraemia> two additional participants, both receiving abc/3tc + atv, experienced virological failure, one shortly after therapy switch (day 14) and one at week 36 . Multiple viral resistance mutations, including tams, were detected in virus from both participants, and the k103n nnrti mutation was detected in one participant . As tams are not selected by treatment with either tdf / ftc or abc/3tc, these results are suggestive of these participants having previously experienced treatment failure on either zidovudine or stavudinecontaining regimens, although no prior exposure was reported for either participant . There are several limitations of this trial that should be considered when interpreting results from this study . This simplification treatment strategy is restricted to certain nrti backbones such as abc/3tc that can be used in combination with unboosted atv . The study enrolled virologically suppressed, therapyexperienced patients and was designed to exclude those patients with prior virological failure that could have resulted in selection for drug resistanceassociated viral mutations affecting resistance to the investigational products . Plasma concentrations of atv in the absence of ritonavir boosting are lower, and therefore this regimen may be more susceptible to patient adherence issues, especially if archived resistanceassociated mutations are present . While the markers of bone density examined are considered accepted markers of bone turnover, this study did not directly measure bone density . Over 48 weeks, discontinuation of ritonavir after switching from a regimen of tdf / ftc + atv / r to abc/3tc + atv maintained viral suppression in study participants and led to improvements in cd4 cell count, the bone biomarkers pth, bap, ctelopeptide and osteocalcin, and the renal 2m: creatinine ratio . The abc/3tc + atv treatment group also had significantly fewer laboratory abnormalities than the tdf / ftc + atv / r treatment group without increasing other fasting lipid levels or cardiovascular biomarkers of inflammation and thrombogenesis . Substantial contributions to study conception and design were made by daw, dam, hhz, llr and mss . Substantial contributions to the analysis and interpretation of the data were made by hhz, daw, llr, dam and mss . Substantial contributions to acquisition of patient clinical study data were made by daw, lb, cbs, he, ed and wgw . All authors (daw, dam, hhz, llr, mss, lb, cbs, he, ed and wgw) had full access to the data and vouch for the accuracy and completeness of the data and analyses . The manuscript was written and approved by all of the authors, each of whom contributed to the drafts and revisions.
Extrahepatic portal vein obstruction is one of causes of portal hypertension.1 varices at sites other than the esophagogastric region have been reported more frequently in patients with portal hypertension associated with portal vein obstruction than in those with cirrhosis.2 especially in patients who have performed the hepaticojejunostomy, extrahepatic portal vein obstruction leads to the afferent loop varices around anastomosis site.3 extrahepatic portal vein obstruction after pylorus - preserving pancreatoduodenectomy (pppd) has been explained by the following: postoperative portal vein thrombosis; adhesion and inflammation around the portal vein due to lymph node dissection or anastomosis leakage; and tumor recurrences around the portal vein.4 afferent loop varices may cause recurrent and massive gastrointestinal bleeding, but the early detection of the bleeding focus is difficult.5 the treatments of varices in this area include surgical, endoscopic or angiographic therapies.46 we report 2 cases of afferent loop jejunal varix bleeding at the hepaticojejunostomy site after pppd . The second patient underwent a meso - caval shunt operation after the failure of portal vein stenting . She had undergone pppd due to stage t1n0 bile duct cancer arising as a choledochal cyst 3 years prior . Postoperative adjunctive chemo - radiation therapy was not performed . To detect the bleeding focus, gastroscopy, colonoscopy, and red blood cell (rbc)-bleeding scan were performed, but no active bleeding was found . However, computed tomography (ct) showed the progressive enlargement of the afferent loop of the jejunal varices around the hepaticojejunostomy with a portal vein total obstruction from one year prior (fig . 1). Furthermore, the superior mesenteric arterial portography showed an extrahepatic portal vein obstruction and marked cavernous transformation around the hepaticojejunostomy . With these examinations, a bleeding of the jejunal varix at the afferent loop therefore, percutaneous transhepatic portography was attempted for the diagnosis and treatment of the portal vein obstruction the passing of a guidewire through the main portal vein was performed successfully (fig . 2). Next, a 6 mm - sized balloon dilatation by a metallic stent insertion (10 mm in diameter and 6 cm in length) were performed to recanalize the obstructed extrahepatic portal vein (fig ., the pressure gradient between the proximal and distal parts of the stent was checked with 0 mmhg . Jejunal varices after the stent insertion showed a remarkable decrease pressure; no further bleeding occurred for 32 months so far (fig . 4). A 71-year - old man presented at our hospital complaining of dizziness and melena . The patient had undergone a pppd due to a stage t3n0 bile duct cancer 7 years before . Postoperative concurrent chemoradiation therapy had been performed for treatment of his condition at that time . When he came to our emergency room, his hemoglobin level was 4.4 g / dl . He has been followed suffering from recurrent bleeding at the jejunal varices around hepatojejunostomy for the 4 years prior to this visit . Extrahepatic portal vein obstruction and collaterals were detected by ct and transhepatic portography (fig . A portal vein stent insertion was attempted, but failed due to severe stenosis of the portal vein . Then, a side - to - side anastomosis of the superior mesenteric vein (smv) and the inferior vena cava (ivc) was performed to reduce the flow of hepatopetal collaterals . This meso - caval operation was technically difficult because of severe adhesions around the anastomosis site . Ivc venography and ct revealed that the meso - caval shunt was intact without obstruction, but the major portion of the portal vein flow was draining through the variceal vein (fig . Therefore, the patient was started on propranolol for treatment; the patient experienced no bleeding for 8 months so far . She had undergone pppd due to stage t1n0 bile duct cancer arising as a choledochal cyst 3 years prior . Postoperative adjunctive chemo - radiation therapy was not performed . To detect the bleeding focus, gastroscopy, colonoscopy, and red blood cell (rbc)-bleeding scan were performed, but no active bleeding was found . However, computed tomography (ct) showed the progressive enlargement of the afferent loop of the jejunal varices around the hepaticojejunostomy with a portal vein total obstruction from one year prior (fig . 1). Furthermore, the superior mesenteric arterial portography showed an extrahepatic portal vein obstruction and marked cavernous transformation around the hepaticojejunostomy . With these examinations, a bleeding of the jejunal varix at the afferent loop therefore, percutaneous transhepatic portography was attempted for the diagnosis and treatment of the portal vein obstruction the passing of a guidewire through the main portal vein was performed successfully (fig . 2). Next, a 6 mm - sized balloon dilatation by a metallic stent insertion (10 mm in diameter and 6 cm in length) were performed to recanalize the obstructed extrahepatic portal vein (fig ., the pressure gradient between the proximal and distal parts of the stent was checked with 0 mmhg . Jejunal varices after the stent insertion showed a remarkable decrease pressure; no further bleeding occurred for 32 months so far (fig . The patient had undergone a pppd due to a stage t3n0 bile duct cancer 7 years before . Postoperative concurrent chemoradiation therapy had been performed for treatment of his condition at that time . When he came to our emergency room, his hemoglobin level was 4.4 g / dl . He has been followed suffering from recurrent bleeding at the jejunal varices around hepatojejunostomy for the 4 years prior to this visit . Extrahepatic portal vein obstruction and collaterals were detected by ct and transhepatic portography (fig . A portal vein stent insertion was attempted, but failed due to severe stenosis of the portal vein . Then, a side - to - side anastomosis of the superior mesenteric vein (smv) and the inferior vena cava (ivc) was performed to reduce the flow of hepatopetal collaterals . This meso - caval operation was technically difficult because of severe adhesions around the anastomosis site . Ivc venography and ct revealed that the meso - caval shunt was intact without obstruction, but the major portion of the portal vein flow was draining through the variceal vein (fig . 6). Therefore, the patient was started on propranolol for treatment; the patient experienced no bleeding for 8 months so far . It is not uncommon for portal hypertension to induce collateral vessels in the distal esophagus and gastric fundus in order to decompress the increased portal pressure.7 in rare cases, intestinal varices are developed as new collaterals accompanying portal hypertension with extrahepatic portal obstruction, postoperatively.8 in particular, the varices at the afferent loop around the hepaticojejunostomy are induced by the extrahepatic portal obstruction, which is caused by the inflammatory changes due to the leakage of hepaticojejunostomy or lymph node dissection of the hepatic hilum.4 similar to the present cases, radical lymph node dissection around hepatic hilum can be a rare risk factor for extrahepatic portal vein obstruction . The consequences from the afferent loop varices are rupture due to engorged veins and bleeding.9 mild and intermittent bleeding can be observed on close follow - up, however, severe and frequent bleeding should be managed by the intervention or surgery.10 the diagnosis of ectopic variceal bleeding is difficult, because of its intermittent nature and small amount . Some authors recommended that enteroscopy was a useful method for detecting varices in the small bowel.5 in addition, varices in the gallbladder and the common bile duct were seen as mural filling defects on endoscopic retrograde cholangiopancreatography.11 however, the varix bleeding at the afferent loop is limited for the diagnosis with endoscopy due to angulation of the jejunojejunostomy and hardness of technical handling . Some authors reported that duplex ultrasonography demonstrated the varices with the presence of serpentine vessels and a low - velocity venous signal.12 to date, the useful methods for detecting ectopic variceal bleeding have been known as ct, rbc scan, and conventional angiography, such as superior mesenteric artery portography and percutaneous transhepatic portography.3461314 in the present case, jejunal varices were suspected by the multidetector contrast - enhanced ct angiography, which were confirmed with percutaneous transhepatic portography . After the procedure and surgery, ct was a useful and safe method to evaluate the change in collateral vessels . The treatment of jejunal varices around the hepaticojejunostomy have not been well established, but stenting has been the preferred treatment modality (table 1). The resection of jejunum containing the varices was reported.15 however, the resection was not a good modality, because it caused massive intraoperative varix bleeding and recurrent jejunal varices . Paquet et al.16 reported that a meso - caval interposition shunt was successfully performed to treat jejunal varices in the afferent loop after a billroth - ii resection and hepaticoduodenostomy, and no recurrence after 24 months . In the case 2 presented here, we performed the meso - caval shunt operation between the smv and the ivc, resulting in current control of this patient's varix bleeding . However, this shunt operation has a risk for occlusion, the possibility of encephalopathy, and operative morbidities . Therefore, chen et al.17 performed a proximal splenic - left intrahepatic portal shunt operation for extrahepatic portal vein obstruction . This procedure is considered in cases for which the adhesion is not severe and the intrahepatic portal vein and splenic vein are patent . However, the shunt operation and resection are aggressive, causing more complications than interventional approaches . Therefore, the percutaneous transhepatic balloon dilatation with stent placement for extrahepatic portal vein obstruction has been preferred in the clinical practice since harville et al.18 performed this procedure for the treatment of colon varices . Although the long - term patency of the stent has not yet been established and the rupture risk during the procedure has existed, it has been preferred because it is far less invasive and has favorable results.1418 in the case 1, the patient was treated with the insertion of a stent and showed no bleeding for 32 months . On the other hand, however, the recurrence was common and several times additional procedures were required in order to control the bleeding . At last, the transcatheter embolization was considered to occlude the jejunal vein to supply the afferent loop.1618 this procedure had the advantage of selecting the collateral vein and preserving the hepatopetal flow . However, it required a laparotomy and had the chance of relapse . In conclusion, the variceal bleeding of the afferent loop after pppd can trigger massive and life - threatening bleeding . After detecting the varix bleeding using ct and conventional angiography, the treatment of varices in this area is recommended with less invasive approach, such as the angiographic interventions of balloon dilatation and stent insertion . Surgical shunt operation can be considered when noninvasive approaches have failed.
Parkinson s disease (pd) is an age - related degenerative disorder of the central nervous system . It is often characterized by muscle rigidity, tremor, slowing of physical movement (bradykinesia) and, in extreme cases, loss of physical movement (akinesia). The symptoms of pd are attributed to the loss of pigmented dopamine - secreting (dopaminergic) neurons in the pars compacta region of the substantia nigra (sn) and a subsequent striatal deficiency of dopamine . Idiopathic pd is also pathologically characterized by the presence of cytoplasmic neuronal inclusions, called lewy bodies, in the affected region of the brain . The etiology of the disease has been poorly understood, and development of novel non - dopaminergic therapeutic strategies has remmained challenging since initial description of the disease by james parkinson in 1817 . Although pharmacological dopamine replacement strategies provide temporary symptomatic relief, there are at present no therapeutic methods for halting progressive neuronal cell loss . Mptp neurotoxicity develops only after metabolization to mpdp+ by mao - b in glial cells and then to mpp+, the active toxic compound . Animal models are an important tool in elucidating mechanisms involved in the pathological process and in investigating new therapeutic strategies for pd . Currently, both genetic and toxic models of pd are available, but use of neurotoxins, such as 6-hydroxydopamine (6-ohda), paraquat, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (mptp) and rotenone, is still the most popular means of modeling destruction of the nigrostriatal dopaminergic neurons seen in pd . Among these neurotoxins, 6-ohda and mptp have been more extensively used by investigators to produce pd models despite their limitations . The neurotoxin 6-ohda is a structural analogue of catecholamines, dopamine and noradrenaline, and exerts its toxic effects on catecholaminergic neurons . Since 6-ohda does not penetrate the blood brain barrier in adult rats, it must be administrated stereotactically into the substantia nigra or striatum to damage the dopaminergic nigrostriatal system . Recognition of mptp as a neurotoxin occurred early in 1982, when several young drug addicts mysteriously developed a profound parkinsonian syndrome after intravenous use of street preparations of meperidine analogs that, unknown to anyone, were contaminated with mptp, an incidental byproduct during the chemical synthesis of a meperidine analog (fig . It is also well known that mptp depletes striatal dopamine and causes damage to the substantia nigra pars compacta (snpc) dopaminergic neurons in non - human primates and several species of rodents . Recently, a great deal of interest has been focused on stem cell therapies for pd . In addition to fetal nigral transplantation, the capability of self - repair in the central nervous system (cns) in the adult mammalian has also given a new perspective in the cell - based approach for treatment of neurodegenerative disorders . In pd or its animal models, much attention has been attracted to whether the depletion of dopaminergic neurons triggers activation of neural stem cells and their subsequent migration into the damaged area, finally resulting in repopulation of dopaminergic neurons in the sn . This review will provide the body of evidence available for endogenous neurogenesis in response to neurotoxin damage in animal models of pd . Schematic representations of the migratory pattern of newly generated a cells in the svz (up) and the cell populations in the sgz of the dg (bottom left) and svz (bottom right) in the adult rodent brain (adapted from ref . Neurogenesis continues into adult life in the brains of rodents, nonhuman primates, and humans . It is confined largely to two discrete areas, the subventricular zone (svz) and the subgranular zone (sgz) of dentate gyrus (dg) (fig . The svz, located throughout the inner wall of the lateral ventricle, is the largest germinal region and harbors neural stem cells that retain the capacity to generate multiple cell types . The svz is composed of neural progenitor cells (migrating neuroblasts, a cells), neural stem cells (astrocytes, b cells), neural precursor cells (rapidly dividing transit amplifying cells, c cells) and ependymal cells (fig . 2). Neural stem b cells divide to give rise to clusters of precursor c cells, which in turn generate neuroblast a cells . Newly generated a cells in the svz migrate through a network of tangential pathways in the lateral wall of the lateral ventricle and then converge onto the rostral migratory stream (rms) to enter the olfactory bulb (ob) via a chain migration behavior, in which they differentiate into granule cells and interneurons (fig . It has been suggested that prokineticin 2 (pk2) serves as a chemoattractant for svz - derived neuroblast a cells, which appears to guide the migration of a cells from the svz through the rms to their final layers in the ob . The multipotential a cells residing in the svz and rms are eliminated through apoptosis to maintain a balance for proper development of the mammalian nervous system . Metalloproteinases (mmps) may also play a crucial role in the migration of individual a cells since their migration rates are reduced by the presence of inhibitors of mmps . The architecture and function of the adult human svz differs significantly from that described in other mammals . There are four layers of varying thicknesses and cell densities throughout the lateral ventricular wall: a monolayer of ependymal cells (layer i), a hypocellular gap (layer ii), a ribbon of cells (layer iii) composed of astrocytes and a transitional zone (layer iv) into the brain parenchyma . Neuronal progenitors in the adult mammalian hippocampal dg reside in the sgz, which is located in the hilus immediately beneath the granular layer of the dg . Neurogenesis in the dg was first demonstrated 40 years ago by autoradiography in rodents and thereafter was further demonstrated in all mammalian species including human and nonhuman primates . Two types of neural progenitors can be identified in the sgz according to their specific morphologies and expression of unique sets of molecular markers (fig.2). The primary progenitors (type-1 cells) have the appearance of radial glia, which also express glial fibrillary acidic protein (gfap). They share similar features with type the b cells residing in the adult svz and are suggested to be a putative stem cell population . Type-2 cells (intermediate progenitors) are gfap - negative and highly proliferative, sharing similar features with the neuroblast a cells residing in the adult svz . Type-2 cells may arise from type 1 cells, but direct evidence delineating this lineage relationship is still lacking . Newborn type-2 cells disperse and migrate a short distance into the granule cell layer where they differentiate, extend axons and express neuronal marker proteins . The migration of newborn neurons in the dentate gyrus may also be controlled by guidance cues, as these cells only migrate to the hilus or molecular layer under pathological conditions, such as in animal models of temporal lobe epilepsy . Dopamine plays important roles in many physiological functions, including motor control, mood and the reward pathway, and as a neurotransmitter, it binds to the five types of dopamine receptor, d1, d2, d3, d4 and d5, and their variants . The d1 and d5 receptors are members of the d1-like family (d1l), whereas the d2, d3 and d4 receptors are members of the d2-like family (d2l). It is known that both pd and neurotoxin - induced pd animal models are mainly characterized by a hallmark of dopamine depletion . Neurogenesis is a key event during both physiological and pathological processes and is regulated by a variety of stimuli, such as hormones, intrinsic growth factors, neurotransmitters, exogenously applied agents, environmental factors, exercise and age . The activation of dopamine receptors influences cell proliferation in the lateral ganglionic eminence (lge) and the neuroepithelium of the frontal cortex in embryonic mice . Dopamine has attracted a lot of attention concerning the role it plays in adult neurogenesis in recent years . In pd animal models, the first thing that needs to be clarified is whether decreased / increased neurogenesis is associated with the dopamine - involved signaling pathway or is a response to destruction of the nigrostriatal system . By using immunohistochemical and ultrastrctural analyses, hglinger et al . Have provided anatomical evidence that d2l receptors in the svz are expressed predominately on c cells, whereas a cells express both d1l and d2l receptors in the mouse brain . Dopaminergic fiber has also been confirmed to contact precursor cells in the svzs of adult humans and primates . Thus, dopamine seems to play a key role in the regulation of adult mammalian neurogenesis . Findings from in vitro experiments show that activation of the d2l receptor directly stimulates proliferation of svz precursor cells . This is supported by a recent study, in which stimulation of dopamine d2 receptors increased the proliferation of neural progenitor cells both in vivo and in vitro . The depletion of nigrostriatal dopamine reduces precursor cell production in the svzs of mice and aged primates and further leads to impaired neurogenesis in the ob . Based on the finding that acute administration of sch23390, a d1 receptor antagonist, reduces the number of 5-bromodeoxyuridine (brdu)-positive cells in the sgz of the dg, suzuki et al . Suggest that adult neurogenesis in the dg may be regulated naturally by dopamine via d1-like receptors . A very recent report demonstrated that dopamine regulates adult neurogenesis by a mechanism of modulating ciliary neurotrophic factor (cntf) expression in svz astrocytes . The findings of van kampen et al . Indicate that administration of the dopamine d3 receptor agonist, 7-hydroxy - n, n - di - n - propyl-2-aminotetralin (7-oh - dpat), significantly increases the proliferation of neural stem cells (nscs) in the adult rat brain but not in the adult mouse brain . Furthermore, chronic intraventricular administration of 7-oh - dpat triggers a profound induction of cell proliferation in the rat sn and promotes adoption of a neuronal phenotype in some of these newly generated cells . On the other hand, it has been suggested that treatment with 7-oh - dpat does not affect the proliferation, survival or neurogenesis of murine and human neural progenitor cells derived from the fetal midbrain in vitro . Moreover, chronic treatment with the antipsychotic drug haloperidol leads to increased nsc numbers, resulting in more progenitors and more new neurons and glia in the adult rat brain due to a mechanism of antagonizing dopamine at the d2 receptors on nscs . Mptp neurotoxicity develops only after metabolization to 1-methyl-4-phenyl-2,3-dihydropyridinium (mpdp+) by an enzyme, monoamine oxidase (mao)-b, and further to 1-methyl-4-phenylpyridinium (mpp+), the active toxic compound (fig . Since mpp+ is selectively transported into presynaptic dopaminergic nerve terminals through the dopamine transporter (dat) and the absence of the dat on dopaminergic neurons confers complete protection against mptp toxicity, the neurotoxicity of mptp is suggested to be selective to dopaminergic neurons in the sn . Mptp is mainly used in non - human primates and rodents, and the latter are less sensitive to mptp neurotoxicity . In rodents, c57bl/6 mice have been used in a great deal of reports for creation of a pd animal model for understanding the disease pathogenesis, despite their limitions . First provided morphological evidence that administration of mptp leads to a 2-fold increase of brdu incorporation in nigral dopaminergic neurons . Newly generated dopaminergic neurons has been demonstrated to be derived from the cells lining the ventricular system . By using the nestin second intron enhancer - controlled lacz reporter transgenic mouse model coupled with the mptp lesion system, shan et al . Demonstrated that there is increased dopaminergic neurogenesis in the sn, supporting the findings of zhao et al . As described above . In rat and macaque monkey hemi - parkinsonian models treated stereotatically with mpp+, the dopamine - depleted hemisphere showed more polysialic acid (psa)-positive cells, candidates for newly differentiated young neurons, than the intact side, and a small number of tyrosine hydroxylase (th)-positive cells were demonstrated to be psa - positive . In the striatum of the mptp - treated aged macaque, although there is an increase in the number of th - immunoreactive neurons, the increase has been suggested to be derived from pre - existing gabaergic interneurons (phenotype shift), not neurogenesis . Performed a retroviral vector - based method to evaluate neurogenesis in the obs of mptp - treated mice, and the results showed that dopaminergic neurogenesis can be enhanced in the ob after dopaminergic neuron loss . Increased neurogenesis has also been confirmed in a very recent paper, in which mptp lesions increased the incorporation of brdu as well as the number of cells that co - expressed brdu and the immature neuronal marker doublecortin (dcx) in the dg, svz and striatum, but not in the sn of the mptp - treated mouse, although the differentiation of newly generated cells into dopaminergic neurons was not investigated . On the other hand, demonstrated that proliferation of transit amplifying cells (c cells) is impaired in the svz and sgz in the mptp mouse model for pd . C cells are the target of a dopaminergic innervations, and impaired neurogenesis in the svz is thus suggested to be mediated by mptp administration - induced dopamine depletion . Furthermore, reduced c cells lead to a subsequent decrease in a cells . In adult macaques, mptp - induced dopamine depletion also results in decreases in the number of proliferating cell nuclear antigen - positive (pcna) cells and a cells in the svz, suggesting that intact dopaminergic nigro - subventricular innervation is crucial for sustained neurogenesis in aged primates . The majority of brdu - positive cells in the svz are rapidly depleted at 2 days, and only few brdu - positive cells migrate into the ob at 7 days after mptp administration . In recent years, the selective neurotoxicity of mptp in the dopaminergic system of the adult brain has been challenged . A number of studies have demonstrated that mptp also destroys forebrain migrating neuroblasts and nigrostriatal glial cells (astrocytes) in the adult mouse brain . In our previous studies, the number of apoptotic cells in the svz and rms peaked at 24 hours after mptp injections and decreased thereafter, paralleling the changes in number of cleaved caspase-3-positive cells . The cells undergoing apoptosis in the svz, rms and ob were identified as a cells using immunohistochemistry and ultrastructural analyses, while a few were astrocytes (b cells), and none were transit - amplifying procursors (c cells). The decrease in a cell numbers was most marked on day 2 and lasted to day 8 after administration . We also demonstrated that mao - b inhibitors, such as deprenyl or n-(2-aminoethyl)-4-chlorobenzamide (ro 16 - 6491) completely protected a cells against mptp neurotoxicity, suggesting that mptp neurotoxicity in a cells is also mediated by the conversion of mptp into mpp+ by mao - b . A cells are main populations in the svz and rms of the adult brain, and proliferation of a cells may contribute to dult endogenous neurogenesis . Using brdu labeling, we provided evidence that mptp injury leads to transiently impaired neurogenesis in the adult mouse svz and ob . The majority of brdu - positive cells in the svz are rapidly depleted by mptp, and only a few brdu - positive cells migrate into the ob thereafter (fig . 3). Thus, impaired neurogenesis is more likely to be due to an mptp - induced decrease in a cells rather than a mechanism through dopamine depletion as reported in previous studies . Impaired neurogenesis persists for about two weeks in the mptp - injured mouse brain and is followed by recruitment of cells in the svz (he et al ., unpublished data). The self - repairing process for the damaged germinal region is evidenced by regeneration of a cells (he et al ., unpublished data). Thus, neurogenesis in the nigrostriatal system in response to mptp damage, if it does occur, may be a later event in the self - repairing process of the injured svz . In a recent study, increases in brdu - positive cells and brdu - dcx double - labeled cells in the svz were observed 14 d after acute mptp treatment . This, however, may have been due to an activated self - repairing process in the svz rather than a response to nigrostriatal damage . Since 6-ohda does not penetrate the blood brain barrier in adults, it must be administrated stereotactically into the substantia nigra or striatum to damage the dopaminergic nigrostriatal system . Although administration of 6-ohda produces a decreased striatal dopamine level and loss of dopaminergic neurons in the sn in the adult brain, the 6-ohda model does not mimic any clinical or pathological features of pd . In adult rats, when the right mesencephalic dopaminergic neurons are ablated by stereotactical injection of 6-ohda into the right nigrostriatal pathway, the number of pcna - positive cells in the svz ipsilateral to the lesion decreases . Impaired proliferation of cells in the svz has also been suggested to be mediated by a mechanism of dopamine depletion as suggested in mptp animals . Evidence from c57bl/6 mice injected unilaterally with 6-ohda into the midbrain part of the medial forebrain bundle also show impaired neurogenesis, and 6-ohda - induced dopaminergic denervation of the striatum reduces cntf mrna levels in the svz, which further contributes to negative regulation of the proliferation of cells . Although the proliferation of nscs in the svz and dg in the adult brain is an important neurogenesis process and has been demonstrated in some neurodegenerative diseases, it remains controversial whether those proliferated nscs migrate into the damaged area and repopulate the lost functional neurons . Treatment of 6-ohda also leads to a transient decrease in neurogenesis in the olfactory granule cell layer, but, in contrast, an increase of neurogenesis is present in the glomerular layer . The increased neurogenesis in the glomerular layer is characterized by more newly generated neuronal nuclear antigen (neun)- and th - expressing neurons, indicating a shift in cell fate decision for newly generated neuronal precursors targeted at the glomerular layer . Detected increases in cell proliferation in the sn in rats injected with 6-ohda into the median forebrain bundle; however, none of the newly born cells expressed a dopaminergic phenotype, and there is no evidence of neural stem cells emanating from the cerebroventricular system and migrating to the substantia nigra . Growth factors, such as platelet - derived growth factor (pdgf - bb) and brain - derived neurotrophic factor (bdnf), can induce striatal neurogenesis in adult rats with 6-ohda lesions, and there are no indications of any newly born cells differentiating into dopaminergic neurons following growth factor treatment both in the striatum and in the sn . Surprisingly, in a salamander 6-ohda model for pd, robust and complete regeneration of the mesencephalic and diencephalic dopamine system occurs after elimination of dopaminergic neurons . This 6-ohda salamander model gives new insight into the animal models for pd and may be helpful for understanding the molecular mechanism of dopaminergic neurogenesis . The findings of steiner et al . Indicate that the absolute numbers of newborn cells in the sn are not affected by dopamine depletion in the 6-ohda rat model for pd . Instead, 6-ohda lesions induce a specific downregulation of generation of newborn nigral astrocytic cells . The replacement of lost neurons in a damaged brain area is the best direction for therapeutic development in relation to neurodegenerative diseases . Based on all of the available evidence, neurogenesis is still a most controversial issue in studies of neurodegenerative diseases and their animal models . In mptp animal models for pd, we have demonstrated that mptp destroys a cells in the svz and ob in the adult brain and then transiently impairs neurogenesis; however, it remains to be investigated whether and how multiple potential neural stem cells migrate into the nigrostriatal system and replace the lost dopaminergic neurons . With increased attention to dopaminergic neurogenesis in neurotoxin - induced animal models for pd, it is expected that the models will become useful as tools for understanding the mechanisms for neurogenesis in pd and that many questions regarding neurogenesis in neurodegenerative diseases will be resolved.
Teaching physicians effectively about low probability, high consequence medical conditions, such as anaphylaxis, is challenging . Medical education curricula emphasize more common high stakes conditions (e.g., stroke) where misdiagnosis or mismanagement leads to poor outcomes . Physicians may lack opportunities to gain firsthand clinical experience or to reinforce their limited learning of infrequent conditions . Clinicians face several challenges when dealing with anaphylaxis, a potentially life - threatening allergic reaction requiring immediate identification and treatment . First, there are no universally accepted diagnostic criteria for anaphylaxis [1, 2]. A comprehensive clinical definition of anaphylaxis from an nih expert panel has not achieved widespread acceptance among physicians despite high reported sensitivity and negative predictive value [1, 3, 4]. Physicians may overlook the diagnosis because the clinical presentation may vary among patients, even in the same patient with a history of multiple episodes [1, 5]. Third, physicians may not consider anaphylaxis when patients do not present stereotypically (e.g., laryngeal edema after bee sting). Furthermore, the usual probability - based methods of clinical reasoning and decision - making are difficult to apply to anaphylaxis [6, 7]. Estimates of anaphylaxis prevalence and incidence are unclear due to the lack of symptom recognition, poor physician awareness of diagnostic criteria [1, 2], and paucity of robust, validated methods for identifying anaphylaxis diagnoses using currently available administrative claims [8, 9]. Thus, it is unsurprising that medical personnel underrecognize and undertreat anaphylaxis [1, 2]. Anaphylaxis diagnosis and management (adam) by physicians need improvement, regardless of the stage of training [1022]. Despite the establishment and dissemination of treatment guidelines, medical providers consistently underutilize or incorrectly administer and dose epinephrine, which is accepted as first - line treatment [1027]. Instead of prompt epinephrine administration, practitioners continue to utilize second - line agents, such as antihistamines and glucocorticoids, contrary to evidence - based recommendations [1029]. Allergists are particularly well suited to address this gap, as evidenced by the few published studies reporting successful allergist - led interventions [3436]. We developed, implemented, and evaluated an educational program consisting of face - to - face didactic session and hands - on training conducted by allergy trainees or attending physicians in the proper use of epinephrine autoinjectors . We hypothesized that this allergist - led intervention would improve residents' knowledge, competence, and perceived confidence in anaphylaxis diagnosis and management . Allergists and likely other nonallergist providers can adapt our simple and resource nonintensive intervention in a variety of settings to educate other providers about evidence - based adam . This longitudinal study examined full - time resident physicians at all training levels who were enrolled in an accreditation council for graduate medical education, accredited training program in internal medicine (n = 204), pediatrics (n = 153), or emergency medicine (n = 40), from july 2010 to june 2013, at tertiary care university hospitals in two health systems . Residents were recruited during an hour - long educational conference in which they received an explanatory letter about the study and asked to participate . To maximize participation and account for differences in academic schedules, the institutional review boards at both institutions approved this study and waived the need to obtain written informed consent from participants . At the recruitment session, residents completed an anonymous questionnaire which queried participants' demographics, prior clinical experience, perceived competency, and comfort with adam, as well as a 10-item multiple choice pretest that assessed baseline knowledge of anaphylaxis . Attending physicians (artemio m. jongco and susan j. schuval) and/or trainees (sheila bina and robert j. sporter) from the division of allergy and immunology from the respective institutions presented a 45-minute evidence - based didactic lecture using powerpoint (microsoft, redman, wa), followed by hands - on practice with needleless epinephrine autoinjector trainers (epipen trainer). Study personnel observed participants' technique, provided constructive criticism when appropriate, and answered participants' questions related to the educational content or autoinjector use . Immediately following the presentation approximately 12 weeks later, during another nonanaphylaxis conference, residents completed a similar 10-item follow - up quiz and questionnaire . To foster a safe and nonpunitive learning environment, no identifying information was collected from the participants, nor were identifiers recorded on quizzes or questionnaires . Hence, linking individual's responses at different time points or connecting an individual's performance to his / her identity was not possible . Examples of the questionnaires and quizzes are provided in supplementary material available online at http://dx.doi.org/10.1155/2016/9040319 . The authors developed the quizzes, consisting of clinical scenarios that evaluated knowledge of evidence - based anaphylaxis diagnosis and management . To ensure that quiz questions were roughly equivalent in complexity, the scientific content was identical from one quiz to another, with minor modifications (e.g., clinical parameters, order of questions, and answer choices)., the authors reviewed the scientific content of the educational intervention, quizzes, and answers . The authors pilot - tested the quiz questions on a small group of rotating residents in the division of allergy & immunology at hofstra northwell school of medicine . Achieving 8 correct answers on the quiz was considered to be the minimum level of competence for medical knowledge . Graphs were generated using prism 6 (graphpad software, san diego, ca). The results of the descriptive analysis were shown as mean standard deviation with 95% confidence intervals and as percentages . The chi - square test was used to measure the association between the categorical variables, and wilcoxon rank sum test or kruskal - wallis test was used to compare the groups on the continuous variables . The two academic health centers employed a total of 397 residents that were eligible to participate (204 internal medicine, 153 pediatrics, and 40 emergency medicine residents). A total of 159 residents participated in the pretest (response rate of 40.05%). One hundred and fifty - two residents of the original 159 (95.60%) completed the posttest, and 86 residents (54.09%) were available for the follow - up test . Since chi - squared analysis failed to reveal a significant difference by site (p = 0.86) or by specialty (p = 0.95) over time, data were combined in subsequent analyses . Chi - squared analysis revealed that the proportion of participants having demonstrated epinephrine autoinjector use increased from baseline to follow - up (p = 0.006). The proportion of participants who had diagnosed (p = 0.06) or managed (p = 0.06) anaphylaxis or used an epinephrine autoinjector (p = 0.08) in the past did not differ significantly from baseline to follow - up . Of the participants who reported having managed anaphylaxis prior to the study, the most common venue was the emergency department (42.35%), followed by general ward (20.59%), intensive care unit (24.71%), and then allergy office (2.35%) (data not shown). Also, the proportion of participants who self - reported being confident in their ability to diagnose (p <0.001) or manage (p <0.001) anaphylaxis increased from baseline to follow - up . Table 3 summarizes participants' self - reported behaviors and attitudes at follow - up . During the interval since the intervention, despite increased self - reported confidence in adam, residents appear to have had few opportunities to utilize what they have learned about anaphylaxis, or to refer patients with anaphylaxis to allergists . The mean pretest score of 7.31 1.50 (95% ci 7.087.54) was lower than the posttest score of 8.79 1.29 (95% ci 8.589.00) and follow - up score of 8.17 1.72 (95% ci 7.808.54). Figure 1(b) shows that the distribution of pretest scores is significantly lower than posttest scores and follow - up scores (p <0.001 for both). The distribution of follow - up scores is significantly lower than that of posttest scores (p = 0.0086). These results were significant even after bonferroni adjustment was made for multiple comparisons (= 0.017). Furthermore, our intervention appears to have helped the participants achieve the medical knowledge competency threshold of 8 correct answers . Quiz scores at the three time points did not significantly differ by specialty (p = 0.59) or by training level (p = 0.62). Moreover, the quiz scores did not vary according to their self - reported experience of past anaphylaxis diagnosis (p = 0.10), management (p = 0.09), past use (p = 0.49), demonstration of epinephrine autoinjector (p = 0.16), or past referral to allergist (p = 0.37). However, quiz scores did significantly differ depending on residents' reported confidence in the ability to diagnose (p = 0.01) or manage (p = 0.004) anaphylaxis . In this study, we demonstrate that allergist - led didactic lectures and hands - on practice with epinephrine autoinjectors are effective educational interventions that enhance short - term resident knowledge of evidence - based adam . These findings corroborate the literature which shows that the continuing opportunities to apply knowledge and to practice skills are essential to maintain knowledge and competency [34, 35]. Indeed, the majority of participants reported having limited opportunity to apply or utilize their new knowledge or skills in the 12-week interval between intervention and follow - up . We suspect that resident performance would have continued to decline in the absence of educational reinforcement if reevaluated after 12 weeks . There are several findings, which failed to reach statistical significance, that further underscore the importance of continuing medical education and ongoing opportunities to practice clinical skills in order to maintain adam proficiency . There were trends to suggest an increase in the proportion of participants who had diagnosed (p = 0.06) or managed (p = 0.06) anaphylaxis or used an epinephrine autoinjector (p = 0.08) at follow - up compared to baseline . Moreover, past anaphylaxis diagnosis (p = 0.10) and management (p = 0.09) are likely covariates of quiz score . Further research is needed to identify the optimal frequency and modality of continuing medical education that will result in maximal retention of knowledge and competency . Interestingly, study participants reported high levels of confidence in diagnosing or managing anaphylaxis at baseline and follow - up, despite limited clinical experience . In fact, the levels of self - reported confidence increased from baseline to follow - up . This observed disconnect between physician self - assessment and objective measures of competence is unsurprising since physicians have a limited ability for self - assessment . It may be beneficial to help physicians at all training levels to become more cognizant of this disconnect . Moreover, training programs should consider restructuring current educational endeavors to include increased allocation of time and resources for educating trainees about low probability, high consequence conditions like anaphylaxis, since simple, non - resource - intensive interventions, such as the one described in this paper, can lead to measurable improvements in resident knowledge, and possibly clinical competence . We acknowledge that our medical knowledge competency threshold score of 8 is somewhat arbitrary and may not necessarily reflect clinical competence . Clinical competence, which relies upon a foundation of basic clinical skills, scientific knowledge, and moral development, includes a cognitive function (i.e., acquiring and using knowledge to solve problems); an integrative function (i.e., using biomedical and psychosocial data in clinical reasoning); a relational function (i.e., communicating effectively with patients and colleagues); and an affective / moral function (i.e., the willingness, patience, and emotional awareness to use these skills judiciously and humanely). Although evaluating medical knowledge through a quiz represents an incomplete assessment of clinical competence at best, it is still reasonable to hypothesize that medical knowledge correlates with clinical competence to some degree and that lower levels of medical knowledge may negatively impact the quality and efficacy of care delivered by the provider . Thus, the observation that the follow - up quiz scores trended back down towards baseline is worrisome for a possible concomitant decline in the quality of care delivered by the residents . Whether the residents demonstrated any change in their clinical practice after the intervention is unknown and outside the scope of the current study . More research is needed to determine the effect of educational interventions such as this in real - life clinical practice . First, only 40% of all eligible participants were included in the study, and only about half of these participated in the 12-week follow - up evaluation . This is likely due to scheduling difficulties and competing demands on resident time, although we cannot exclude the possibility of participation bias . Notably, our participation rate is similar to other studies of physicians and residents [3941]. Second, since we did not collect identifying information, we could not ensure that recruited participants completed the entire study, track individual performance over time, or give participants personalized feedback on their quiz performance . Third, extensively validated quiz questions (e.g., questions from previous certification examinations) were not used due to the lack of access . The content of each question on the quizzes was directly linked to each one of our evidence - based learning goals, thus serving as a measure of face validity . Further, there was consensus among the board certified allergists / content experts who developed, verified, and honed the quiz questions, thereby providing us with a measure of content validity . Finally, because the quizzes were utilized at more than one site, in more than one clinical department, and on a modest sample size, we believe that the generalizability of the instrument was attained to a respectable degree . Finally, this study only utilized traditional educational modalities of didactic lecture and hands - on practice . More research is needed to evaluate the efficacy of various educational interventions, especially with regard to long - term knowledge retention, improved performance on objective measures of clinical competence, and actual patient outcomes . Simulation - based education may hold promise in this regard [3032, 35]. Also, the larger sample size of this study and the longer follow - up interval distinguish this study from other published studies of educational interventions . Furthermore, since the intervention is relatively simple and not resource - intensive, it can be adapted and implemented in a variety of educational settings . Physicians, regardless of the stage of training, underdiagnose and undertreat anaphylaxis . Teaching providers about evidence - based adam is challenging . The allergist - led face - to - face educational intervention described above improves residents' short - term knowledge, competence, and perceived confidence in adam . Lack of clinical experience and/or educational reinforcement may contribute to knowledge and competence decline over time . Thus, continuing medical education, coupled with ongoing opportunities to apply knowledge and practice skills, is necessary . Innovative educational interventions are needed to improve and maintain resident knowledge and clinical competence regarding evidence - based adam . More research is also needed to determine the impact of such interventions on patient outcomes.
Sexually transmitted infections (stis), including human immunodeficiency virus (hiv), continue to present major health, social, and economic problem in the developing world, leading to considerable morbidity, mortality, and stigma . Unprotected sex with an infected partner is by far the most important risk factor for sti / hiv infection . They show various trends in different parts of the country and constitute a major public health problem for both developing and developed countries . Stis increases the risk of transmission of hiv infection causing immense need to understand the patterns of stis prevailing in the regions of a country for proper planning and implementation of sti control strategies . Due to the lack of adequate laboratory infrastructure in the country, information regarding the profile of stis relies essentially on syndromic diagnosis . The availability of baseline information on the epidemiology of stis and other associated risk behaviors remain essential for the designing, implementing, and monitoring successful targeted interventions . To study the pattern of common stis and prevalence of hiv infection in patients attending the sti clinic of a tertiary care hospital in northern part of india using a syndromic approach . To study the pattern of common stis and prevalence of hiv infection in patients attending the sti clinic of a tertiary care hospital in northern part of india using a syndromic approach . A retrospective analysis of data collected from the clinical records of 2700 patients over a period of 21 months (july 2012 to march 2014) was carried out . The data were collected from individuals attending the sti clinic at the skin and vd department of sms hospital, jaipur . Detailed history, demographical data, and clinical features were recorded from all the patients . All patients were tested for hiv by elisa / rapid tests as recommended by the national aids control organization (naco). Stis were categorized in different syndromes as depicted by naco in the syndromic management of stis . The syndromes depicted by naco were urethral discharge, vaginal discharge, genital ulcer disease herpetic and nonherpetic (gud - h and gud - nh), inguinal bubo, lower abdominal pain, scrotal swelling, etc . Stis, which were not included in the syndromic management such as molluscum contagiosum, condyloma acuminata, and balanoposthitis were detected clinically . In the study population, 78% (2108/2700) were males, 22% (592/2700) were females with male to female ratio being 3.5:1 . The majority of patients attending sti clinic belonged to the age group 25 - 44 years 57.47% (1552/2701), followed by the 20 - 24 years 20.7% (559/2700),> 44 years 15.84% (428/2700), and <20 years 5.92% (161/2700) [figure 1]. The overall most common sti was balanoposthitis (39.62%) followed by genital herpes (17.5%), vaginal / cervical discharge (13.4%), genital molluscum (11.74%), genital warts (10.77%), gud - nh (4.59%), lower abdominal pain (2.66%), and urethral discharge (2.55%) in decreasing order . Balanoposthitis = bp; genital ulcer disease herpetic = gud - h; vaginal/ cervical discharge = vd / cd; anogenital warts = ca; molluscum contagiosum = mc; genital ulcer disease nonherpetic = gud - nh; lower abdominal pain = lap; urethral discharge = ud; scrotal swelling = ss; inguinal bubo = ib among the male patients, balanoposthitis 50.75% (1070/2108) was the most common sti, followed by gud - h 19.87% (419/2108), condyloma acuminata 12.28% (259/2108), and molluscum contagiosum 9% (190/2108). In the females, the most common sti was combination of cervical and vaginal discharge 61.04% (362/593) followed by molluscum contagiosum 20.2% (120/593), gud - h 9.1% (54/593), and condyloma acuminata 5.3% (32/593). Of the 4.59% (123/2700) patients of gud - nh, 62.6% (77/123) patients were rapid plasma reagin (rpr) positive with 56 males and 21 female patients . Among the study population, 2.55% (69/2700) were found to be hiv - positive, in which 84% (58/69) were males and 16% (11/69) were females [figures 3 and 4]. Pie chart showing prevalence of hiv positivity among the sti patients pie chart showing male and female distribution among hiv positivity patients the pattern of stds differs from country to country and from region to region, especially in large countries such as india . They are responsible for a significant proportion of infertility in both sex, morbidity, economic loss to the family, and increased susceptibility to hiv infection . Sti are a major contributor to fetal deaths, abortions, and the delivery of low birth weight babies . Early diagnosis and appropriate treatment will definitely curb the transmission of hiv / aids . To achieve this, it is an approach where the health care providers diagnose and treat patients on the basis of signs and symptoms (syndrome) rather than specific stis . In the present study, males accounted for 78% cases, females were 22% (m: f = 3.5:1), which was almost similar to the north eastern study where males constituted 75.45% and females constituted 24.24% (m: f = 3.09:1). The most common sti was balanoposthitis (39.62%) majority of which were fungal in origin, followed by genital herpes (17.5%), vaginal / cervical discharge (13.4%), genital molluscum (11.74%), genital warts (10.77%), gud - nh (4.59%), lower abdominal pain (2.66%), and urethral discharge (2.55%) in decreasing order . This was in contrast to the north eastern study and study conducted at medical college trivandrum . In the eastern study, gud - h (38.1%) was the most common, followed by vaginal / cervical discharge (18.6%), urethral discharge (13.8%), and molluscum contagiosum (4.7%) while in study at trivandrum, the commonest std was syphilis, followed by herpes genitalis and condyloma acuminata . Our data compared well with a previous study at a regional std center in new delhi were changing trends of the profile of stis and hiv seropositivity over a 15-year period were analyzed . The sti profile and hiv seropositivity were compared between 1990 and 1993, 1994 - 1997, 1998 - 2001, and 2002 - 2004 . Similar to our study, that is, the viral stis such as molluscum contagiosum, gud - h, condyloma acuminata, etc ., were much more prominent than the bacterial stis such as urethral discharge and gud - nh which were more prominent in our institution 10 years back . In the present study, the prevalence of hiv among sti patients was 2.55% (males - 84%, females - 16%) with male to female ratio being 3.5:1 which was in concordance with the epidemiological analysis of reported aids cases where disease is common in males than females, ratio being 3:1 . And with the national average (2.5%) as per recent naco estimates . There was a wide variation for seropositivity for hiv among sti patients, 8.21% in zamzachin et al, study, 9.62% in jaiswal et al, study, 17.2% in saikia et al, study, and 10.3% in choudhry et al, study . This difference could be attributed to the high prevalence of hiv infection and intravenous drug abuse in the north - eastern part of india . Rpr reactivity was seen in 12.8% of the total ulcerative sti patients, which is contrary to the reports of vora et al, and mewada et al, where the incidence of vdrl reactivity was 19.41% and 53.3%, respectively . Hiv and stis are perfect examples of epidemiologic synergy as they are core transmitters of each other . The presence of multiple stis is a risk factor for increased rate of transmission of hiv . This can be controlled by promoting the strategies to reduce high - risk behavior, encouraging condom use, strengthening sti clinics and family health awareness programs, and imparting sex education and awareness regarding sti / hiv among the masses and vulnerable population . Our study showed that the most common presenting complaint of the patients was balanoposthitis (39.62%) followed by genital herpes (17.5%), vaginal / cervical discharge (13.4%), genital molluscum (11.74%), genital warts (10.77%), gud - nh (4.59%), lower abdominal pain (2.66%), and urethral discharge (2.55%) in decreasing order . Viral stis such as molluscum contagiosum, herpes genitalis, and condylomata acuminata are on the rise among sti / rti clinic attendees due to the occurrence of asymptomatic shedding, partial treatment or modified course of the bacterial stds, thereby leading to apparent reduction in the total number of cases of stds attending std clinics, as well as a decrease in the proportion of bacterial to viral stds.
Acromegaly is serious endocrinological derangement which, left untreated, reduces life expectancy and results in physiological derangements and complications that may negatively affect a patient's quality of life . Those patients with residual or recurrent disease are often treated with medication to decrease growth hormone secretion or block its action on peripheral tissues . These treatments are not universally effective for patients and are sometimes contraindicated . As an adjunct treatment, and sometimes as an alternative treatment, radiosurgery has proven to be an attractive therapy . It is noninvasive, has few side effects, and is available in many centers internationally [13]. In the current paper, we first describe the pathophysiology of acromegaly, the existing surgical and medical treatments, and then introduce radiosurgical methods . In particular, we will focus on gamma knife radiosurgery (gks) since it has a broader base of supporting literature than alternative forms of stereotactic radiosurgery . Finally, the overall efficacy of gks is described, along with its reported morbidities . Acromegaly is a syndrome caused by elevated levels of circulating growth hormone (gh). The most common cause of the disorder, accounting for about 98% of cases, is a gh - secreting pituitary adenoma . Rare nonpituitary causes of acromegaly include diverse entities such as hypothalamic hamartomas, small - cell lung cancers, pheochromocytomas, and bronchial carcinoids . Among gh - secreting pituitary adenomas, roughly 60% are pure gh - secreting somatotrope adenomas, while the remainder are mixed mammosomatotropes, which secrete both gh and prolactin (prl) and sometimes thyroid - stimulating hormone (tsh). The symptoms of increased gh are mediated by both the direct effects of gh binding to the gh receptor, activating the jak / stat pathway, and indirectly via insulin - like growth factor 1 (igf-1). The combined effects of gh and igf-1 on target tissues lead to bony and soft tissue growth, noted by a characteristic constellation of signs including frontal bossing, prognathism, widely spaced teeth (due to mandibular growth), increased shoe or ring sizes, skin tags, carpal tunnel syndrome, and coarse facial features . These externally recognizable signs in themselves are not as important to overall morbidity as internal changes, including cardiomegaly and visceromegaly [4, 5]. Diabetes mellitus occurs in roughly 25% of patients, and cardiomyopathy with arrhythmia, hypertension, and diastolic dysfunction occurs in 30% . Additionally, colonic polyps are more frequent, as is sleep apnea which occurs in 60% of patients, presumably secondary to soft tissue expansion and macroglossia . Multiple studies through the 1920s to 1990s agreed on a prevalence of roughly 60 per million, a mean age of onset of 44 years, symptoms lasting on average 8 years before diagnosis, and no difference in incidence between men and women . However, more recent studies place the prevalence at 86 per million, 124 per million, and a remarkably high 1034 per million . However, in addition to being geographically and demographically restricted (primary care patients in germany), this last study was based on a biochemical definition of acromegaly (elevated igf-1 and gh) rather than a syndrome definition, and therefore will include many patients who would otherwise not seek treatment . Such a more liberal definition might be one method of discovering at - risk patients earlier in the course of their disease and permitting more time to mitigate the accruing morbidity . Surgery is typically the first line of treatment for acromegaly due to pituitary adenomas provided there are no surgical contraindications . Residual disease is then managed medically with somatostatin analogues, dopamine agonists, or gh receptor antagonists . Most adenomas are resected via the transsphenoidal approach (transsphenoidal adenomectomy, tsa). Using either a microscope or endoscope, the surgeon enters the sphenoid sinus transnasally, then penetrates the sella, and finally debulks and removes the tumor, sparing as much of the normal pituitary as possible . For microadenomas (<10 mm in diameter), tsas lead to correction of gh levels in ~70% of patients . Only 50% of patients with macroadenomas achieve normalization . For those patients who do not experience normalization of gh levels after surgery, or who are not surgical candidates, the lar (long - acting release) formulation of octreotide uses polymeric microspheres and is injected monthly . Lanreotide atg (autogel) is the only formulation of lanreotide currently available in the united states and is suspended in aqueous solution in microsyringes for subcutaneous delivery by the patient . The two formulations appear to be equally efficacious and normalize igf - i levels in 5060% of patients [1316]. Somatostatin analogues appear to induce tumor shrinkage in approximately 42% of patients, when data are pooled across studies . Interestingly, however, tumor shrinkage appears more pronounced in primarily treated patients (52%) as opposed to patients receiving adjunctive, postsurgical treatment (21%). Dopamine agonists appear to work best in patients whose tumors also secrete prolactin . Among dopamine agonists, cabergoline appears to be the most efficacious, resulting in normalization of igf - i levels in 34% of patients . However, no dopamine agonist alone is as effective as a somatostatin analogue, although there might be synergistic effects when used in conjunction with somatostatin analogues [2124]. Pegvisomant is a pegylated growth hormone analogue that is subcutaneously injected by patients . In one study, treatment for 12 months led to normalized igf-1 levels in 97% of patients, and treatment for 24 months led to normalization in 76.3% of patients in a different study . However, the side effects of pegvisomant include transaminitis, lipodystrophy at injection sites, and, most worrisome, possible tumor progression, due to blocking of the normal inhibitory feedback of growth hormone levels of the adenoma [2729]. Simply put, resective surgery is a means of removing unwanted tissue from the body . Radiosurgery (rs) achieves the identical goal but, rather than directly removing cells, induces cell death instead . This cell death can be induced either directly, via necrosis or apoptosis, or indirectly, by damaging the tissue blood supply . When inducing cell death, rs uses ionizing radiation, wherein charged particles or photons strip electrons from atoms and molecules, thereby damaging dna, proteins, and other molecules within cells and in the extracellular matrix . When this damage cannot be repaired, cells undergo either apoptosis or necrosis [30, 31]. Various types of radiosurgery are available, but each works by emitting charged particles or photons . Proton beams, for example, generated by particle accelerators and can be used to target structures deep within the cranium . This is due, in part, to the characteristic peak and subsequent drop - off of radiation intensity in proton beams the bragg peak . The depth of this peak can be modulated and is used to focus the effects of the radiation on particular structures, sparing healthy ones . The drawback to proton therapy, however, is the high cost and relative scarcity of facilities capable of providing therapeutic proton beams . The two major techniques are linear accelerators and radioactive isotopes . Linear accelerators, like the cyberknife (accuray inc ., sunnyvale, ca), produce photons which are then aimed toward deep structures within the brain or spine . To limit damage along the path of the beam thus, the target of the beam is constant, always receiving radiation, but the intervening structures are exposed only briefly [3032]. The gamma knife (elekta ab, stockholm, sweden), developed by lars leksell in 1968, each source generates a beam of photons as the cobalt decays, and these beams are focused on a central target within the brain through the use of collimators . Because each individual beam is weak, intervening tissue is exposed to far less radiation than the central target, which is the common focus of all 201 beams . The gamma knife has been around longer than most linear accelerator radiosurgical devices and is therefore a better - studied tool for use by neurosurgeons, though direct comparisons between the gamma knife and linear accelerators will undoubtedly change the prevailing practices in the future . A large number of small case series have been carried out to evaluate the effects of gamma knife surgery (gks) on acromegaly (table 1). Most use remission criterion of a normal igf-1 level and many add the criterion gh <1 to 2 ng / ml, but it should be noted that remission criteria vary across studies . These criteria are roughly in line with the criteria set forth by the acromegaly consensus group on 2010 (normal igf-1 and gh <1 ng / ml). Also variable between studies is the follow - up time, along with radiation dose, targeting protocol, and, most critically, pre - gks therapy . This last note is particularly important since most series include patients who have already received transsphenoidal surgical resection as an inadequate treatment . Unfortunately, the data are not presented in the reviewed papers in such a way as to separate out response rates in patients receiving prior treatment versus those who were treated primarily with gks . Overall, however, the results from these studies suggest that gks is an effective treatment for acromegaly . Across the 29 studies and 964 cases examined, 43% of patients achieved remission . The time to remission is not reliably reported, but most studies agree that the further out from rs patients are examined, the more likely they are to achieve a cure . For example, vik - mo et al . Showed an increase from 58% of patients with normal igh-1 levels to 86% of patients over the time span of 5 to 10 years after rs . How exactly the effects of rs continue to evolve over such a protracted time is unknown, but consistent with how rs affects other diseases, like epilepsy and vestibular schwannoma . Interestingly, there is some (still debated) evidence that the use of antiacromegalic medicines prior to irradiation attenuates the effects of gks . That is, using somatostatin analogues or gh receptor antagonists has been shown in select studies to decrease a patient's chances of remission [3739]. Though this has not been thoroughly examined, the mechanism is believed to be suppression of tumor cell cycle, thereby making the cells less prone to radiation - induced damage . There does not appear to be a correlation between radiation dose and rates of remission (figure 1). However, the heterogeneity in study design, follow - up, and definition of remission makes such conclusions fraught . The most common complication of gks for acromegaly is hypopituitarism, presumably from damage to the normal gland during irradiation, ranging from 0 to 43% (table 2). However, the degree to which such damage is a pure result of gks versus prior surgery, if done, is unclear . Moreover, there is no discernible relationship between radiation dose and the incidence of this complication (figure 2), though these are very heterogeneous studies and it is possible that better - controlled or larger studies would uncover such relationships if they exist . Other complications are inadequately documented but include headache, epilepsy, carotid artery stenosis, and, more frequently, cranial nerve palsies or neuropathies (including trigeminal neuralgia and visual decline) (table 2). The rate of visual disturbances seems to be, in the worst case, 11% [40, 41], though most trials either do not report these adverse events or show them to be on the order of 06% (table 2). Nevertheless, these complications should be viewed in light of the complications inherent to uncontrolled acromegaly or, if being used as an alternative to surgery, the morbidity of an endonasal neurosurgical procedure . While surgical resection remains the first line of treatment, stereotactic radiosurgery is proving itself a feasible alternative therapy (when surgery might be contraindicated) and adjunct (when surgical resection is unsuccessful in leading to complete disease remission). Ultimately, further studies are needed to delineate which patients are most likely to receive benefit from gks, along with ways to improve gks outcomes.
Most human disease pathways are evolutionarily conserved with other organisms . For example, the nematode worm caenorhabditis elegans, which is relatively distant in phylogeny from humans, is used as a model system to study human parkinson's disease (1). Despite limited functional mimicry of some human diseases (2) and recent advances in patient - based disease genetics due to genome - wide association studies (gwas) and disease genome sequencing, non - human model organisms remain indispensable in human disease research, because (i) disease - associated dna variants typically explain only a small proportion of disease heritability; (ii) detailed molecular mechanisms of disease processes often cannot be studied directly in humans for ethical reasons (3). While model organisms will remain critical for human disease research into the future, the functional relevance of pathways conserved between humans and other species can sometimes be nonobvious (4), hampering identification of new human disease models in other, more experimentally tractable organisms . The identification of human disease - relevant pathways conserved in model organisms will allow new opportunities for studying diseases, disease genes and drug candidates, and often allow for genetic manipulations of the disease phenotype to illuminate molecular mechanisms of disease progression . Therefore, bioinformatics tools that can efficiently translate the biology of model organisms into new insights about human diseases are critical to connect model organism observations to human disease research . Here, we present a new web - based tool for prioritizing the human diseases most relevant to a given set of model organism genes . Morphin (model organisms projected on a human integrated gene network) harnesses model organisms to investigate human disease by performing an orthology - based projection of model organism pathway genes onto a human integrated functional gene network (humannet (5)). Genome - scale functional gene networks have proven useful in prioritizing novel candidate genes for phenotypes in diverse species, including for human diseases (6). A typical use case for morphin is as follows: suppose a user obtained a list of worm (c. elegans) genes involved in, for example, dauer induction, and wants to identify the human diseases most relevant to the worm dauer induction pathway . The user can submit the worm genes for dauer induction to the morphin server, which then returns the following results: (i) human orthologs of the worm query genes, (ii) a list of human diseases significantly associated with the worm dauer induction pathway, based upon gene set enrichment or network - based closeness, (iii) gene networks from humannet between human orthologs of the worm genes for dauer induction and the associated human disease genes, (iv) a list of prioritized human orthologs of worm genes most relevant to each associated human disease . The user can actively link the model organism pathway to the most relevant human diseases, and thus potentially identify new disease models and find novel candidate genes for those human diseases . Morphin substantially differs from other tools for mapping model - to - disease pathway associations, which are based on either intersection of orthologous genes (e.g. Phenologs (4), kobas (7)) or semantic similarity (e.g. Phenomenet (8), phenodigm (9)). For example, morphin employing the fisher's exact test is equivalent to searching for phenologs for the model organism gene set . However, morphin additionally uses not only overlap but also functional links between orthologous genes from pathways of model species and human to measure their association, by projecting orthologous genes of the model species on a human gene network, enabling detection of functional association between pathways with no overlap (10). This extra detection powered by a network algorithm, riddle (reflective diffusion and local extension), may be particularly beneficial if annotation of a model pathway or a human disease is largely incomplete . Moreover, gene functional links within and between pathways allow prioritization of the model pathway genes for a disease, and may provide new molecular insights about pathogenesis . Once a user submits a set of model organism query genes, morphin performs the following analyses . First, morphin identifies human orthologs of the submitted model organism query genes using the inparanoid algorithm (11). Morphin then measures associations between human disease pathways and the query genes, calculated by overlap - based fisher's exact test and network - based riddle algorithm, and returns all significantly associated human diseases . Third, morphin visualizes gene networks depicting the functional couplings between the query genes and human disease genes . Finally, morphin returns a list of query genes prioritized by relevance to each of the associated human diseases . Figure 2 shows representative screenshots of the morphin web interface . The overall design of the morphin web server . Once a user submits a set of query genes for one of nine supported model organisms, morphin performs the following analyses: morphin first identifies human orthologs of the submitted model organism genes using inparanoid (11). Morphin then searches for related human disease pathways to the query genes using fisher's exact test (12) and riddle (10). Third, for each significantly associated human disease pathways, morphin displays the gene network between the query genes and disease genes by humannet links . (b) a table of significantly associated human disease pathways ranked by the fisher's exact test . The p - value represents the statistical significance by fisher's exact test; the q - value represents the adjusted significance for multiple hypotheses test; the morphin web server currently supports nine model organisms: c. elegans (worm), danio rerio (zebrafish), dictyostelium discoideum (social ameba), drosophila melanogaster (fruit fly), mus musculus (mouse), rattus norvegicus (rat), saccharomyces cerevisiae (budding yeast), schizosaccharomyces pombe (fission yeast) and xenopus laevis (african clawed frog). The protein sequences of human, zebrafish, mouse, rat were downloaded on 25 february, 4 december, 9 september 2013 from the ncbi reference sequence (refseq) database (http://www.ncbi.nlm.nih.gov/refseq) (13), worm ws239 from wormbase (http://www.wormbase.org) (14), social ameba on 28 january 2014 from dictybase (http://dictybase.org) (15), fruit fly release 5.54 from flybase (http://flybase.org) (16), budding yeast on 25 november 2013 from saccharomyces genome database (http://www.yeastgenome.org) (17), fission yeast on 28 january 2014 from pombase (http://www.pombase.org) (18) and african clawed frog on 28 january from uniprot (http://www.uniprot.org) (19). Users can submit query genes using gene names or unique systematic ids, although we generally recommend using unique systematic ids . Details of supported gene ids are available on the morphin tutorial page . To identify human orthologs for the model organism query genes, morphin employs the inparanoid 4.1 standalone algorithm (http://inparanoid.sbc.su.se) (11), which allows multiple human orthologs for a given query gene by considering not only the best ortholog but also its functionally similar paralogs (in - paralogs) (20). This algorithm achieves a balance between sensitivity and specificity in identifying orthologs across two species (21,22) by distinguishing in - paralogs duplicated after speciation from out - paralogs duplicated before speciation . This score indicates the relative similarity to the two - way best - hit orthologs and ranges from 0 to 1, where 1 indicates the maximum likelihood of orthology . The suggested default threshold is 0, which maximizes sensitivity at the expense of specificity . However, the in - paralog score threshold can be increased so as to use only the most confident orthologs in subsequent analyses . Notably, where gene expansions have occurred, inparanoid allows for multiple human orthologs for each model organism gene, each associated with its own in - paralog score (figure 2a). Morphin shows go annotations for each human ortholog of the query genes, from which we may quickly capture functional properties of query genes in a human context . For more extensive functional characterization of the query genes in human contexts, morphin also returns a list of the human genes closely connected to the query genes in humannet along with their go annotations . Such annotations supplement the group - wise analysis search for functionally associated human disease pathways . Morphin returns those human diseases significantly associated with the query gene set as determined by two algorithms: gene set enrichment by fisher's exact test (12) (figure 2b) and riddle, a measure of network proximity between two gene sets (10) (figure 2c). Fisher's exact test is a classic overlap - based enrichment analysis, which measures the statistical significance of the observed overlap between two gene sets . When adjusted for multiple hypotheses (assessing significance as a q - value), this test requires the presence of common member genes between two gene sets . Considering that current annotations for many pathways are still largely incomplete, we might thus anticipate associations between pairs of gene sets to be missed due to failure to observe the overlap . To overcome this limitation, we previously developed a network - based measure of association between two gene sets, riddle (10). Riddle determines functional closeness between two gene sets using a set - wise distance on an integrated functional human gene network, humannet (5). Because riddle measures association based upon network connections between two gene sets, not their overlap in gene content, it can also detect relationships between gene sets which have no overlap, thus increasing its power to identify relevant human diseases . While fisher's exact test cannot detect associations between gene sets with no overlap, it showed slightly better performance for sets with overlap in a previous study using simulated test data (10). In addition, we anticipate that the two methods may be somewhat complementary since they employ entirely different methodologies . Therefore, morphin provides the search results from both methods in two tables of candidate human diseases, one ranking pathways with q - value <0.1 by fisher's exact test and the other ranking pathways with fdr (false discovery rate) <0.01 by riddle (limited to the top 1000 pathways). Morphin currently tests gene sets from seven databases of human pathways, including five databases cataloging human disease genes: (i) disease ontology (http://disease-ontology.org downloaded on 4 april 2014) (24), (ii) genetic association database (gad, http://geneticassociationdb.nih.gov, downloaded on 14 december 2013) (25), (iii) genome - wide association study catalog (gwas catalog, http://geneticassociationdb.nih.gov, downloaded on 5 december 2013) (26), (iv) human phenotype ontology (hpo, http://www.human-phenotype-ontology.org/ downloaded on 7 april 2014) (27), (v) online mendelian inheritance in man (omim, http://omim.org, downloaded on 4 december 2013) (28) and two databases for pathways or biological processes: (i) kyoto encyclopedia of genes and genomes (kegg, http://www.kegg.jp, downloaded on 18 december 2013) (29) and gene ontology biological processes (gobp, http://www.geneontology.org, downloaded on 17 december 2013) (30). Particularly, gobp annotations are provided with various types of evidence including exclusively computational annotation (inferred from electronic annotation). To achieve high specificity in morphin analysis, we used only highly reliable annotation with experimental or literature evidence: inferred from direct assay, expression pattern, genetic interaction, mutant phenotype, physical interaction and traceable author statement . Users also need to be aware of potentially inaccurate gene - to - disease associations by gwas due to its mapping strategy, based on a gene's physical proximity to functional genetic variation affecting diseases . Network representations of human disease genes associated with model organism query genes can help interpret the associations and better prioritize genes of interest . Morphin provides network visualizations for each candidate human disease significantly associated with the model organism query genes . Clicking on the network icon beside each human pathway name opens a web - based network view (figure 2d) displayed using cytoscape web, an interactive network browser (http://cytoscapeweb.cytoscape.org/) (31). The cytoscape web browser requires flash player to be installed on the local client machine . (note that visualizing particularly large networks may be problematic depending on the performance of the local client machine .) Genes are grouped into three categories, represented as boxes: query genes (orange nodes), disease genes (blue nodes) and overlap genes between two gene sets (red genes), and are linked by humannet functional associations . Morphin shows both group - level connections (black edges) and gene - level connections (blue edges) (figure 2d). The riddle algorithm can find connections between a group of query genes and a group of disease genes with no overlapping genes (see figure 3 for examples). Sometimes two groups are connected in the absence of gene - level connections between them . This is possible because riddle measures closeness between two groups of genes using not only direct connections but also indirect ones . Clicking on a network link shows detailed information about that link including its supporting evidence and confidence scores (lls, log likelihood score) (32,33). Clicking on a node provides detailed information for that gene including its name, category (query gene, disease gene or overlap gene) and total connection score to the disease genes, represented as a weighted sum of lls as calculated in (33). Networks between human orthologs of the 11 worm genes linked to an increased number of fat associated organelles (query genes) and human homocystinuria (a) or hyperhomocysteinemia (b) genes (disease pathway genes). Despite no overlap between query genes and disease pathway genes (there is no box for overlap genes) riddle detected statistically significant association between them by using humannet - based connections between genes from the two gene sets . Although a group of query genes from a model organism is found to be significantly associated with a human disease, individual query genes may not be equally relevant to the disease . Hence, prioritizing the query genes for the disease by functional relevance will be useful for follow - up functional studies focusing on key candidate genes . Query genes that overlap with the human disease genes might be considered to be top candidates, which will be listed in the first table . The rest of the query genes are prioritized for relevance to the disease based on network connectivity scores to the disease genes and listed in the second table (figure 2e). For more reliable candidate selection, morphin employs two complementary ranking systems: group ranks and global ranks . Group ranks are assigned among the group of query genes, while global ranks are assigned for all genes in humannet . Both group and global ranks of each query gene are presented in the table, allowing the user to assess both relevance within the query gene set and relevance relative to all other human genes . A query gene highly ranked among its group as well as among all human genes may be a particularly strong candidate for the disease . Lastly, morphin returns all human genes prioritized for the disease by total connectivity scores to the disease genes in the third table for users who want to see disease candidate genes other than the query genes . In order to evaluate the capability of morphin for disease model discovery, we assessed its power to prioritize gene - to - disease relationships annotated by two distinct methods: manual curation and semantic similarity . To construct validation sets of gene disease relationships, we downloaded 149 and 88 manually curated gene - omim sets from wormbase (14) and mouse genome informatics (34), respectively, on 14 april 2014 . For fly and zebrafish, such annotations by manual curation we therefore compiled 68 and 152 gene - omim sets for fly and zebrafish, respectively, from phenomenet (8), which is a cross - species phenotype ontology network based on semantic similarity (selecting pairs with similarity score> 0.1). In all four species, morphin, with the default in - paralog score threshold, outperformed the conventional fisher's exact test in prioritizing reference gene - omim sets (figure 4). Morphin that employs not only the overlap - based fisher's exact test but also the network - based riddle algorithm significantly improved identification of the reference gene - omim sets over the use of using fisher's exact test only in mouse, worm, fly and zebrafish; 3.4 percentage points(pp), 12.1pp, 8.8pp and 3.3pp more matches in the top 10 ranks, respectively . The network - based method was critical particularly for fly and zebrafish, in which the overlap - based fisher's exact test could identify none or only a few reference gene - omim relationships . The different degree of contribution of riddle across the four species may be attributable to the differences in cross - species phenotype association approaches . Manual curation generally uses literature information, which mostly contains experimental data, and the majority of traditional disease models have been established from orthology - based hypotheses . Disease models by manual curation are likely to contain orthologous overlaps between model species and human . In contrast, semantic approaches of gene disease modeling do not consider genetic components of testing phenotypes, allowing associations between phenotypes of model species and human diseases even in the absence of orthologous genes in common . The reciprocal of the rank of the matching subset of reference gene - omim disease pairs is shown for fisher's exact test (fet) and morphin in mouse (a), worm (b), fly (c) and zebrafish (d). Morphin identified 85.2%, 67.1%, 8.8% and 5.3% of the reference gene - omim pairs, while the conventional fet method identified 81.8%, 55.0%, 0.0% and 2.0% in the top 10 ranks for mouse, worm, fly and zebrafish, respectively . The riddle algorithm improved the performance of morphin by 3.4pp, 12.1pp, 8.8pp and 3.3pp in the four species, respectively . To demonstrate application of morphin in identification of new disease models, here we present two case studies using worm genes annotated according to the worm phenotype ontology (wpo) (35). First, morphin shows a pre - computed example using six worm genes modulating dauer induction (wpo:0001539; f52d10.3, c54d1.3, y110a7a.10, f52b5.5, r13h8.1, f55a3.3), which is known as an animal model of human diabetes (36). Both fisher's exact test and riddle identified diabetes - related terms among the top ranked human diseases (figure 2b and c). Fisher's exact test identified type ii diabetes mellitus (kegg) in the fourth rank and riddle identified pregnancy in diabetics (gad) in the third rank and type ii diabetes mellitus (kegg) in the 16th rank . Dauer formation is known to be regulated by insulin signaling pathways in worm (37). Riddle also identified two insulin - related biological processes, cellular response to insulin stimulus (gobp) and insulin - like growth factor receptor signaling pathways (gobp), tied in the fourth rank, while fisher's exact test returns the most relevant term insulin signaling pathway (kegg) as the 41st rank . Another example is a set of 11 worm genes associated with an increased number of fat associated bodies (wpo:0001888; f54c9.7, c54h2.5, c17g10.5, r11h6.1, c02a12.4, f01g10.3, zk622.3, f01g10.2, k04e7.2, zc416.6, c49f5.1). High levels of homocysteine in blood and urine confer risk of cardiovascular diseases (38). Animal studies have suggested that a high fat diet is associated with an elevated homocysteine level (39). In contrast, riddle identified not only the related pathway homocysteine metabolic process (gobp) but also the related diseases homocystinuria (hpo) and hyperhomocysteinemia (hpo) as third and fifth ranks, respectively, suggesting some of the query genes may be relevant to defects of homocysteine metabolism . There are no overlapping genes between the query genes and each of the two diseases (figure 3), explaining why only the network - based riddle method was effective at identifying the association with such diseases, which now suggest potential future experimental directions . Morphin identifies human diseases most relevant to model organism pathway genes using highly sensitive and statistically robust methods incorporating a human gene network . For each significant human disease association, morphin also prioritizes the query genes for disease relevance and visualizes the gene network comprised of query and disease genes . Morphin thus facilitates connecting model organism discoveries with relevant human diseases by (i) identifying potential new model systems for disease research, (ii) prioritizing new disease gene candidates for follow - up studies and (iii) using network data and visualization to promote insight about underlying biology of human disease . Morphin will be updated as significant changes in annotations for human disease pathways and new versions of humannet become available in the future . National research foundation of korea (20100017649, 2012m3a9b4028641, 2012m3a9c7050151 to i.l . ); national institutes of health, national science foundation, cancer prevention research institute of texas, u.s.
Western health services are characterized by increasing demands for effectiveness, production, and financial profit and economizing . This has an impact on professional standards and clinical nursing values and constitutes a source of job - related stress and unrest for the nurses . Norwegian studies from hospitals and local health services show that lack of resources and heavy work pressure lead to ethically difficult prioritizations and a lowering of nursing standards . European studies demonstrate that professional ethical codes become unattainable ideals for many nurses due to the lack of resources and heightened effectiveness in the healthcare environment . Lack of resources and the lowering of priorities cause considerable strain on healthcare professionals both in hospitals and the local health services . This strain can be understood as moral distress, a concept associated with the ethical dimension in practice and with issues related to difficulties in practice in terms of maintaining professional values, responsibility, and duties [711]. Moral distress, even though it is understood differently in different studies, has shown to have negative consequences and contributes to emotional discomfort, such as, for example, anger, frustration, withdrawal, insecurity or poor quality in patient care, reduced job satisfaction, and occupational fatigue [1115]. We took our point of departure in the national core values in the norwegian specialist health services: respect, quality, and safety . The norwegian national core values for the specialist health services were introduced with the health reform in 2002 . We wanted to explore whether these values after ten years are present and of importance in nurses' daily practice and how this affects the nurses' daily practice and professional wellbeing . The object of this study was to examine nurses' experience of how ethical values are expressed in daily practice in a norwegian hospital where health reforms have been introduced as well as market thinking . The collection of data was carried out by conducting focus group interviews with a total of 20 nurses from different somatic and psychiatric bed units in a medium large hospital in norway . The ages of the participants were from 27 to 60 years, with 2 to 38 years of experience as nurses, the majority around 20 years . The request for participants for the study was sent to the administration in each clinic, and the participants were then selected and asked by their immediate superior if they were willing to take part in the study . The selection of participants was convenient as the request to participate was made to all the nurses in the units without any criteria due to, for example, gender or work experience . Focus groups are a qualitative method where a group of people with certain common qualities are gathered together to discuss a given, well - defined topic, in permissive and nonthreatening surroundings . Focus groups should be informal and the questions should be direct, comfortable, and simple but not guiding . One of the foremost advantages of focus groups is the interaction between the participants which may generate more profound and richer data than individual interviews . Three interviews were conducted with nurses from different somatic units and one interview with nurses from different psychiatric units . After three interviews in the somatic units, we reached a point of saturation in the data and decided to conduct a last interview with participants from psychiatric units to look for comparable results . The second author was moderator during three of the interviews, and this provided both continuity and experience in the moderator role . The interviews lasted 90 minutes, they were tape recorded, and ongoing notes were taken by the assistant moderator . The focus groups took place in the hospital's meeting room during the participants' working hours . A semistructured topic guide ensured that the same key topic was under discussion in the different focus groups . The topic guide's starting point was the national core values in the norwegian specialist health service: quality, safety, and respect, and how these values were expressed or challenged in everyday work . Other topics were whether the core values or other ethical values functioned as guidance in daily practice and whether reflection about ethics and values took place in the daily work . The aim of the study was to explore attitudes, thoughts, feelings, experiences, and knowledge among nurses about core values and other ethical values . The method must be adapted to the research question and thus is a qualitative method chosen [19, 20]. The main key to content analysis is that the words in the text are classified into smaller content categories . We experienced that the participants were greatly committed and candid in the focus groups, with the result that the material obtained was highly informative and comprehensive . In the analysis we emphasized the findings that generated most engagement and discussion among the participants . The analysis started after each interview with listening to the tape and making a verbatim transcription of each interview . Then the results from the three interviews from the somatic units were considered across and analyzed as a whole . The interview from the psychiatric units was separately analyzed similarly to the three others, and one looked for comparable results . We chose to consider the manifest content, that is, what the text says [21, 22]. The next step was to find meaning units, condense, and then encode them . The codes were analyzed and systematized, and five main categories appeared, and the codes were sorted into these main categories . (2) the system . (3) management . (4) patient . (5) nursing . The codes and the content in all the main categories were analyzed, condensed, and placed into subcategories, and a brief abstract was written summarizing the main content from the different subcategories . These abstracts were considered across, and further meaning compacting was done in order to systematize and further abbreviate the text . In this process we identified two main themes containing and presenting the results: values and reflection are important for the nurses, time pressure and nursing frustrations . Values and reflection are important for the nurses, time pressure and nursing frustrations . The validity, or authenticity and trustworthiness of a study, is seen in two dimensions, internally and externally . This enabled us during the entire process to discuss and verify perceptions and conclusions which enhanced the internal validity of the study . The study was conducted with a homogeneous group, nurses, and special nurses without leader responsibility and with daily patient contact . If the study had been carried out with nurse managers in the group, the results might have been different . The study's preliminary findings have been presented for experienced nurses and other researchers who found the results recognizable . The external validity of the study could have been strengthened if corresponding studies had been carried out in several hospitals . The study was carried out in a medium sized hospital in norway with nurses from different somatic and psychiatric units . It represents diversity both in terms of fields of study and nurses' previous experiences and can thus be transferable to other nurses in other hospitals . The project has been reported to the norwegian social science data services (nsd). The participants received verbal and written information about the study and agreed in writing to voluntary participate . As earlier described, two main themes were identified in the analysis, and the results are presented with sub - themes under each main theme: values and reflection are important for the nurses, time pressure and nursing frustrations in daily work . Values and reflection are important for the nurses, time pressure and nursing frustrations in daily work . The nurses from all units describe how ethics, values, and reflection on values are of great importance for the quality in nursing . The informants see the need for values that are formulated in writing and the need for specific aims for their workplace . Yet they feel that the values are solidly rooted even though they are not formulated in writing . The informants from somatic units say that they are aware of when the values are absent in practice and that it is their task to speak up about undignified conditions.it would have been fine if it was like that, that we all went along the same road with values at the bottom . I feel, and can see it among my colleagues, that these values are solidly rooted . It has surely to do with the training and what one sort of has learnti would say that there is often more focus on the hustle and bustle and lack of space, so one would like to be reminded of the values . One needs it even though it lies deeply in us it would have been fine if it was like that, that we all went along the same road with values at the bottom . I feel, and can see it among my colleagues, that these values are solidly rooted . It has surely to do with the training and what one sort of has learnt i would say that there is often more focus on the hustle and bustle and lack of space, so one would like to be reminded of the values . The informants say that being nurses they need to have basic values and that what is most important is to see the patient and to treat him with respect . The informants say that their work is based on the basic nursing values learned in their training and from colleagues with good values . The nurses in somatic units experience that values are repressed and may be neglected in their daily work due to time pressure, lack of resources, and great challenges . Nurses from somatic units prioritize helping patients in grave and acutely life - threatening conditions . With limited resources, this can result in undignified conditions for other patients whose illnesses are not life - threatening . These patients, who, for example, have to wait a long time without anyone keeping an eye on them, are given insufficient or unsatisfactory nursing . In these cases, the nurses feel that the job they are doing is not good enough . These values vanish into thin air when it is a matter of whether the patient breathes or does not breathe, and so the prioritizing becomes tough and at that point i cannot exactly manage to see these values, well, and even if they are there in the tiny moment when you are with the patient, i do believe that there are many of us who think that we could have done a better job it has often to do with resources, unfortunately . At times these values vanish into thin air when it is a matter of whether the patient breathes or does not breathe, and so the prioritizing becomes tough and at that point i cannot exactly manage to see these values, well, and even if they are there in the tiny moment when you are with the patient, i do believe that there are many of us who think that we could have done a better job the quality varies . The nurses in the somatic units experience the quality in nursing as varying on account of lacking resources and time pressure in their daily work . The informants state that the quality is good when patients' basic needs are taken care of, when they do not have to lie in the corridor but are given a room and when one has time to talk with them and their families . If the nurse finds time to do this little extra for the patient and does not have to run from room to room, she will feel that she has done a good job which will make her feel good inside . The informants from the psychiatric units report that good quality in nursing depends on evidence - based knowledge and a flexible and individually adjusted program for psychiatric treatment, with client participation . The informants tell us that the quality is totally dependent on the composition of the staff and that experienced nurses can help to create an atmosphere of quiet and security in the milieu and also, for example, in a coerced treatment situation.it is about offering flexible psychiatric treatment . That's what we are preoccupied with, the fact that no one is alike that's what we are preoccupied with, the fact that no one is alike the informants from somatic units inform us that they will go to great lengths for quality and that they feel it is important to adapt nursing measures to the patient's needs rather than focusing on procedure . Nurses from all the units emphasize that procedures cannot be followed in all situations and that one cannot produce recipes or procedures for interpersonal relationships.it has to do with interpersonal relationships . We cannot write it, provide a recipe or a procedure to be followed in all situations . Because things can really go wrong then, since every situation is unique and there are so many facets in what we do . Experience and basic values and ethical principles are required, and good child rearing, that is always welcome it has to do with interpersonal relationships . We cannot write it, provide a recipe or a procedure to be followed in all situations . Because things can really go wrong then, since every situation is unique and there are so many facets in what we do . Experience and basic values and ethical principles are required, and good child rearing, that is always welcome safety is not always there . Respect, genuine presence, and good communication with patients and relatives generate safety and is described as decisive for quality as a whole . At times the informants from the somatic units experience insecurity and lack of control in their work situation . They feel that it all is haphazard and that they have to rely on sheer luck when there is a lack of both time and resources.i'm thinking a little about it in terms of safety and procedures . One can of course say that when they are busy developing it, it is a form of safety, but it happens quite often that when i am working i feel that things are not safe . When it is too much for you to cope when there are and there can be long periods between each time you are with a patient, so there is not much safety to speak about one can of course say that when they are busy developing it, it is a form of safety, but it happens quite often that when i am working i feel that things are not safe . When it is too much for you to cope when there are so many you should give care to . And there can be long periods between each time you are with a patient, so there is not much safety to speak about in psychiatry, safety was described as a safe workplace which will not be closed down, where there is enough staff, and the technical and administrative systems are functioning . A candid and honest environment where nothing is too stupid to say among colleagues all the nurses confirm that professional and ethical reflection is important for nursing professionals, but in the somatic units formal arenas and time for reflection are lacking . The informants in the somatic units often have informal talks about reflection with other nurses and physicians in the course of their workday but are continually interrupted because things are hectic and one certainly cannot stop the running of the ward.well, the things that we know would be good for the quality we do not get done in practice because one has to keep things going . We haven't got the time to stop and even if the hospital maybe has taken a patient's life, we do not have time for reflection and certainly not together with a surgeon well, the things that we know would be good for the quality we do not get done in practice because one has to keep things going . We haven't got the time to stop and even if the hospital maybe has taken a patient's life, we do not have time for reflection and certainly not together with a surgeon only two of the informants from somatic units have been offered clinical nursing supervision . The nurses from the somatic units have requested clinical nursing supervision, but regular offers have neither been made nor has there been a tradition for it . The nurses say that one can ventilate ideas, get feedback, and have one's values with you to the supervision, and this they believe may turn you a better nurse.i'm in clinical nursing supervision and that is very useful since there are many cases that touch one deeply . It is mostly a place for letting it all come out, and then you get feedback from the others about what they are thinking, because they too have often been in the same situation i'm in clinical nursing supervision and that is very useful since there are many cases that touch one deeply . It is mostly a place for letting it all come out, and then you get feedback from the others about what they are thinking, because they too have often been in the same situation the informants from the psychiatric units had experienced formal clinical nursing supervision . In addition, they described a culture and an organizing of the daily work which provided adequate time for reflection and supervision . The nurses from the psychiatric units say that it is important to be observant and available to new staff who need clinical supervision and that clinical supervision has been important and essential for their own choice to continue in this profession.i am thinking about the report between the shifts, well, it becomes partly a kind of clinical supervision plus that when one is at work one keeps the discussions going well, it becomes partly a kind of clinical supervision plus that when one is at work one keeps the discussions going the nurses from the psychiatric units describe a reflective workday with discussions about right or wrong, values, and reflection on values in most situations . The nurses from the psychiatric units confront many ethical dilemmas and say that it is important and necessary that several people take part in these discussions.i do not think we say that today we are evaluating values in our unit, but it goes on all the time . A lot of it in the staff room, but also at morning meetings, team meetings, where things are taken up and discussed whether this is right, or should we do it differently i do not think we say that today we are evaluating values in our unit, but it goes on all the time . A lot of it in the staff room, but also at morning meetings, team meetings, where things are taken up and discussed whether this is right, or should we do it differently constant guilty conscience . The informants from the somatic units are often feeling frustrated and worn out on account of considerable work and time pressure . They say they always have to go to greater lengths and run faster and that this work pressure gives them a feeling of mass production and of having to calm things down in their daily work which they feel are both unsatisfactory and tiring.the pressure is huge, and there is quite simply too much to do . One does what one can, but it is perhaps not quite what one actually wants to do or manages to do . And one has a constant bad conscience, well, one feels that one should have done more and that's a strain the pressure is huge, and there is quite simply too much to do . One does what one can, but it is perhaps not quite what one actually wants to do or manages to do . And one has a constant bad conscience, well, one feels that one should have done more and that's a strain rock - hard priorities . Some of the informants from the somatic units fear that they might turn into indifferent and superficial nurses because of the frequent rock - hard priorities given to basic needs in their daily work that as much as possible should be done in the shortest time . The great number of corridor patients also contributes to the fact that patients have to wait for a long time before the nurses can find time for them.in my opinion there is rock - hard prioritizing in our daily work . First of all, one must prioritize the things that are most important for the patient, be it shaving, taking a shower or medicine, but then it has also to do with this great number of corridor patients, the corridors have actually been filled up, and then there are patients with newly discovered cancer diagnoses who have to lie out there in the corridor . One does not feel like going home and knowing that this is where she will be lying tonight, and she is 50 years old and has pulmonary cancer . Even if the patient seems to feel alright about it, it is clear that she only does so because she has cancer after all and that is much worse than lying in the corridorthe ethical values are actually challenged according to the way our employer organizes us within the existing staff resources . When there is sickness during the day one mustn't call anyone for the first one who is sick, but we are then supposed to go to greater lengths, run faster, we have to be everywhere at once and that is at the expense of safety and quality and care and in my opinion there is rock - hard prioritizing in our daily work . First of all, one must prioritize the things that are most important for the patient, be it shaving, taking a shower or medicine, but then it has also to do with this great number of corridor patients, the corridors have actually been filled up, and then there are patients with newly discovered cancer diagnoses who have to lie out there in the corridor . One does not feel like going home and knowing that this is where she will be lying tonight, and she is 50 years old and has pulmonary cancer . Even if the patient seems to feel alright about it, it is clear that she only does so because she has cancer after all and that is much worse than lying in the corridor the ethical values are actually challenged according to the way our employer organizes us within the existing staff resources . When there is sickness during the day one mustn't call anyone for the first one who is sick, but we are then supposed to go to greater lengths, run faster, we have to be everywhere at once and that is at the expense of safety and quality and care and the informants from the surgical units report that patients with cancer or chronic wounds have to wait because the picking up and bringing of patients to surgery are prioritized, and this is experienced by nurses as an ethically difficult prioritizing . The informants from the somatic units speak about the great gap between theory and practice, and they feel frustrated when reality is so far from the ideals they were taught in their training . The nurses also state that these conditions affect their professional pride and that they are discouraged and feel that they barely can keep their heads above water.i feel like taking care of the poor person who was admitted to our hospital six months ago and is getting more and more depressed . At the same time . You must pick up someone from post - op . All the time, while you are standing there confused, you actually want to go to that patient but you did get that phone - call . I can never manage to do what i ought to do with the cancer patients . They see all the names on the list but they do not quite understand what it feels like to be in the centre of the anthill i feel like taking care of the poor person who was admitted to our hospital six months ago and is getting more and more depressed . At the same time you have this pressure all the time . You must pick up someone from post - op . All the time, while you are standing there confused, you actually want to go to that patient but you did get that phone - call . I can never manage to do what i ought to do with the cancer patients . They see all the names on the list but they do not quite understand what it feels like to be in the centre of the anthill when time pressure became the topic in the psychiatric units, the informants pointed out that in psychiatry there should be sufficient time . In psychiatry, the nurses claim that a possible demand from the management for greater efficiency is not consistent with the way they are working.it is all right to be busy, but never so busy that you feel that you are now losing your grip on someone . On the whole we need that in psychiatry because we cannot go fast here, that is obvious . It is obvious that if the management comes with demands for far greater efficiency, then it will go all wrong, there will be a collision it is all right to be busy, but never so busy that you feel that you are now losing your grip on someone we need that in psychiatry because we cannot go fast here, that is obvious . It is obvious that if the management comes with demands for far greater efficiency, then it will go all wrong, there will be a collision they feel that they do not have a say in decisions, are not being heard, and know that they would have much to contribute with if they were permitted to do so.we would have welcomed being permitted to take some part in the decisions . Because it feels like we have no influence on anything, it would have been absolutely fantastic if we were permitted to take part and speak up about some matters . I do not believe they are aware of what we are doing during the day . I feel that there won't be much quality in the long run, i really do we would have welcomed being permitted to take some part in the decisions . Because it feels like we have no influence on anything, it would have been absolutely fantastic if we were permitted to take part and speak up about some matters . I do not believe they are aware of what we are doing during the day . I feel that there won't be much quality in the long run, i really do the nurses from somatic units describe how the management does not set aside sufficient time for talking about values or focusing on them, for example, in staff meetings . The leaders are especially responsible for creating targets in their units and what should be focused on but as said before, we are all responsible for supporting and complying with it the leaders are especially responsible for creating targets in their units and what should be focused on but as said before, we are all responsible for supporting and complying with it the system cannot be changed . The informants from the somatic units place the responsibility for present conditions on the system without defining more specifically who or what the system is . The nurses say that, when the system does not function, it is to the patient's detriment . The system and its financial limitations are stated as the reason why ethical values are challenged by structural issues . One has the impression that the system is almost impossible to change or do anything about.we talk about quality and about what we can do about it, like with procedures, and deviations, but there is a lot in this system that has to do with quality that we cannot do anything about . . We could certainly have sat in a group discussing values, how to put them to best use, but a lot has to do with having more staff in order to be able to include quality and safety in our work, and respect that we hopefully have in any case we talk about quality and about what we can do about it, like with procedures, and deviations, but there is a lot in this system that has to do with quality that we cannot do anything about . Like economy, that there is too little funding and all that . We could certainly have sat in a group discussing values, how to put them to best use, but a lot has to do with having more staff in order to be able to include quality and safety in our work, and respect that we hopefully have in any case all the nurses in this study experience that ethics, values, and reflecting on values are of great importance for the quality in nursing . The ability to be compassionately affected and empathic is anchored in ethics, as the basis for value susceptibility and depth of intentionality and motivation behind the nurses' actions . Emotions are of importance for the normative demarcation between impartial justice and utility maximization on the one hand and compassion on the other . The nurses in our study say that it becomes obvious to them when the ethical values are absent in practice and that it is their duty to speak up about unworthy conditions . The fact that they are aware of the lack of values and unworthy conditions confirms their moral and ethical sensitivity which again rests on sensibility and the ability to be accessible in human encounters . Our study demonstrates that the nurses' ability to be accessible in human encounters in their work is threatened by time pressure and lack of staff . The nurses in our study give expression to this by stating that they fear becoming superficial and indifferent nurses because their work load is too heavy for satisfactory nursing . This is a challenge involving professional healthcare education, the attitude of the entire national health service, politics, and the financial structures which have an impact on this setting . They witness that there is a big difference between theory and practice and this gives rise to frustration . It is consistent with research around this phenomenon which shows that reality shock is traumatic; it is a reaction in newly trained nurses who cannot practice what they have been trained to do and wish to do . In the opinion of our informants, what is most important is to see the patient and have respect for him, and they consider this to be a fundamental nursing value which they learnt in their training and from colleagues who have good values . None of the nurses refer to or mention codes for professional ethics as a basis for values . This is consistent with european studies that show that nurses lack knowledge about ethical codes and do not use them to evaluate moral aspects in practice . Nurses from the somatic units state that there is little focus on ethical values on the part of the management, and they maintain that it is the management that is mainly responsible for what is brought into focus . Bondas has done research on nursing management and taken part in the development of caritative leadership theory . Caritative leadership (cl) has been developed from a caring science perspective with the caritas motive as its point of departure, human love, and mercy, focused on ministering to the patient . Cl strives for an effective and sustainable development of nursing and caring, with a holistic view on patients, relatives, and staff . Together with the staff, the leader creates a caring culture with the patient at its centre . In this type of culture the ethical values must be included and focused on, both by staff and leaders . The nursing management's focus on human dignity, human love, and mercy may be set aside by the introduction of a market oriented leader style derived from industry and business . In her research on first line nursing management bondas found that these leaders, together with the staff, have to prepare the ground for nursing care . Preparing the ground for nursing care is a continuous process for the nursing leaders which is important for the unit's core function, for creating direction and quality in nursing, and with the patient as main focus . In our study, the nurses in somatic units repeatedly describe a culture where it seems that the managers do not prepare the ground for nursing care but are concentrating on production objectives, cost savings, and effectiveness instead of on human dignity and compassion . This is illustrated by the fact that the nurses have to prioritize transport of remunerative elective surgery patients, that is, part of the hospital's production, rather than giving care to cancer patients and other patients who are not part of the same production . They also experience varying support from the managers and that the management does not understand what is going on or what it feels like to be a nurse such practice is not compatible with a caritative, nurturing culture which should prepare the ground for good nursing care in the best interest of the patient, relatives, and the nurse [1, 27]. The nurses from the somatic units have little opportunity to get formal clinical supervision or to reflect on their practice, not even after serious incidents . In most somatic units the nurses who do have this offer are extremely pleased with it; they find it useful, and say that it can turn you into a better nurse . The nurses from the psychiatric units described a reflective workday where the ethical values and reflection on them were present in most situations . It was an internalized way of working which facilitated good conditions for the patients as well as for the nurses . The nurses in psychiatry said that they were caught up in many ethical dilemmas and that it was important and correct that several people took part in the discussion of these issues . Confronting the suffering of the depressed and mentally ill patient activates the nurses own feelings of pain, and they seek help from colleagues when they experience the relationship to the patient as painful . They need a forum which enables them to verbalize and explore their own feelings, where they are met with acceptance and response where feelings are set into a professional context . The nurses from the psychiatric units had all had clinical nursing supervision, and their workday was organized in such a manner that there were time and room for constructive reflection on the practice . This contributed to an improvement of the practice and prevented the nurses from taking incidents and problems from their practice home with them . Some of the informants said that supervision had been decisive for their choice to continue in their profession . To be able to discuss feelings and experiences from the practice with other nurses will help the nurse to understand her own moral stance in terms of other nurses and the organization . Clinical supervision can have a positive effect on the nurses' experience of their own wellbeing in as much as psychic symptoms such that anxiety is reduced and the feeling of being in control grows . Summarized research on clinical supervision demonstrates that clinical supervision can be both advantageous for professional practice and also for the patients . It is the health organizations who are responsible for developing and maintaining clinical supervision for nurses . Clinical supervision is a reflective practice where nurses are given support and supervision on practice, which then will contribute to the nurses' professional and personal development . The nurses from the somatic units told about high work pressure, rock - hard prioritizations, varying quality, and sometimes a lack of safety . They experienced that the values vanished into thin air, that is, became insignificant in what they referred to as rock - hard prioritizations of basic needs . As a result of their work situation, the informants felt frustrated, worn - out, and feared becoming superficial . They describe a busy day with constant bad conscience and a feeling of not being adequate . Several studies from norwegian hospitals and nursing homes show that lacking resources lead regularly to suboptimal professional standards for nursing and medical treatment . The tendency is to lower the standard in order to safeguard the patient's vital needs, and this is at the expense of good practice and all - round care . The nurses have to reduce their professional obligations and adapt to reality, and this creates a state in the nurses that can be understood as moral distress . The nurses in our study tell about crowded corridors and patients with newly diagnosed cancer who must lie in the corridor . This is in accordance with studies showing that crowded rooms and corridors make it difficult to safeguard the patient's dignity and private life, and that the nurse feels moral unease for not being able to give the patient care in accordance with professional ideals . Research on moral distress shows that it can arise on the basis of clinical situations and internal factors in the nurse, for example, a sense of powerlessness and lack of control over the situation, as well as external factors in the culture or organization, for example, inadequate staff . Research shows that moral distress is linked to stress of conscience, while moral stress is seen in a more psychological perspective . Moral distress and related concepts are linked to moral and ethical sensitivity and the ethical climate which forms the basis for the nurse's moral freedom of action . Compromises with ethical values or duties may have prolonged and negative effects causing the staff to become insensitive to the moral aspect of their work, or they may leave their profession . The nurses in our study experienced that they did not have a say in the matter and that there was nothing to be done with the system which caused these difficult conditions . They describe a feeling of powerlessness and lack of hope in terms of being able to influence and improve the conditions . None of our informants talked about leaving the profession, but they were discouraged and felt that they were barely keeping their head above water . Research shows that the concern about nurses leaving their profession as a result of moral distress has created a greater awareness among health leaders and that the need for changes in the system is necessary as a partial response to the increasing level of moral distress in the health service [9, 11]. As shown before, the nurses in the psychiatric units had access to clinical nursing supervision and constructive professional reflection, and none of these expressed frustration or a guilty conscience as a result of time pressure and lacking resources . As shown before, reflective practice promotes moral practice and we therefore believe that formal clinical nursing supervision and reflection, in addition to changes in the system, will also promote the values in practice and thus contribute to a reduction in moral distress . Recent studies show that there is a high degree of moral distress among health professionals in many countries . Health professionals themselves need to reaffirm the values and convictions that are important for their practice . The healthcare environment must be organized in a way that supports and promotes the health staff's moral considerations . If the healthcare environment is not seen as a moral community but rather as simulated market places, then the health staff's moral freedom of action will be reduced and make them more vulnerable to moral distress . The results of the study demonstrate that all the nurses believe that ethical values are of decisive importance for the quality in nursing . The nurses from the psychiatric units have a more reflective workday where the values are internalized in their work . The nurses from the somatic units experience that the values are repressed and become meaningless words in a busy workday with time pressure and resource shortage . These nurses have to lower their professional standard, thus compromising their clinical nursing values and ideals . The nurses from somatic units are worn out and feel frustrated; they are constantly troubled by a guilty conscience and a sense of powerlessness . Financial and organizational structures contribute to the fact that the values are repressed in the nurses' practice and that this, together with insufficient time for clinical supervision and reflection, can be the basic reason for moral distress . We suggest a change of system based on the caritative leadership theory which may contribute to a combining of nursing and administration in such a way that it prevents suffering in the patient and will also further the development of nursing and care, humane and professional, on all levels in the organization . We also consider formal and regular clinical nursing reflection and supervision as crucial for safeguarding moral practice and thus preventing the development of moral distress.
Cancer is the leading cause of deaths worldwide with breast cancer being second in the list of cancer related deaths.1 among all of the available treatment options, chemotherapy is the treatment of choice for advanced stage breast cancer that is refractory to hormonal therapy.2,3 although traditional breast cancer chemotherapy regimens include combinations of cyclophosphamide, methotrexate, and 5-flurouracil,4 anthracycline- and taxane - based chemotherapy is being commonly used nowadays for the treatment of breast malignancy due to its better performance in patient s overall survival rate.57 though breast cancer is considered to be highly chemosensitive with response rates as high as 80%,8 the majority of initially chemoresponsive tumours develop resistance to once effective chemotherapeutic agents.9 therefore, a switch to other chemotherapy regimens is ineffective because of the tumour s cross - resistance to multiple structurally and functionally unrelated chemotherapy drugs.10 abc transporter represents the key component of energy - dependent efflux transport system contributing to the development of multidrug - resistant phenotype in cancer.11 abcb1 transporter or p - glycoprotein, the first cytotoxic drug efflux pump to be identified,12 functions by actively extruding drugs including anthracyclines, taxanes, and vinca alkaloids out of cancer cells, thus reducing intracellular drug concentration below the threshold level required for cell killing.10,11 the poor chemotherapy response in breast cancer is generally correlated to the extent of abcb1 gene expression.1316 a second type of abc transporter discovered in a multidrug resistant breast cancer cell line is abcg2,17 though there are no conclusive data currently available to correlate its expression with chemotherapy efficacy in breast cancer.18 however, judging from the ability of abcg2 transporter in extruding doxorubicin, a very commonly used anthracycline drug,19 it might be reasonable to correlate abcg2 expression with development of clinical multidrug resistance in cancer . Although low molecular weight pharmacologically active compounds have been developed to circumvent multidrugresistant phenotypes by either directly or indirectly modulating the abc transporter efflux activity,20 the first generation modulators failed in clinical trials because of their inherent toxicities21 while the second generation molecules were associated with undesirable pharmacokinetic interactions.22,23 thus, no drug has been clinically approved for the reversal of cancer multidrug resistance.24 a new promising approach to enhance chemosensitivity without undesirable side effects, other than the structure - based drug design, is to prevent the biosynthesis of abc transporters by selectively inhibiting the expression of abc transporters through gene silencing technologies.25 in the last decade, ribozymes2628 and antisense oligonucleotides2931 were introduced to modulate cancer multidrug resistance through inhibition of abc transporter gene expression . Very recently, small interfering rnas (sirnas), a double - stranded rna of between 2128 nucleotides that selectively degrade mrna and thus block production of a particular protein,32 have been applied in vitro for reversal of abc transporter - mediated drug efflux by targeting both abcb1 and abcg2 transporters . A pioneering study using exogenous sirna demonstrated suppression of abcb1 expression in conjunction with reversal of doxorubicin and paclitaxel resistance in human breast cancer cells.33 reversal of multidrug - resistant abcg2 phenotype was also investigated with a sirna - mediated knockdown study in several human cancer cells.34 currently, we have established ph - sensitive carbonate apatite as a potential tool to efficiently deliver sirna across the cell membrane and silence cyclin b1 gene transcript, thus inducing apoptosis of cervical cancer cells in synergy with anti - cancer drugs.35,36 here, we reveal that carbonate apatite - mediated delivery of the sirna targeting either abcg2 or abcb1 gene transcript in mcf-7, a human breast cancer cell line constitutively expressing abcg2 and abcb1 transporters, led to an increased chemosensitivity to doxorubicin, paclitaxel, and cisplatin, depending on the doses of the individual drug . Moreover, co - delivery of two specific sirnas targeting abcb1 and abcg2 transcripts caused a dramatic enhancement of chemosensitivity activity in mcf-7 cells, indicating the reversal of abc transporter - mediated multidrug resistance . Dulbecco s modified eagle medium (dmem) was purchased from biowhittaker (walkersville, md). Dmem powder, fetal bovine serum (fbs) and trypsin - ethylenediamine tetraacetate (trypsin - edta) were obtained from gibco brl (carlsbad, ca). Calcium chloride dihydrate (cacl22h2o), sodium bicarbonate, diammineplatinum(ll) dichloride, dimethyl sulphoxide (dmso), doxorubicin hydrochloride, paclitaxel, and thiazolyl blue tetrazolium bromide (mtt) were from sigma - aldrich (st louis, mo). The sirna sequence targeting abcg2 (genbank accession no nm_004827) and abcb1 (genbank accession no nm_000927) purchased from qiagen (valencia, ca) correspond to coding regions of these genes . One target sequence (5-ctggtctaatttattaatcta-3) was selected for abcg2 (abcg2_v1) and one (5-gacagaaagcttagtaccaaa-3) for abcb1 (abcb1_v4). The 21-nucleotide sirnas having a 3-dtdt extension were chosen based on recommendations made by others.37,38 sirna was supplied in lyophilised form and upon delivery, the sirna (1 nmol) was reconstituted with rnase - free water provided by manufacturer to obtain a stock solution of 20 m . Human breast cancer cell line mcf-7 was grown in 25 cm culture flask in dmem, supplemented with 10% heat - inactivated fbs in a humidified atmosphere containing 5% co2 at 37c . Once the monolayer cells had reached 80%90% confluency, the medium was removed and the cells were washed with phosphate - buffered saline (pbs). Trypsin - edta was added to detach the cells from the flask and the suspended cells were collected in a fresh medium and transferred to new 25 cm flasks . The cells were then sub - cultured at densities of 1.05.0 10 cells/10 ml every 3 to 4 days . Exponentially growing mcf-7 cells (log phase) were trypsinised and fresh medium was added to wash remaining cells from the bottom of the culture flask . The cells were counted using hemocytometer and appropriate dilutions were made using culture medium to produce a cell suspension with a concentration of 5.0 10 cells / ml . One ml of the prepared cell suspension was subsequently added into each of the wells in 24-well plate and allowed to attach overnight at 37c and 5% co2 before sirna transfection . On the day of sirna transfection, 100 ml of dmem was prepared using 1.35 g of dmem powder and 0.37 g of sodium bicarbonate . Ph of the prepared dmem solution was adjusted to 7.4 using 0.1 m hydrochloric acid . Dmem was then filtered using 0.2 a m syringe filter in a laminar flow hood, followed by transferring 1 ml of the filtered medium into 1.5 ml microcentrifuge tubes . 3 l of 1 m calcium chloride was then added into the microcentrifuge tubes, followed by addition of 1 or 10 nm sirna35,36 and incubation at 37c for 30 minutes . After the incubation, ten nm to 100 m of a drug (doxorubicin, paclitaxel, or cisplatin) prepared from serial dilution using 1 mm stock solution was then added into the respective microcentrifuge tubes . Culture medium from the wells seeded one day before was aspirated and replaced with 1 ml of the prepared medium containing sirna - loaded carbonate apatite nanoparticles and free drugs . Plates were then incubated at 37c and 5% co2 for 2 consecutive days . Following two days of sirna transfection, the fraction of viable mcf-7 cells was determined using mtt assay as previously described.39 briefly, 50 l of mtt (5 mg / ml in pbs) was added aseptically into each of the wells in sirna transfected plates, followed by incubation at 37c and 5% co2 for 4 hours . After the incubation period, medium containing mtt was aspirated and the purple formazan crystals at the bottom of each well were dissolved by mixing with 300 l of dmso solution . Absorbance of the resulting formazan solution was then determined spectrophotometrically at wavelength 595 nm using a microplate reader (dynex technologies inc . Each experiment was performed in triplicate and the data were plotted as mean standard deviation (sd) of three independent experiments . Dulbecco s modified eagle medium (dmem) was purchased from biowhittaker (walkersville, md). Dmem powder, fetal bovine serum (fbs) and trypsin - ethylenediamine tetraacetate (trypsin - edta) were obtained from gibco brl (carlsbad, ca). Calcium chloride dihydrate (cacl22h2o), sodium bicarbonate, diammineplatinum(ll) dichloride, dimethyl sulphoxide (dmso), doxorubicin hydrochloride, paclitaxel, and thiazolyl blue tetrazolium bromide (mtt) were from sigma - aldrich (st louis, mo). The sirna sequence targeting abcg2 (genbank accession no nm_004827) and abcb1 (genbank accession no nm_000927) purchased from qiagen (valencia, ca) correspond to coding regions of these genes . One target sequence (5-ctggtctaatttattaatcta-3) was selected for abcg2 (abcg2_v1) and one (5-gacagaaagcttagtaccaaa-3) for abcb1 (abcb1_v4). The 21-nucleotide sirnas having a 3-dtdt extension were chosen based on recommendations made by others.37,38 sirna was supplied in lyophilised form and upon delivery, the sirna (1 nmol) was reconstituted with rnase - free water provided by manufacturer to obtain a stock solution of 20 m . Human breast cancer cell line mcf-7 was grown in 25 cm culture flask in dmem, supplemented with 10% heat - inactivated fbs in a humidified atmosphere containing 5% co2 at 37c . Once the monolayer cells had reached 80%90% confluency, the medium was removed and the cells were washed with phosphate - buffered saline (pbs). Trypsin - edta was added to detach the cells from the flask and the suspended cells were collected in a fresh medium and transferred to new 25 cm flasks . The cells were then sub - cultured at densities of 1.05.0 10 cells/10 ml every 3 to 4 days . Exponentially growing mcf-7 cells (log phase) were trypsinised and fresh medium was added to wash remaining cells from the bottom of the culture flask . The cells were counted using hemocytometer and appropriate dilutions were made using culture medium to produce a cell suspension with a concentration of 5.0 10 cells / ml . One ml of the prepared cell suspension was subsequently added into each of the wells in 24-well plate and allowed to attach overnight at 37c and 5% co2 before sirna transfection . On the day of sirna transfection, 100 ml of dmem was prepared using 1.35 g of dmem powder and 0.37 g of sodium bicarbonate . Ph of the prepared dmem solution was adjusted to 7.4 using 0.1 m hydrochloric acid . Dmem was then filtered using 0.2 a m syringe filter in a laminar flow hood, followed by transferring 1 ml of the filtered medium into 1.5 ml microcentrifuge tubes . 3 l of 1 m calcium chloride was then added into the microcentrifuge tubes, followed by addition of 1 or 10 nm sirna35,36 and incubation at 37c for 30 minutes . After the incubation, ten nm to 100 m of a drug (doxorubicin, paclitaxel, or cisplatin) prepared from serial dilution using 1 mm stock solution was then added into the respective microcentrifuge tubes . Culture medium from the wells seeded one day before was aspirated and replaced with 1 ml of the prepared medium containing sirna - loaded carbonate apatite nanoparticles and free drugs . Following two days of sirna transfection, the fraction of viable mcf-7 cells was determined using mtt assay as previously described.39 briefly, 50 l of mtt (5 mg / ml in pbs) was added aseptically into each of the wells in sirna transfected plates, followed by incubation at 37c and 5% co2 for 4 hours . After the incubation period, medium containing mtt was aspirated and the purple formazan crystals at the bottom of each well were dissolved by mixing with 300 l of dmso solution . Absorbance of the resulting formazan solution was then determined spectrophotometrically at wavelength 595 nm using a microplate reader (dynex technologies inc . Each experiment was performed in triplicate and the data were plotted as mean standard deviation (sd) of three independent experiments . Since abc transporter gene expression is essential in causing multidrug - resistant phenotype in breast cancer cells, knockdown of this gene might enhance sensitivity of the cancer cells towards chemotherapeutic agents, and possibly reverse cancer multidrug resistance . Therefore, mcf-7, the widely used human breast model cell line intrinsically expressing abcg2 (alternatively called brcp or breast cancer resistance protein),17 was transfected with the target sirna / carbonate apatite complexes in presence or absence of traditionally used chemotherapeutic agents (doxorubicin, paclitaxel, or cisplatin) for a consecutive period of 48 hours prior to cell viability assessment by mtt assay . As shown in figure 1a, while the viability of the cancer cells was reduced to 36% due to the potent cytoxicity of 100 nm doxorubicin, the concerted effect of sirna delivery and drug exposure resulted in 26% and 24% reduction in cell viability for 1 nm and 10 nm of sirna initally used for complex formation, respectively . Thus, the combination of sirna and doxorubicin killed a much higher number of cells than the drug and the sirna alone, suggesting that silencing of the abcg2 gene might have inhibited to a significant extent the efflux of doxorubicin across the membrane, and raised intracellular concentration of the drug for effective killing of the cancer cells (table 1). Abcg2 gene knockdown similarly accelerated chemosensitivity of paclitaxel (100 nm) particularly at the higher dose of sirna (10 nm), while demonstrating an insignificant response with cisplatin (figure 1b, table 1). The apparent enhancement of chemosensitivity to cisplatin, as shown in table 1, was virtually due to the silencing effect of sirna (figure 1c) alone . Indeed, the decrease in viability of the cells treated with sirna / nanoparticle complexes in comparison with those untreated or treated with nanoparticles alone indicates the role of abc expression in delaying apoptosis of cancer cells.4042 based on the notion that anti - cancer drugs could dose - dependently influence the expression profile or susbtrate specificity of abc transporters, a range of relatively lower concentrations of the drugs was investigated in the knockdown study of abc transporters using 1 nm of target sirnas . As shown in figure 2 and table 2, with drug doses ranging from 10 pm to 1 nm for doxorubicin as well as paclitaxel and 100 pm to 10 nm for cisplatin, silencing of abcg2 gene expression resulted in significant enhancement of chemosensitivity towards doxorubicin and paclitaxel in all of the drug concentrations used, whereas a weak response to 100 pm of cisplatin was observed probably due to the poor cytotoxic effect of cisplatin at the particular concentration . The finding thus suggests that the chemosensitivity of mcf-7 breast cancer cells due to the knockdown of abcg2 gene is virtually similar among the three drugs in respect to the maximum enhancement and dependent on the dose of the invidual drug . The enhancement of chemosensitivity to the popular anti - cancer drugs by virtue of silencing abcg2 gene expression triggered us to carry out a similar study in the same breast cancer cells using the sirna targeting the gene of abcb1, the most extensively studied abc transporter involved in development of multi - drug resistance in various cancer cells including mcf-7.13 as shown in figure 3, in synergy with 100 nm of paclitaxel, anti - abcb1 sirna at both 1 and 10 nm led to killing of much more cancer cells than the target sirna (1 and 10 nm) or the drug (100 nm) alone, resulting in 14% and 33% enhancement of chemosensitivity respectively . On the contrary, the apparent enhancement of c hemosensitivity (to the same extent) towards both 100 nm of doxorubicin and cisplatin following knock - down of abcb1 transporter (table 3) was solely due to the effect of gene silencing killing more cells than the individual drugs or the target sirna alone (figure 3). However, when the doses of the individual drugs were reduced to 10 pm to 1 nm for doxorubicin and paclitaxel, sirna - mediated cleavage of abcb1 gene transcript surprisingly increased chemosensitivity towards each of the two drugs (table 4) with demonstration of significantly higher level of cytotoxicity than sirna or drugs alone (figure 4), suggesting the broader substrate - specificity of abcb1 transporter at lower concentrations of the individual cancer drugs . On ther other hand, the apparent increase in chemosensitivity for 100 pm to 10 nm of cisplatin was statistically insignificant, which is consistent with the fact that cisplatin is not a substrate for abcb1 . Regardless of the type of sirna (abcg2 or abcb1) used in this study, the enhancement of chemosensitivity after single sirna treatment was only modest . This observation was similar to that made by other groups, where no complete reversal of cancer multidrug resistance was noted even at sirna concentration as high as 200 nm.33,43 such incomplete reversal of cancer multidrug resistance may be attributed to the inherent nature of the target protein under study . Due to its long half - life (from 16 hours44 to 72 hours45) and high cellular abundance, abc transporter protein remains a challenging target for sirna silencing.46 delivery of single sirna targeting either abcg2 or abcb1 transporter with carbonate apatite nanoparticles demonstrated a modest 15% to 30% enhancement in c hemosensitivity in mcf-7 cells (tables 14). To explore whether the combined delivery of two specific sirnas simultaneously targeting the two different transporters could further improve the chemo - sensitivity towards the anti - cancer drugs, mcf-7 cells were transfected with the sirnas (at 1 nm each) targeting the gene transcripts of abcg2 and abcb1 in presence of 1 nm of the individual drugs . As shown in figure 5, nanoparticle - facilitated intracellular delivery of single sirna targeting a particular transporter killed fewer cells than co - delivery of the two target sirnas, suggesting that both of the transporters, which are intrinsically expressed in mcf-7 cells,13,17 are important for survival of the cells.4042 on the other hand, simultaneous delivery of the two target sirnas into mcf-7 cells in presence of cisplatin, paclitaxel, or doxorubicin, was found to dramatically enhance cytotoxicity with enhancement of chemosensitivity by 50%, 49% and 43%, respectively (table 5), in comparison to the individual drugs and the sirnas co - delivered without a drug . This suggests that both abcg2 and abcb1 are fully functional at a time in extruding any of those popular anti - cancer drugs from the cytoplasm of mcf-7 cells and inhibition or knockdown of a single transporter could only partly block the cellular efflux process, allowing drug efflux to continue through the other channel(s). Since abc transporter gene expression is essential in causing multidrug - resistant phenotype in breast cancer cells, knockdown of this gene might enhance sensitivity of the cancer cells towards chemotherapeutic agents, and possibly reverse cancer multidrug resistance . Therefore, mcf-7, the widely used human breast model cell line intrinsically expressing abcg2 (alternatively called brcp or breast cancer resistance protein),17 was transfected with the target sirna / carbonate apatite complexes in presence or absence of traditionally used chemotherapeutic agents (doxorubicin, paclitaxel, or cisplatin) for a consecutive period of 48 hours prior to cell viability assessment by mtt assay . As shown in figure 1a, while the viability of the cancer cells was reduced to 36% due to the potent cytoxicity of 100 nm doxorubicin, the concerted effect of sirna delivery and drug exposure resulted in 26% and 24% reduction in cell viability for 1 nm and 10 nm of sirna initally used for complex formation, respectively . Thus, the combination of sirna and doxorubicin killed a much higher number of cells than the drug and the sirna alone, suggesting that silencing of the abcg2 gene might have inhibited to a significant extent the efflux of doxorubicin across the membrane, and raised intracellular concentration of the drug for effective killing of the cancer cells (table 1). Abcg2 gene knockdown similarly accelerated chemosensitivity of paclitaxel (100 nm) particularly at the higher dose of sirna (10 nm), while demonstrating an insignificant response with cisplatin (figure 1b, table 1). The apparent enhancement of chemosensitivity to cisplatin, as shown in table 1, was virtually due to the silencing effect of sirna (figure 1c) alone . Indeed, the decrease in viability of the cells treated with sirna / nanoparticle complexes in comparison with those untreated or treated with nanoparticles alone indicates the role of abc expression in delaying apoptosis of cancer cells.4042 based on the notion that anti - cancer drugs could dose - dependently influence the expression profile or susbtrate specificity of abc transporters, a range of relatively lower concentrations of the drugs was investigated in the knockdown study of abc transporters using 1 nm of target sirnas . As shown in figure 2 and table 2, with drug doses ranging from 10 pm to 1 nm for doxorubicin as well as paclitaxel and 100 pm to 10 nm for cisplatin, silencing of abcg2 gene expression resulted in significant enhancement of chemosensitivity towards doxorubicin and paclitaxel in all of the drug concentrations used, whereas a weak response to 100 pm of cisplatin was observed probably due to the poor cytotoxic effect of cisplatin at the particular concentration . The finding thus suggests that the chemosensitivity of mcf-7 breast cancer cells due to the knockdown of abcg2 gene is virtually similar among the three drugs in respect to the maximum enhancement and dependent on the dose of the invidual drug . The enhancement of chemosensitivity to the popular anti - cancer drugs by virtue of silencing abcg2 gene expression triggered us to carry out a similar study in the same breast cancer cells using the sirna targeting the gene of abcb1, the most extensively studied abc transporter involved in development of multi - drug resistance in various cancer cells including mcf-7.13 as shown in figure 3, in synergy with 100 nm of paclitaxel, anti - abcb1 sirna at both 1 and 10 nm led to killing of much more cancer cells than the target sirna (1 and 10 nm) or the drug (100 nm) alone, resulting in 14% and 33% enhancement of chemosensitivity respectively . On the contrary, the apparent enhancement of c hemosensitivity (to the same extent) towards both 100 nm of doxorubicin and cisplatin following knock - down of abcb1 transporter (table 3) was solely due to the effect of gene silencing killing more cells than the individual drugs or the target sirna alone (figure 3). However, when the doses of the individual drugs were reduced to 10 pm to 1 nm for doxorubicin and paclitaxel, sirna - mediated cleavage of abcb1 gene transcript surprisingly increased chemosensitivity towards each of the two drugs (table 4) with demonstration of significantly higher level of cytotoxicity than sirna or drugs alone (figure 4), suggesting the broader substrate - specificity of abcb1 transporter at lower concentrations of the individual cancer drugs . On ther other hand, the apparent increase in chemosensitivity for 100 pm to 10 nm of cisplatin was statistically insignificant, which is consistent with the fact that cisplatin is not a substrate for abcb1 . Regardless of the type of sirna (abcg2 or abcb1) used in this study, the enhancement of chemosensitivity after single sirna treatment was only modest . This observation was similar to that made by other groups, where no complete reversal of cancer multidrug resistance was noted even at sirna concentration as high as 200 nm.33,43 such incomplete reversal of cancer multidrug resistance may be attributed to the inherent nature of the target protein under study . Due to its long half - life (from 16 hours44 to 72 hours45) and high cellular abundance, abc transporter protein remains a challenging target for sirna silencing.46 delivery of single sirna targeting either abcg2 or abcb1 transporter with carbonate apatite nanoparticles demonstrated a modest 15% to 30% enhancement in c hemosensitivity in mcf-7 cells (tables 14). To explore whether the combined delivery of two specific sirnas simultaneously targeting the two different transporters could further improve the chemo - sensitivity towards the anti - cancer drugs, mcf-7 cells were transfected with the sirnas (at 1 nm each) targeting the gene transcripts of abcg2 and abcb1 in presence of 1 nm of the individual drugs . As shown in figure 5, nanoparticle - facilitated intracellular delivery of single sirna targeting a particular transporter killed fewer cells than co - delivery of the two target sirnas, suggesting that both of the transporters, which are intrinsically expressed in mcf-7 cells,13,17 are important for survival of the cells.4042 on the other hand, simultaneous delivery of the two target sirnas into mcf-7 cells in presence of cisplatin, paclitaxel, or doxorubicin, was found to dramatically enhance cytotoxicity with enhancement of chemosensitivity by 50%, 49% and 43%, respectively (table 5), in comparison to the individual drugs and the sirnas co - delivered without a drug . This suggests that both abcg2 and abcb1 are fully functional at a time in extruding any of those popular anti - cancer drugs from the cytoplasm of mcf-7 cells and inhibition or knockdown of a single transporter could only partly block the cellular efflux process, allowing drug efflux to continue through the other channel(s). Due to the heterogenous nature of tumours where several drug resistance mechanisms may coexist and simultaneously contribute to the development of cancer multidrug resistant - phenotype, 11 the use of sirna combinations might represent a potential mechanism for circumventing this problem . Additionally, the concern about saturating the cellular rna interference machinery by exogenous addition of two sirnas may have been potentially overcome by combining sirnas at the lowest possible concentration . Thus, the synergism achieved with combination of abcg2 and abcb1 targeting sirnas would provide valuable insight for complete reversal of clinical cancer multidrug resistance otherwise deemed impossible to be achieved by currently available therapeutics.
As a strategy for antipsychotic treatment of schizophrenia, monotherapy is clearly optimal when both effective and tolerated . When a patient fails to respond to an adequate dose of an antipsychotic, however, the alternatives include switching, administering a dose higher than the licensed dose, polypharmacy or clozapine . Clozapine is the only option with established efficacy, but is less manageable than other antipsychotics, with a relatively high frequency of clozapine - induced agranulocytosis . Little evidence has been accumulated from acute - phase practice, as enrolling acute psychotic and agitated patients in randomized clinical trials is challenging, particularly when using a double - blind design . However, most clinical guidelines are based on findings from double - blind trials, which include ideal patients.1) such guidelines are therefore not necessarily useful in actual clinical practice . From this practical perspective, strategies for early non - response to an antipsychotic drug in acute - phase schizophrenia are discussed (fig . How long an antipsychotic should be trialed before being viewed as ineffective is a key unanswered question in clinical trials for patients with schizophrenia.2) although previous studies have identified early non - response as a robust predictor of subsequent non - response with continued treatment of the same medication,3,4,5,6,7) those studies were retrospective in nature . The first prospective study was performed by kinon et al.8) finding early response / non - response to risperidone at 2 weeks as a reliable clinical marker of subsequent clinical outcomes (table 1). We examined whether early response / non - response to risperidone according to the clinical global impressions - improvement scale (cgi - i) at 2 weeks could predict subsequent response in the treatment of acute - phase schizophrenia.9) at 4 weeks, 81% of risperidone early responders achieved 50% response, whereas only 9% of early non - responders staying on risperidone achieved 50% response, with a negative likelihood ratio of 0.057 . In contrast, the negative likelihood ratio for the prediction of 50% response at 4 weeks based on early response status to olanzapine at 2 weeks was 0.28, suggesting this prediction was not necessarily applicable to olanzapine . Levine and leucht10) recently reanalyzed data from a double - blind, randomized, multicenter, international clinical trial comparing the effectiveness of treatment with olanzapine and haloperidol in recent - onset psychotic patients, in which patients were followed up to 84 weeks.11) they reported that early response (i.e., within 2 weeks) is marked by up to 39 weeks of longer subsequent symptom response than non - response (i.e., in the initial 4 weeks), and infrequently differs to delayed - response (i.e., 3 - 4 weeks). As half of patients were allocated to receive olanzapine, results from the reanalysis may not necessarily be applicable to risperidone . Furthermore, participants were defined as patients with recent - onset psychosis with an interval since first onset of psychotic symptoms of 1 month but 60 months prior to study entry, indicating that patients were not necessarily in the acute phase . Mean total scores for the positive and negative syndrome scale (panss) at baseline were relatively low, i.e. : early - responder, 57.24; delayed - responder, 61.93; and non - responder, 78.32, also indicating that most cases were not in the acute phase . Derks et al.12) reported data from 299 first - episode patients who completed the full 12-month european first - episode schizophrenia trial, finding that remission status was correctly predicted in 61% of patients based on baseline and 2-week assessments, and this percentage increased to 63% and 68%, respectively, with the inclusion of 4- and 6-week assessments, respectively . Accordingly, they concluded that earlier 2-week measures of response are associated with remission, and that as the increase in prediction accuracy by including 4- and 6-week assessments is modest, whether such improvement is clinically relevant remains uncertain . Focusing on acutely psychotic patients, o'gorman et al.13) reported that from two separate pooled analyses of two placebo - controlled and two active comparator (risperidone and olanzapine) randomized trials of ziprasidone in schizophrenia, non - improvement in weeks 1 or 2 was highly predictive of non - response, and week 2 improvement was more reliably predictive of subsequent outcome than week 1 improvement, with high sensitivity and specificity . Giegling et al.14) reported that a panss reduction 16% at 1 week predicts non - response at 3 weeks of treatment with haloperidol (specificity 92%, sensitivity 82%), and, conversely, a panss reduction 23% at 1 week of treatment predicts response at 3 weeks, with 84% specificity and 86% sensitivity . Considering our results that non - response to risperidone at 2 weeks can predict subsequent response in newly admitted patients with acute schizophrenia, and that significant response to olanzapine does not seem to occur until 4 weeks,9) the duration for which an antipsychotic should be trialed before being viewed as ineffective might depend on the kind of antipsychotic, such as affinity and specificity for dopamine d2 receptors . According to these findings, an antipsychotic may be able to be viewed as ineffective within 1 - 4 weeks in acute - phase practice, although some differences may exist among specific antipsychotics . Before we presume that identifying early non - responders minimizes prolonged exposure to suboptimal or ineffective treatment strategies, alternative treatment strategies such as " switching ", " augmentation ", and " high - dose " antipsychotics should be evaluated . First, we reviewed whether patients who are early non - responders to an antipsychotic and are then switched to another antipsychotic would show significantly greater improvement in psychopathology, compared with those staying on the initial antipsychotic . In a switching study of first - episode patients with schizophrenia who showed residual symptoms following treatment with risperidone, takahashi et al.15) reported the rate of responders to olanzapine was 29.3% . They also reported that in another switching study of first - episode patients with schizophrenia who experienced residual symptoms following treatment with olanzapine, the rate of response to risperidone was 35.3%.16) however, the patients enrolled were those who were able to complete 12 weeks of treatment with the initial antipsychotic, indicating that they were not in the acute phase . Furthermore, those studies lacked a control group of patients staying on the initial antipsychotic, and we therefore cannot conclude that switching is indeed a beneficial option . Although essock et al.17) and rosenheck et al.18) reported no benefit of switching antipsychotic medications in analyses of the clinical antipsychotic trials of intervention effectiveness (catie) trial, which included a control group, those analyses were post hoc exploratory nature . The first randomized, double - blind study of whether' switching' early non - responders to another antipsychotic represents a better strategy than' staying' was reported by kinon et al.8) they showed that switching to risperidone in early non - responders to olanzapine at week 2 resulted in a small but significantly greater reduction in panss total score and in depressive symptoms . The significant difference in panss total score at end point mean change at week 12 was reportedly 3.49 . We failed to show any robust advantage of the switching strategy for early non - responders within the ordinary doses of risperidone or olanzapine in acute - phase clinical practice, probably due to the lack of statistical power for the randomized phase of the study.9) agid et al.19) reported that from data gathered from a treatment algorithm implemented in patients with first - episode schizophrenia that employs two antipsychotic trials at increasing doses before clozapine, the percentage response for subjects who switched from olanzapine to risperidone was 4.0%, compared to 25.7% for those who switched from risperidone to olanzapine . Thus, whether a switching strategy is effective might depend on the initial antipsychotic administered and the antipsychotic switched to . A substantial proportion of schizophrenia patients receive more than one antipsychotic.20,21,22) the current problem is that the degree of polypharmacy being practiced seems far in excess of the supporting data.2) in a systematic review of 19 randomized studies, the pooled odds ratio suggested a small effect favoring combination treatment, and positive effects appear to have been associated with studies using clozapine combinations.23) however, clozapine is not tolerated by some patients . Studies combining non - clozapine second - generation antipsychotics with each other and with the first - generation antipsychotics utilized most in clinical practice are thus required.23) kotler et al.24) indicated no significant differences in changes to positive or negative symptomatology between patients receiving a combined regimen of olanzapine with sulpiride augmentation and those receiving olanzapine monotherapy among chronic schizophrenia patients unresponsive to olanzapine . Kane et al.25) reported that addition of aripiprazole to either risperidone or quetiapine in 323 patients showed no greater efficacy than placebo added to either risperidone or quetiapine . In contrast, essock et al.26) reported that patients assigned to a switch to monotherapy displayed shorter times to all - cause treatment discontinuation than those assigned to remain on polypharmacy . These studies were indicators of what could happen with antipsychotic combinations in chronic - phase patients . We have presented the first randomized clinical trial of olanzapine augmentation of risperidone in patients non - responsive to risperidone monotherapy in the acute phase.27) in the study, early response was defined as cgi - i 3 following 2 weeks of treatment, and early non - responders were then allocated to receive either augmentation with olanzapine (ris+olz group) or increased risperidone dose (ris+ris group). Although time to treatment discontinuation for any cause was significantly shorter in the ris+ris group than in early responders to risperidone, no significant difference was evident between the ris+olz group and early responders to risperidone . These outcomes justify the inclusion of augmentation arms in additional, larger studies comparing strategies for early non - responders in the treatment of acute - phase schizophrenia . In clinical practice, nearly 50% of olanzapine prescriptions in the united states was reportedly above 20 mg / day28) and the median recommended dose for olanzapine by u.s . Experts was 30 mg / day.29) the upper limit of olanzapine dose in the catie study, in which olanzapine was the most effective in terms of rates of discontinuation, was designed to be 30 mg / day.30) in chronic schizophrenia inpatients showing suboptimal response to treatment, one randomized controlled trial allowed the use of high doses of olanzapine.31) in that study, clozapine and olanzapine were superior to haloperidol, but no obvious superiority of risperidone over haloperidol was seen . In patients with treatment - resistant schizophrenia, olanzapine at higher - than - customary doses reportedly demonstrated similar efficacy to clozapine32) or was less effective than clozapine.33) for acute - phase patients, we presented the first randomized clinical trial that examined whether olanzapine at 40 mg / day would be superior to risperidone at 12 mg / day.34) as the numbers of patients allocated to each treatment group did not reach the required numbers set by power analysis, the results were not conclusive . Although time to treatment discontinuation due to any cause did not differ between treatment groups, more than half of cases that were non - responsive to conventional doses showed moderate improvement on subsequent treatment with high doses . In the study, serum olanzapine concentrations at the time of oral 20 mg / day could be obtained from 5 out of 7 patients who subsequently required high - dose olanzapine . All values were> 30 ng / ml, which were appropriate with regard to a therapeutic range of 20 - 50 ng / ml . The reason for requiring high - dose olanzapine in the treatment of acute - phase schizophrenia thus cannot be explained simply based on pharmacokinetics . Furthermore, 2 of 5 patients who subsequently required high - dose olanzapine, and from whom serum olanzapine concentrations could be obtained, were antipsychotic - nave, suggesting that the etiology of dopamine supersensitivity psychosis was not applicable in those cases.35) how long an antipsychotic should be trialed before being viewed as ineffective is a key unanswered question in clinical trials for patients with schizophrenia.2) although previous studies have identified early non - response as a robust predictor of subsequent non - response with continued treatment of the same medication,3,4,5,6,7) those studies were retrospective in nature . The first prospective study was performed by kinon et al.8) finding early response / non - response to risperidone at 2 weeks as a reliable clinical marker of subsequent clinical outcomes (table 1). We examined whether early response / non - response to risperidone according to the clinical global impressions - improvement scale (cgi - i) at 2 weeks could predict subsequent response in the treatment of acute - phase schizophrenia.9) at 4 weeks, 81% of risperidone early responders achieved 50% response, whereas only 9% of early non - responders staying on risperidone achieved 50% response, with a negative likelihood ratio of 0.057 . In contrast, the negative likelihood ratio for the prediction of 50% response at 4 weeks based on early response status to olanzapine at 2 weeks was 0.28, suggesting this prediction was not necessarily applicable to olanzapine . Levine and leucht10) recently reanalyzed data from a double - blind, randomized, multicenter, international clinical trial comparing the effectiveness of treatment with olanzapine and haloperidol in recent - onset psychotic patients, in which patients were followed up to 84 weeks.11) they reported that early response (i.e., within 2 weeks) is marked by up to 39 weeks of longer subsequent symptom response than non - response (i.e., in the initial 4 weeks), and infrequently differs to delayed - response (i.e., 3 - 4 weeks). As half of patients were allocated to receive olanzapine, results from the reanalysis may not necessarily be applicable to risperidone . Furthermore, participants were defined as patients with recent - onset psychosis with an interval since first onset of psychotic symptoms of 1 month but 60 months prior to study entry, indicating that patients were not necessarily in the acute phase . Mean total scores for the positive and negative syndrome scale (panss) at baseline were relatively low, i.e. : early - responder, 57.24; delayed - responder, 61.93; and non - responder, 78.32, also indicating that most cases were not in the acute phase . Derks et al.12) reported data from 299 first - episode patients who completed the full 12-month european first - episode schizophrenia trial, finding that remission status was correctly predicted in 61% of patients based on baseline and 2-week assessments, and this percentage increased to 63% and 68%, respectively, with the inclusion of 4- and 6-week assessments, respectively . Accordingly, they concluded that earlier 2-week measures of response are associated with remission, and that as the increase in prediction accuracy by including 4- and 6-week assessments is modest, whether such improvement is clinically relevant remains uncertain . Focusing on acutely psychotic patients, o'gorman et al.13) reported that from two separate pooled analyses of two placebo - controlled and two active comparator (risperidone and olanzapine) randomized trials of ziprasidone in schizophrenia, non - improvement in weeks 1 or 2 was highly predictive of non - response, and week 2 improvement was more reliably predictive of subsequent outcome than week 1 improvement, with high sensitivity and specificity . Giegling et al.14) reported that a panss reduction 16% at 1 week predicts non - response at 3 weeks of treatment with haloperidol (specificity 92%, sensitivity 82%), and, conversely, a panss reduction 23% at 1 week of treatment predicts response at 3 weeks, with 84% specificity and 86% sensitivity . Considering our results that non - response to risperidone at 2 weeks can predict subsequent response in newly admitted patients with acute schizophrenia, and that significant response to olanzapine does not seem to occur until 4 weeks,9) the duration for which an antipsychotic should be trialed before being viewed as ineffective might depend on the kind of antipsychotic, such as affinity and specificity for dopamine d2 receptors . According to these findings, an antipsychotic may be able to be viewed as ineffective within 1 - 4 weeks in acute - phase practice, although some differences may exist among specific antipsychotics . Before we presume that identifying early non - responders minimizes prolonged exposure to suboptimal or ineffective treatment strategies, alternative treatment strategies such as " switching ", " augmentation ", and " high - dose " antipsychotics should be evaluated . First, we reviewed whether patients who are early non - responders to an antipsychotic and are then switched to another antipsychotic would show significantly greater improvement in psychopathology, compared with those staying on the initial antipsychotic . In a switching study of first - episode patients with schizophrenia who showed residual symptoms following treatment with risperidone, takahashi et al.15) reported the rate of responders to olanzapine was 29.3% . They also reported that in another switching study of first - episode patients with schizophrenia who experienced residual symptoms following treatment with olanzapine, the rate of response to risperidone was 35.3%.16) however, the patients enrolled were those who were able to complete 12 weeks of treatment with the initial antipsychotic, indicating that they were not in the acute phase . Furthermore, those studies lacked a control group of patients staying on the initial antipsychotic, and we therefore cannot conclude that switching is indeed a beneficial option . Although essock et al.17) and rosenheck et al.18) reported no benefit of switching antipsychotic medications in analyses of the clinical antipsychotic trials of intervention effectiveness (catie) trial, which included a control group, those analyses were post hoc exploratory nature . The first randomized, double - blind study of whether' switching' early non - responders to another antipsychotic represents a better strategy than' staying' was reported by kinon et al.8) they showed that switching to risperidone in early non - responders to olanzapine at week 2 resulted in a small but significantly greater reduction in panss total score and in depressive symptoms . The significant difference in panss total score at end point mean change at week 12 was reportedly 3.49 . We failed to show any robust advantage of the switching strategy for early non - responders within the ordinary doses of risperidone or olanzapine in acute - phase clinical practice, probably due to the lack of statistical power for the randomized phase of the study.9) agid et al.19) reported that from data gathered from a treatment algorithm implemented in patients with first - episode schizophrenia that employs two antipsychotic trials at increasing doses before clozapine, the percentage response for subjects who switched from olanzapine to risperidone was 4.0%, compared to 25.7% for those who switched from risperidone to olanzapine . Thus, whether a switching strategy is effective might depend on the initial antipsychotic administered and the antipsychotic switched to . A substantial proportion of schizophrenia patients receive more than one antipsychotic.20,21,22) the current problem is that the degree of polypharmacy being practiced seems far in excess of the supporting data.2) in a systematic review of 19 randomized studies, the pooled odds ratio suggested a small effect favoring combination treatment, and positive effects appear to have been associated with studies using clozapine combinations.23) however, clozapine is not tolerated by some patients . Studies combining non - clozapine second - generation antipsychotics with each other and with the first - generation antipsychotics utilized most in clinical practice are thus required.23) kotler et al.24) indicated no significant differences in changes to positive or negative symptomatology between patients receiving a combined regimen of olanzapine with sulpiride augmentation and those receiving olanzapine monotherapy among chronic schizophrenia patients unresponsive to olanzapine . Kane et al.25) reported that addition of aripiprazole to either risperidone or quetiapine in 323 patients showed no greater efficacy than placebo added to either risperidone or quetiapine . In contrast, essock et al.26) reported that patients assigned to a switch to monotherapy displayed shorter times to all - cause treatment discontinuation than those assigned to remain on polypharmacy . These studies were indicators of what could happen with antipsychotic combinations in chronic - phase patients . We have presented the first randomized clinical trial of olanzapine augmentation of risperidone in patients non - responsive to risperidone monotherapy in the acute phase.27) in the study, early response was defined as cgi - i 3 following 2 weeks of treatment, and early non - responders were then allocated to receive either augmentation with olanzapine (ris+olz group) or increased risperidone dose (ris+ris group). Although time to treatment discontinuation for any cause was significantly shorter in the ris+ris group than in early responders to risperidone, no significant difference was evident between the ris+olz group and early responders to risperidone . These outcomes justify the inclusion of augmentation arms in additional, larger studies comparing strategies for early non - responders in the treatment of acute - phase schizophrenia . In clinical practice, nearly 50% of olanzapine prescriptions in the united states was reportedly above 20 mg / day28) and the median recommended dose for olanzapine by u.s . Experts was 30 mg / day.29) the upper limit of olanzapine dose in the catie study, in which olanzapine was the most effective in terms of rates of discontinuation, was designed to be 30 mg / day.30) in chronic schizophrenia inpatients showing suboptimal response to treatment, one randomized controlled trial allowed the use of high doses of olanzapine.31) in that study, clozapine and olanzapine were superior to haloperidol, but no obvious superiority of risperidone over haloperidol was seen . In patients with treatment - resistant schizophrenia, olanzapine at higher - than - customary doses reportedly demonstrated similar efficacy to clozapine32) or was less effective than clozapine.33) for acute - phase patients, we presented the first randomized clinical trial that examined whether olanzapine at 40 mg / day would be superior to risperidone at 12 mg / day.34) as the numbers of patients allocated to each treatment group did not reach the required numbers set by power analysis, the results were not conclusive . Although time to treatment discontinuation due to any cause did not differ between treatment groups, more than half of cases that were non - responsive to conventional doses showed moderate improvement on subsequent treatment with high doses . In the study, serum olanzapine concentrations at the time of oral 20 mg / day could be obtained from 5 out of 7 patients who subsequently required high - dose olanzapine . All values were> 30 ng / ml, which were appropriate with regard to a therapeutic range of 20 - 50 ng / ml . The reason for requiring high - dose olanzapine in the treatment of acute - phase schizophrenia thus cannot be explained simply based on pharmacokinetics . Furthermore, 2 of 5 patients who subsequently required high - dose olanzapine, and from whom serum olanzapine concentrations could be obtained, were antipsychotic - nave, suggesting that the etiology of dopamine supersensitivity psychosis was not applicable in those cases.35) to establish strategies for early non - response to an antipsychotic drug in the treatment of acute - phase schizophrenia, evidence for antipsychotic switching, augmentation, or high - doses has gradually been accumulating . More studies performed in actual clinical practice with minimal bias are required to assist clinicians in making rational treatment decisions.
The difficulty in making the correct diagnosis because of the multitude of coinfections and comorbidities in the hiv population led the authors to a first erroneous diagnosis almost compromising the results of treatment . This experience is discussed further in the text . The patient wpo, from the city of so paulo (brazil), a 75-year - old caucasian male, had been receiving antiretroviral treatment in hospital das clnicas of so paulo, since being diagnosed with hiv 12 years earlier (now taking lamivudine, tenofovir, atazanavir, and ritonavir for the last three years). He was in a good general condition, with good viral and immunological control (undetectable viral load and cd4 count of 653/ml). Other laboratory tests were normal, and he did not have any other coinfections, had no drug addiction, and no other comorbidities . He sought medical assistance because of a complaint of patellofemoral pain in his right knee that had started one year earlier with increasing local volume and progressive inability to perform active extension . He had a knee extension lag of 30. he was able to walk only with a stick . The increased volume was concentrated below the patella and posterior to the patellar tendon, without local inflammatory signs, history of trauma, and infection . In addition, a tendinopathy with partial chronic lesion of the patellar tendon probably explained the clinical findings . It was decided first to perform a magnetic resonance imaging (mri) [figures 1 and 2]. This revealed the presence of edema and thickening of the infrapatellar fat, with the formation of a nodule measuring 2.01.62.0 cm, with low signal and no impregnation with contrast . The patellar tendon was found thickened, with foci of partial ruptures and with peritendinous edema . Sagital mri image, showing thickening of hoffa's fat pad and adjacent synovial membrane, with foci of partial rupture of the patellar tendon axial mri image, showing a solid fibrous mass in hoffa's fat pad the treatment for this situation is essentially nonsurgical . Since our patient did not show any improvement through physiotherapy and drug treatment, surgical treatment was indicated . This was done with the aim of exploring the patellar tendon and reconstructing it if necessary, along with partial resection of hoffa's fat and the nodule, with pathological analysis of the tissue . The intraoperative appearance of the knee showed an intact patellar tendon with an infiltrated tissue around hoffa's fat, without any local inflammatory reaction [figure 3]. It was decided only to biopsy this tissue and culture for bacteria, mycobacteria, and fungi . Intraoperative aspect of the lesion in hoffa's fat pad, with an intact patellar tendon none of the cultures was positive . On the 14 day after the operation, the results from the anatomopathological examination revealed that the nodule consisted of a high - grade malignant fibrohistiocytoma, without sufficient margins of resection . After ruling out metastasis [by thoracic and abdomen computed tomography (ct) and bone scintigraphy], our patient underwent bloc resection of the entire joint, including the distal femur, proximal tibia, patella, and musculature of the distal quadriceps . The knee joint was reconstructed using an endoprosthesis, which was covered by rotating a flap from the medial gastrocnemius muscle and the extensor mechanism was reconstructed using the sartorius muscle . The patient has a good functional outcome, walking with the aid of one crutch and without any recidive after 18 months of the surgery . The most common causes for this pain are degenerative or traumatic modifications of the patellar and trochlear cartilage, tendonitis, inflammatory processes of hoffa's fat, and patellar dysfunction caused by muscle weakness, shortening, or instability . The pain - specific localization was suggestive of an inflammatory process in hoffa's fat or chronic partial lesion of the patellar tendon, which would explain the active extension deficit . Within the context of considerable increase in life expectancy among patients infected with hiv achieved since the introduction of highly active antiretroviral therapy (haart), certain osteoarticular consequences of prolonged duration of viral infection and its treatment have been observed, such as osteopenia / osteoporosis, osteonecrosis, carpal tunnel syndrome, adhesive capsulitis of the shoulders, modifications of body shape, and changes in lipid and glucose metabolism . A care and study group called the bone - hiv group was created in our hospital to assist patients with hiv / aids presenting any orthopedic complaint . The medical literature on this orthopedic abnormality is very sparse, and we have not found any reports or epidemiological characterization of patellofemoral pathological conditions among this group of patients . One of the few studies on this subject in the literature described abnormalities in hoffa's fat in patients with hiv, which may have an etiological link to patellofemoral pain . So, the typical changes induced by long - term infection and therapy of hiv may have a negative impact on the extensor mechanism of the knee, which led us to an erroneous initial diagnosis, instead of a much more aggressive disease . Malignant fibrohistiocytoma was first described in 1964 and is currently known as high - grade pleomorphic sarcoma in adult life . Malignant fibrohistiocytoma usually presents as a deep painless mass, without other local or systemic symptoms . The staging depends on the histological grade, size, and presence of metastasis, which is the most important prognostic factor regarding survival . Radiotherapy is administered in most cases, with good results relating to local control, either before or after the surgery . The role of chemotherapy is unclear, but it can be administered particularly in cases with a high risk of metastases . In this case, chemotherapy was not used because of unclear evidence of benefit and the risk of side effects . As the case evolved, it became clear that the abnormalities in the patient's knee were unrelated to his chronic hiv infection . However, this case once again raises the issue of lack of knowledge of the long - term changes caused by this disease . Since there have been sporadic reports of abnormalities of hoffa's fat among such patients, along with the nonspecific presentation of the condition in our patient, we were led to believe that his condition could have been related to hiv . We again emphasize that studies on osteoarticular changes (such as bone density, muscular mass, joint mechanics, and cartilage loading) induced by chronic hiv infection need to be conducted, to epidemiologically define this population better and diminish the frequency of erroneous diagnoses among patients with hiv / aids.
(baill .) Is a source plant of traditional chinese medicine . The s. chinensis fruit (sf), which has five - flavored fruits (salty, pungent, bitter, sweet and sour), is called wuweizi in china . It naturally distribute in china, russian, korea and japan and was used in treatment of diseases including liver injury, tumor inhibition, urinary tract disorders, insomnia and palpitation, even inhibit both hiv-1 rt - associated dna polymerase activity and virus replication . In addition to its application as a chinese herbal medicine . It is also used for making beverages, tea, wine, health food industries and cosmetics . Therefore, recent research has been focused on extracting active ingredients and identifying function . Despite of the importance of s. chinensis, the next generation sequencing technology has dramatically improved the efficiency and speed of gene discovery . Two cultivars yanhong (red skin) and jinwuwei (white skin) were grown in orchard located in zuojia town, jilin city, jilin province, china (440647 n 1260718 e) that belong to the plant resources nursery of institute of special wild economic animal and plant science, chinese academy of agricultural sciences . Fours samples, including fruits and skins, were harvested and immediately frozen in liquid nitrogen for further experiments . Total rna was extracted using modified ctab method, library construction and high - throughput sequencing for each sample was performed at a contract sequencing company (novogene, china). The cdna library was sequenced using an illumina hiseq2500 platform . With the purpose of determining the red fruit, white fruit, red skin of red fruit and white skin of white fruit of s. chinensis . We obtained a total of 72.5 million, 55.1 million, 68 million and 69.3 million raw data reads from four sequencing libraries prepared, respectively (table 1),> 94.9% bases has a q value 20 (an error probability of 0.035%). After cleaning and quality checks, the de novo assembly of all sequencing data using the trinity method . It generated 92,415 all - transcripts with an average length of 496 bp and an n50 of 1466 bp; and 71,443 all - unigenes were achieved . Of these, 46,461 (65.0%) were 200500 bp, 11,322 were 5001000 bp, 8612 were 12 kb and the remaining 5078 were> 2 kb (table 2). The results provides us useful information for further explorer the gene synthesis pathways of active ingredients . Two cultivars yanhong (red skin) and jinwuwei (white skin) were grown in orchard located in zuojia town, jilin city, jilin province, china (440647 n 1260718 e) that belong to the plant resources nursery of institute of special wild economic animal and plant science, chinese academy of agricultural sciences . Fours samples, including fruits and skins, were harvested and immediately frozen in liquid nitrogen for further experiments . Total rna was extracted using modified ctab method, library construction and high - throughput sequencing for each sample was performed at a contract sequencing company (novogene, china). With the purpose of determining the red fruit, white fruit, red skin of red fruit and white skin of white fruit of s. chinensis . We obtained a total of 72.5 million, 55.1 million, 68 million and 69.3 million raw data reads from four sequencing libraries prepared, respectively (table 1),> 94.9% bases has a q value 20 (an error probability of 0.035%). After cleaning and quality checks, the de novo assembly of all sequencing data using the trinity method . It generated 92,415 all - transcripts with an average length of 496 bp and an n50 of 1466 bp; and 71,443 all - unigenes were achieved . Of these, 46,461 (65.0%) were 200500 bp, 11,322 were 5001000 bp, 8612 were 12 kb and the remaining 5078 were> 2 kb (table 2). The results provides us useful information for further explorer the gene synthesis pathways of active ingredients.
Periodontitis describes a group of related inflammatory diseases resulting in the destruction of the tissues that support the tooth . Periodontitis has a multifactorial etiology with the primary etiologic agents being pathogenic bacteria that reside in the subgingival area . Treatment of periodontal disease is routinely based on the mechanical debridement of the tooth surface and appropriate and meticulous maintenance of oral hygiene . It alone may fail to eliminate the pathogenic microflora because of their location within the gingival and dental tissues or in other areas inaccessible to periodontal instruments . As an adjunctive approach, systemic or local administration of antimicrobials since the systemic antibiotics therapy has various disadvantages like hypersensitivity reaction, organ toxicity and development of resistant bacteria and also requires higher dosage to attain required gingival crevicular fluid (gcf) concentration at the target site, this lead to use of local drug delivery system . Topical administration of antibacterial agents in the form of mouthwashes, dentifrice or gels can be used effectively in controlling supragingival plaque . Irrigation systems or devices can deliver agents into deep pockets, but are not effective in halting the progression of periodontal attachment loss . As gcf within a periodontal pocket is replaced every 90 s, the effects of antiseptic pocket irrigation are only transient . Local delivery devices are system designed to deliver agents into the periodontal pocket to retain therapeutic levels for a prolonged period of time . The periodic use of local delivery systems in reducing probing depths (pd), stabilizing attachment levels and minimizing bleeding would allow better control of the disease . Controlled release local delivery systems, in which the antimicrobial is available at therapeutic levels for several days, have been evaluated in several forms and using different antimicrobials . Different drugs used for local drug delivery are tetracyclines including doxycycline and minocycline, metronidazole and chlorhexidine (chx). These devices are less invasive treatment options and it requires less time compared to surgical treatment . Hence patients with moderate periodontitis can be treated by non - surgical therapy to halt periodontal disease and limit the extent of surgical intervention needed in the future . Patients in whom surgery is contraindicated due to certain systemic factors in those patients local drug therapy is needful . Chx is one of the most effective topical agents, long been used as an effective antimicrobial agent . It is an antiseptic, which adheres to organic matter and demonstrates low toxicity when applied topically . Its efficacy as a topical rinse to inhibit dental plaque and gingivitis has been well - established in study periods for up to 2 years without evidence of development of any bacterial resistance . It has been found to be effective against subgingival bacteria when delivered through a sustained release device . Chx has been shown to be an effective agent in plaque inhibition because it is well - retained in the oral cavity, reacting reversibly with receptors in the mouth due to its affinity for hydroxyapetite and acidic salivary protein . It has very low systemic toxic activity in humans and has not produced any appreciable resistance to oral microorganism and has not been associated with any teratogenic alterations . Short term use of chx causes a striking reduction in the number of oral microorganisms . In the absence of other oral hygiene measures, chx has been shown to reduce the number of bacteria in saliva . In a developing country with a high prevalence of periodontitis, there are very few studies available for the use of chx in gel form as a local drug delivery system as an adjunct to scaling and root planing . In the present study, an attempt has been made to evaluate and compare the efficacy of gel containing chlorhexidine digluconate (0.5%) and chlorhexidine dichydrochloride (1.0%) as an adjunct to scaling and root planing with scaling and root planing alone . Digluconate is released immediately and dihydrochloride is released more slowly and over a longer period of time . These are bacteriostatic and bactericidal antiseptics, capable of protecting the treated pocket from recolonization of microorganism for at least 2 weeks . A total of 30 subjects, above 30 - 60 years of age comprising of both the gender were randomly selected and diagnosed as suffering from chronic localized or generalized periodontitis, from the outpatient department of periodontics, k. m shah dental college and hospital, vadodara . An informed consent was taken from all the subjects before the start of the study . The ethical committee of the university was presented the synopsis and a clearance was obtained after their approval . Patients between 30 to 60 years of agegood general healthpresence of two periodontal sites located on the same side pd between 5 to 7 mmlocalized or generalized chronic periodontitis patients . Patients between 30 to 60 years of age presence of two periodontal sites located on the same side pd use of systemic or subgingival antimicrobial within the 6 months prior to the studypresence of vertical bone defectuse of anti - inflammatory therapy within the 6 months prior to the studyallergy to chxhabit of tobacco chewing and smokinghistory of systemic diseases that could influence the course of periodontal disease or would require prophylactic antibiotics (not medically compromised)pregnancy and lactationaggressive periodontitisany teeth with furcation involvementhistory of periodontal therapy 6 months back . Use of systemic or subgingival antimicrobial within the 6 months prior to the study presence of vertical bone defect use of anti - inflammatory therapy within the 6 months prior to the study habit of tobacco chewing and smoking history of systemic diseases that could influence the course of periodontal disease or would require prophylactic antibiotics (not medically compromised) pregnancy and lactation aggressive periodontitis any teeth with furcation involvement history of periodontal therapy 6 months back . A special proforma was designed for the present study so as to have a systemic and methodical recording of all the observation and information . Clinical examination was done on a dental chair, under standard conditions of light, using a mouth mirror, university of north carolina-15 (unc-15) probe (hu - friedy) and tweezers and assessment of clinical parameters were carried out . Selected sites were randomly divided into control sites and test sites . Both of these sites should be placed on the same side of the patient mouth . Control sites: 30 sites were treated by scaling and root planing alone . Test sites: 30 sites were treated with scaling and root planing followed by the placement of the chx gel in the periodontal pocket [figure 1]. The following parameters were recorded at baseline (day 0), 6 weeks, 3 months and 6 months using a unc-15 probe (hu - friedy) by a single examiner who was blinded: gingival index (gi)sulcus bleeding indexplaque index (pi)probing pocket depth - measurement to the nearest millimeter of the distance from the gingival margin to the depth of the pocketrelative clinical attachment level (cal). Gingival index (gi) sulcus bleeding index probing pocket depth - measurement to the nearest millimeter of the distance from the gingival margin to the depth of the pocket relative clinical attachment level (cal). Customized acrylic occlusal stents with vertical grooves were prepared for each subject on a study model to standardize the readings . The pocket depths were measured from the crest of marginal gingiva to the base of the pocket using unc-15 probe [figure 2]. Measurement of relative clinical attcahment level and probing depth using a stent relative distance between the base of the pocket and fixed reference point (horizontal notch) on the stent was measured . This was later on used for assessment of clinical attachment gain or loss . After recording all the parameters at the baseline, full mouth scaling and root planing was performed using ultrasonic instruments followed by hand instruments until all supra and subgingival root surfaces felt hard and smooth . Following debridement, target sites were irrigated gently with cold saline and then left for 10 min to achieve hemostasis prior to placement of chx gel (chlosite) and it was applied into the deepest portion of periodontal pocket by means of a thin rounded tip needle [figure 3]. Placement of chlorhexidine gel to ensure that the chx gel stays long enough to be effective in the pocket, a periodontal dressing was placed without pressure over the treated sites [figure 4]. Periodontal pack in place post - operative home care instructions including brushing 2 times daily with a soft brush were given . Subjects were recalled after 7 days for removal of the periodontal dressing and for oral hygiene maintenance instructions . Recall visits were again scheduled after 6 weeks, 3 months and 6 months of placement of the experimental drug for recording of clinical parameters . No subgingival instrumentation was performed at subsequent visits and patients were reinstructed on oral hygiene maintenance . Patients between 30 to 60 years of agegood general healthpresence of two periodontal sites located on the same side pd between 5 to 7 mmlocalized or generalized chronic periodontitis patients . Patients between 30 to 60 years of age presence of two periodontal sites located on the same side pd between 5 to 7 mm localized or generalized chronic periodontitis patients . Use of systemic or subgingival antimicrobial within the 6 months prior to the studypresence of vertical bone defectuse of anti - inflammatory therapy within the 6 months prior to the studyallergy to chxhabit of tobacco chewing and smokinghistory of systemic diseases that could influence the course of periodontal disease or would require prophylactic antibiotics (not medically compromised)pregnancy and lactationaggressive periodontitisany teeth with furcation involvementhistory of periodontal therapy 6 months back . Use of systemic or subgingival antimicrobial within the 6 months prior to the study presence of vertical bone defect use of anti - inflammatory therapy within the 6 months prior to the study habit of tobacco chewing and smoking history of systemic diseases that could influence the course of periodontal disease or would require prophylactic antibiotics (not medically compromised) pregnancy and lactation aggressive periodontitis any teeth with furcation involvement history of periodontal therapy 6 months back . A special proforma was designed for the present study so as to have a systemic and methodical recording of all the observation and information . Clinical examination was done on a dental chair, under standard conditions of light, using a mouth mirror, university of north carolina-15 (unc-15) probe (hu - friedy) and tweezers and assessment of clinical parameters were carried out . Selected sites were randomly divided into control sites and test sites . Both of these sites should be placed on the same side of the patient mouth . Control sites: 30 sites were treated by scaling and root planing alone . Test sites: 30 sites were treated with scaling and root planing followed by the placement of the chx gel in the periodontal pocket [figure 1]. The following parameters were recorded at baseline (day 0), 6 weeks, 3 months and 6 months using a unc-15 probe (hu - friedy) by a single examiner who was blinded: gingival index (gi)sulcus bleeding indexplaque index (pi)probing pocket depth - measurement to the nearest millimeter of the distance from the gingival margin to the depth of the pocketrelative clinical attachment level (cal). Gingival index (gi) sulcus bleeding index probing pocket depth - measurement to the nearest millimeter of the distance from the gingival margin to the depth of the pocket relative clinical attachment level (cal). Customized acrylic occlusal stents with vertical grooves were prepared for each subject on a study model to standardize the readings . The pocket depths were measured from the crest of marginal gingiva to the base of the pocket using unc-15 probe [figure 2]. Measurement of relative clinical attcahment level and probing depth using a stent relative distance between the base of the pocket and fixed reference point (horizontal notch) on the stent was measured . This was later on used for assessment of clinical attachment gain or loss . After recording all the parameters at the baseline, full mouth scaling and root planing was performed using ultrasonic instruments followed by hand instruments until all supra and subgingival root surfaces felt hard and smooth . Following debridement, target sites were irrigated gently with cold saline and then left for 10 min to achieve hemostasis prior to placement of chx gel (chlosite) and it was applied into the deepest portion of periodontal pocket by means of a thin rounded tip needle [figure 3]. Placement of chlorhexidine gel to ensure that the chx gel stays long enough to be effective in the pocket, a periodontal dressing was placed without pressure over the treated sites [figure 4]. Periodontal pack in place post - operative home care instructions including brushing 2 times daily with a soft brush were given . Subjects were recalled after 7 days for removal of the periodontal dressing and for oral hygiene maintenance instructions . Recall visits were again scheduled after 6 weeks, 3 months and 6 months of placement of the experimental drug for recording of clinical parameters . No subgingival instrumentation was performed at subsequent visits and patients were reinstructed on oral hygiene maintenance . Table 1 shows a comparison of mean pd between test and control group at baseline, 6 weeks, 3 months and 6 months with statistically significant results at 3 months and 6 months (i.e., 0.002 and p = 0.002 respectively) for the test group . Comparison of mean pocket probing depth table 2 shows a comparison of mean cal between test and control group at baseline, 6 weeks, 3 months and 6 months with statistically significant results at 6 week, 3 months and 6 months for the test group (i.e., p = 0.000, p = 0.006 and p = 0.014 respectively). Comparison of mean relative clinical attachment level table 3 shows a comparison of mean gi scores between test and control group at baseline, 6 weeks, 3 months and 6 months with statistically significant results at 6 weeks and 6 months for the test group (i.e., p = 0.039 and p = 0.026, respectively). Comparison of mean gingival index scores table 4 shows at baseline, 6 weeks, 3 months and 6 months there was no statistically significant difference between test and control group . Comparison of mean plaque index score table 5 shows comparison of mean bleeding index scores between test and control group at baseline, 6 weeks, 3 months and 6 months with statistically significant results at 3 months and 6 months in the test group (i.e., p = 0.030 and p = 0.009 respectively). Table 1 shows a comparison of mean pd between test and control group at baseline, 6 weeks, 3 months and 6 months with statistically significant results at 3 months and 6 months (i.e., 0.002 and p = 0.002 respectively) for the test group . Table 2 shows a comparison of mean cal between test and control group at baseline, 6 weeks, 3 months and 6 months with statistically significant results at 6 week, 3 months and 6 months for the test group (i.e., p = 0.000, p = 0.006 and p = 0.014 respectively). Table 3 shows a comparison of mean gi scores between test and control group at baseline, 6 weeks, 3 months and 6 months with statistically significant results at 6 weeks and 6 months for the test group (i.e., p = 0.039 and p = 0.026, respectively). Table 4 shows at baseline, 6 weeks, 3 months and 6 months there was no statistically significant difference between test and control group . Table 5 shows comparison of mean bleeding index scores between test and control group at baseline, 6 weeks, 3 months and 6 months with statistically significant results at 3 months and 6 months in the test group (i.e., p = 0.030 and p = 0.009 respectively). The gel is a combination of two chx formulations: 0.5% chlorhexidine digluconate and 1.0% chlorhexidine dihydrochloride incorporated in a saccharidic polymer, xanthan . Cross linking structure of xanthan controls the release of drugs and it exhibits a near zero order drug release . When in contact with water, it forms a three dimensional pseudoplastic reticulum capable of holding and maintaining various substances in suspension . The chx xanthan based gel undergoes a progressive process of imbibition and is physically removed in 10 - 30 days . Chlorhexidine digluconate is liberated in the first day and achieves a concentration> 100 g / ml, which is maintained for an average of 6 - 9 days which is greater than the minimum inhibitory concentration for chx (0.10 g / ml). Chlorhexidine dihydrochloride is released in the following days and maintains the bacteriostatic and bactericidal concentrations for at least 2 weeks and prevents recolonization . The chx gel is supplied with a special needle having a blunt tip and a lateral opening . Periodontal probing is one of the most widely used diagnostic tools for the clinical assessment of connective tissue destruction in periodontal disease . Increased pd and loss of clinical attachment are pathognomonic for periodontitis and hence, serve as primary parameters in diagnosis of periodontal disease and evaluation the success of periodontal therapy . Results of this study are consistent with the results observed by vinholis et al . When they evaluated the effect of subgingival irrigation with a 1% chx collagen gel in periodontal pockets as an adjunct to scaling and root planing . Observed similar reduction of probing pocket depth with both the group that were treated either by scaling and root planing alone or scaling and root planing with controlled release chx chip . Determining change in attachment level (gain or loss) is a primary measure and one of the most practical methods of determining progression of periodontal disease . The results of this study coincide with the study carried out by perinetti et al . Who observed more gain of relative cals in subjects cosyn and wyn observed significant additional gain of clinical attachment with sites treated by scaling and root planing with chx varnish as compared with sites treated by scaling and root planing alone . Furthermore, several investigators have indicated that bleeding from gingiva after light probing is indicative of gingival inflammation and disease activity . Observed significant reduction of gi score with scaling and root planing with chx treated sites when compared with the control (scaling and root planing) site, which is similar to the observation of the present study . Similar results were obtained in the study by he et al . Where significant percentage reduction of gi was observed in the experimental group (scaling and root planing with chx chip) compared with the control group (scaling and root planing). Azmket et al . Observed that the pi decreased in both the control (scaling and root planing alone) and experimental group (scaling and root planing with chx chip)., the reduction of plaque from baseline in the control and experimental group could be due to greater attention to oral hygiene practice by all the selected participants . It is accepted that bleeding upon probing and pd is critical site - specific periodontal parameters evaluating the probability of periodontal disease progression . The observations of the present study are consistent with the findings of heasman et al . Who observed mean reduction of bleeding index score from baseline in the experimental group (scaling and root planing with chx chip) as well as control group (scaling and root planing alone). It can be concluded that experimental local drug delivery system containing 1.5% chx gel can be effectively used as an adjunct to scaling and root planing and is more effective than scaling and root planing alone in the treatment of periodontal pockets . The experimental local drug delivery system is easy to use, noninvasive and requires less chair side time . To elucidate the use of this local drug delivery system in future, a long term study, carried out on a large sample of subjects, in addition, microbiological studies can also be used to correlate with the clinical findings observed . An attempt is required to find out the efficacy of experimental material in comparison to other marketed local drug delivery system.
Neuroendocrine (ne) carcinoma of the breast was first described by cubilla and woodruff in 1977 . Primary ne carcinomas of the breast are rare and represent about 2 - 3% of breast carcinomas . They are much rarer in the male breast occurring in less than 1% of the cases . Ne breast cancers encompass a heterogeneous group of tumors exhibiting morphological features similar to those of ne neoplasms of the gut and lung . They invariably express one or more ne markers (neuron specific enolase, chromogranin, synaptophysin) in at least 50% of the tumor cells . We report one such rare case in male breast diagnosed by fine needle aspiration cytology (fnac). An 80-year - old male presented with a mass in the right breast of 10 years duration with rapid progression in growth since the previous 3 months . Mammography showed a well - defined mass in the right breast with ipsilateral axillary lymphadenopathy . Fine - needle aspiration cytology showed cellular smears with dispersed single cells and loose sheets . Many nuclei showed moderate anisocytosis, irregular nuclear contours, prominent nucleoli and sprinkled chromatin [figure 1a and 1b]. Considering these features, a cytological diagnosis of ne carcinoma of the breast (a) highly cellular smear, dispersed cells and loose sheets (pap, 100). (b) tumor cells with anisocytosis, irregular nuclear borders, prominent nucleoli and sprinkled chromatin (pap, 400) the patient underwent right radical mastectomy with axillary lymph node dissection . Gross examination revealed a tumor measuring 10 cm 8 cm 8 cm, with a solid, grey - white appearance and a focal cystic area [figure 2a]. Histopathological sections studied showed a malignant tumor with cells in solid nests, trabecular and organoid patterns, separated by delicate fibro - vascular connective tissue stroma . A focal area in the tumor also showed features of invasive papillary carcinoma [figure 2c]. Four out of the six resected lymph nodes showed metastatic deposits from the primary breast tumor . (a) gross specimen showing solid, grey - white tumor with a cystic area . (c) focal area in the tumor with features of papillary carcinoma (h and e, 400). (d) tumor cells with cytoplasmic chromogranin positivity (ihc, 400) immunohistochemical staining revealed cytoplasmic positivity for chromogranin which confirmed the ne nature of the tumor [figure 2d]. To exclude a non - mammary primary site, the chest, abdomen and pelvis were thoroughly examined for abnormalities, but none was detected . Final diagnosis of primary pure solid ne carcinoma of the breast with a minor component of papillary carcinoma and metastasis to ipsilateral axillary lymph node was arrived at, following which the patient received adjuvant chemotherapy . Features of primary pure neuroendocrine breast carcinomas (nebc) were considered to be special features within conventional breast carcinomas until recently . In 2003, the world health organization classification of breast tumors definitely established that, the immunohistochemical expression of ne markers in more than 50% of the tumor cell population is the unique requisite for nebc diagnosis . But, invasive papillary carcinoma associated with ne carcinoma in the male breast as seen in the present case is an unusual occurrence . Endocrine hormone related syndromes are rare in primary nebc despite the presence of widespread disease . The tumor cells in ne carcinomas of the breast are argyrophilic in contrast to the gut and lung carcinomas which may be of either argyrophil or argentaffin types and are hence called argyrophilic carcinomas . Argyrophilic carcinomas of the male breast are ne tumors containing predominantly chromogranin b. most argyrophilic tumors show uniform cellularity and expansive growth as in the present case . Despite being a distinct morphological and immunohistochemical entity, no statistically significant differences are seen between argyrophilic and the other commoner types of male breast carcinomas . Stage on an average is higher than for the other breast carcinomas, as in the present case, since more than 50% of the patients show lymph node metastasis at the time of presentation . However, in the present case, the predominant component was ne carcinoma which was associated with a minor component of papillary carcinoma . About 50% of well - differentiated and moderately differentiated ne carcinomas express chromogranin a and b. only 16% express synaptophysin . Mucin as seen in the present case, as also observed by stita et al ., in 26% of cases and was considered to be a favorable prognostic factor by them . Ne breast carcinomas are observed not to exhibit aggressive behavior despite the presence of adverse prognostic factors . Good prognosis in these tumors relates to their intrinsic nature and high rate of treatment responses . Ne carcinoma in male breast is a rare entity but does occur and can be diagnosed on fnac . However, it is important to distinguish the primary nebc from metastatic carcinoma because the management and prognosis differ . Primary ne carcinoma of the breast may pose a cyto - histologic diagnostic challenge due to the rarity of its occurrence and paucity of literature . Nevertheless, it is an entity which no pathologist can afford to miss since it is less aggressive and shows a high rate of treatment response.
The endothelium represents a monolayer of cells that coat the interior surface of the cardiovascular and lymphatic systems . It is the largest secreting organ in the body weighing up to 1.8 kg and extending to a total surface area greater than 4,000 m in the vessel wall of adult humans . In the cardiovascular system, the vessel wall is typically composed of extracellular matrix, connective tissue fibers, and smooth muscle and endothelial cells . The number of endothelial cells (ecs) lining the endothelium is believed to be more than one trillion [2, 3]. Physiologically, the ecs are involved in the maintenance of vascular structure and function including formation of physical barrier between blood elements and vessel wall, as well as biological regulation of smooth muscle cell proliferation, adhesion of inflammatory cells to vessel wall, vascular tone, inflammation, thrombosis, and vascular remodeling . These biological effects of the endothelium are mediated by one or more of the molecules that it secretes such as prostacyclin (pgi2), thromboxane a2 (txa2), thrombomodulin (tm), tissue - type plasminogen activator (tpa), von willebrand factor (vwf), plasminogen activator inhibitor type 1 (pai-1), cd39, angiotensin type 2 (at2), adenosine, endothelin (et), and nitric oxide (no). Among these bioactive molecules, no is likely the most studied molecule in the pathophysiology of the cardiovascular system including its role in vascular tone regulation, angiogenesis and cell proliferation, inflammatory and immune response, blood clotting, and carbohydrate and lipid metabolism . In the chemical industry, nitric oxide or nitrogen monoxide is known as a byproduct released from heat engines upon refinery of raw materials such as coal, oil, natural gas, and metals . However, mammals are squarely dependent on the biological function of no ranging from maintenance of blood pressure to immune defense and neurotransmission . Biochemically, no is synthesized from oxidation of the natural amino acid l - arginine upon catalysis by its three enzymes in the presence of several cosubstrates and cofactors . The first enzyme (nos i; nnos) is actively expressed by resident cells of the central nervous system including neurons and nonneuronal cells such as astrocytes, microglial cells, and oligodendrocytes . The main function of no in the nervous system is its involvement in neurogenesis, neurotransmission, cerebral blood flow, and memory and learning . By contrast, the second nos isoform (nos ii; inos) is mainly expressed by inflammatory cells as an immune defense mechanism including involvement in bacterial killing, cardiovascular inflammation, and neuroinflammation . The endothelial nos- (nos iii; enos-) derived no is the most active vasodilator and is involved in the regulation of blood pressure, vascular smooth muscle cell proliferation, aggregation of platelets, and adhesion of inflammatory cells to vessel wall . Although it can directly activate downstream targets such as soluble guanylate cyclase (sgc) and calcium - dependent potassium channels to influence vascular function, its half - life is incredibly short measuring only in seconds before its oxidation into nitrites (no2) and nitrates (no3). Endogenously, the concentration of cellular no is regulated by several factors including oligomeric structure of the nos enzymes, concentrations of substrate (l - arginine), cofactors (heme, bh4, fmn, and fad), cosubstrates (oxygen molecule, nadph), and intracellular calcium (ca). The concentrations of each of these factors are in turn regulated by other factors including enzymes (e.g., arginase regulates arginine), vasoactive molecules (e.g., histamine, bradykinin, and acetylcholine), lipid particles (e.g., oxidized low - density lipoprotein; oxldl), and reactive oxygen species . In addition, several studies have demonstrated that the endothelium - derived nos (enos) is endogenously regulated by protein - protein interaction with structural, regulatory, and transport proteins (e.g., with calmodulin, caveolin, actin, tubulin, lipoproteins, soluble guanylate cyclase, and cationic amino acid transporters), posttranslational modification (e.g., phosphorylation, s - nitrosylation) triggered by shear stress, hypoxia, inflammatory cytokines (e.g., tnf, il1), growth factors (e.g., vegf), and hormones (e.g., estrogen, erythropoietin, and insulin). Moreover, enos activity is regulated by its competitive inhibitor asymmetric dimethylarginine (adma). Physiologically, adma is synthesized as a result of posttranslational methylation of arginine residues in cellular proteins by a family of enzymes called protein arginine methyltransferases (prmts) in the presence of a methyl - group donor s - adenosyl methionine (sam). Once generated and released into the cytosol, adma competes with l - arginine for the substrate binding site on the enos protein . As a result, the enos - bound adma inhibits the production of no and favors the uncoupling of enos where highly reactive oxygen (superoxide anion; o2) and nitrogen (e.g., peroxynitrite; oono) radicals are generated instead of vasoactive no molecules . The concentration of circulating adma is normally regulated by urinary excretion and enzymatic metabolism through dimethylarginine dimethylaminohydrolase (ddah). In humans generally, the biodistribution of ddah1 appears to be colocalized with the neuronal nos (nos i) while that of ddah2 appears to coexpress with enos in endothelial and cardiac tissues . Studies have, however, shown that both isoforms can express in various cells and tissue types despite the distribution of the noss . For example, in addition to the brain, ddah1 is known to express in the pancreas, skeletal muscle, heart, liver, and kidneys . Recent genetic and biochemical studies report that ddah1 is mainly responsible for the enzymatic breakdown of adma into citrulline and methylamine . The clearance of adma by ddah is however compromised by several cardiovascular risk factors that impair the expression and/or activity of ddah . Preclinical and clinical studies have demonstrated that hypercholesterolemia, hypertension, coronary artery disease, renal failure, insulin resistance, and diabetes mellitus are associated with increased production of reactive oxygen species (ros), decreased expression and/or activity of ddah, and accumulation of adma in cells and tissues . The oxidative stress perpetrated by the cardiovascular risk factors is believed to be the principal cause of impaired ddah activity due to the sensitivity of the ddah enzyme to oxidation at a catalytically active site and subsequent loss of enzymatic activity to metabolize adma . Higher level of circulating adma is a known independent risk factor for major adverse cardiovascular events (mace) including myocardial infarction (mi) and stroke [1315]. Elevated plasma adma could trigger reduction of endothelium - derived no through uncoupling of enos as described above . In addition, the oxidative stress induced by the cardiovascular risk factors in the vascular wall persistently decreases ddah activity and reduces no levels further leading to disturbed hemodynamic compliance . Chronically low level of no is associated with several morbid vascular disorders and increases the risk of mace including cardiovascular - related disability and death . By contrast, increased expression of ddah reduces plasma adma and stimulates no production . Preclinical studies have shown that genetic overexpression of human ddah1 in mice reduces plasma levels of adma by 50% and increases no by about 2-fold . As a result, the ddah transgenic animals show optimal vascular compliance including significantly reduced systemic vascular resistance (svr) and systolic blood pressure (sbp). In addition, cross - breeding of the ddah1 transgenic animals with the hyperlipidemic apoe - deficient mice reduces plasma adma and decreases the development of atherosclerotic plaque . Physiologically, endothelium - derived no (eno) is known to regulate the transport of insulin and uptake of glucose by several tissues including the endothelium, liver, pancreas, and skeletal muscle [19, 20]. No enhances flow - mediated vasodilation and likely improves the delivery of nutrients and other chemicals to these target tissues . Conversely, both high glucose and insulin stimulate the transport of l - arginine and increase no production in vascular endothelial cells . In addition, insulin has been shown to stimulate skeletal muscle blood flow and enhance vasodilation by increasing no release . The transport of glucose to skeletal muscle is also reported to be regulated by no . Taken together thus, understanding the interaction among no, glucose, and insulin as well as defining the role of the nos / adma / ddah pathway in this process is essential towards complete characterization and development of effective preventative strategies and novel therapeutic agents for diabetes and its cardiovascular complications, including insulin resistance . In line with the well - characterized contribution of the nos / adma / ddah pathway in cardiovascular diseases, the volume of the literature deciphering the precise role of this pathway in the development and progression of insulin resistance, type 2 diabetes, and its complications is growing . Pharmacological and genetic studies indicate that the nos pathway is necessary in the transport of insulin and glucose to various tissues . Genetic deletion of the enos or nnos genes in mice triggers vascular impairment and insulin resistance [23, 24] likely due to impairment of insulin - stimulated glucose uptake by skeletal muscle and other insulin sensitive tissues . Furthermore, the enos knockout mice develop diabetic microvascular complication of advanced diabetic nephropathy in multiple preclinical models of diabetes [2528]. Similarly, accumulation of adma reduces nos activity and is associated with insulin resistance . As described above, excessive adma is often a result of ddah impairment . The role of dysfunctional ddah in cardiovascular complications including diabetes and insulin resistance has been studied in several preclinical models [8, 29]. For example, in high - fat - diet - fed type 2 diabetic rats, adipose - tissue - derived ddah / adma is involved in the regulation of insulin sensitivity through modulation of key insulin signaling genes such as insulin receptor substrate 1 (irs-1) and glucose transporter 4 (glut-4). Homozygous ddah1 knockout mice are, for example, hypertensive, showing significantly higher adma level and decreased no production . Moreover, targeted deletion of endothelial ddah1 significantly elevates plasma and tissue adma and increases systemic blood pressure . Several studies have documented that both essential and salt - sensitive hypertension are associated with insulin resistance [33, 34]. By contrast, in ddah1 overexpression mice, blood glucose and plasma insulin levels after glucose challenge were lower and the insulin resistance index was reduced by 50% indicating enhanced insulin sensitivity in the transgenic animals compared to wild type controls . Likewise, the ddah2 overexpression mice show enhanced insulin secretion from pancreas despite high - fat diet . Clinically, a number of prospective studies also show that elevated adma level is correlated with type 1 or type 2 diabetes [3742]. Considering all the data from preclinical models and clinical studies, it is evident that the nos / ddah pathway is involved in the regulation of insulin and glucose metabolism . In addition, many of these studies indicate that plasma adma has a reciprocal relationship with insulin sensitivity . For example, sthlinger et al . Conducted a cross - sectional study evaluating the relationship between plasma adma and insulin resistance . Interestingly, treatment of these subjects with the insulin sensitizing drug rosiglitazone enhanced insulin sensitivity and reduced plasma concentration of adma, a correlation that remained significant even after adjusting for risk factors associated with insulin resistance . Although effective pharmacological approaches and lifestyle - oriented intervention strategies for the prevention and treatment of diabetes have been implemented, the prevalence and incidence of diabetes are still rising at an alarming rate and are particularly worrisome in the developing world . According to the world health organization (who), the global prevalence of type 2 diabetes, the most common type accounting for 90 to 95% of all cases, in 2014 was about 9% of the adult population . One of the major characteristics of type 2 diabetes is relative insulin deficiency (hypoinsulinemia) or decreased response to insulin stimulation (insulin resistance). In insulin resistant subjects, the body fails to properly respond to endogenous insulin and as a result glucose accumulates within the body instead of being absorbed . This phenomenon overwhelms the insulin - secreting pancreatic beta - cells resulting in gradual loss of the ability to produce sufficient insulin that can dispose the accumulated glucose in the bloodstream . Although insulin resistance alone may not lead to type 2 diabetes, it significantly increases the risk of developing type 2 diabetes . When left uncontrolled, patients with type 2 diabetes are frequently burdened with life - threatening comorbidities including atherosclerosis, hypertension, nephropathy, retinopathy, and neuropathy . Results from several clinical trials including the diabetes control and complications (ddct), epidemiology of diabetes interventions and complications (edic), and the uk prospective diabetes study (ukpds) revealed that conventional glycemic control treatment can reduce the risk of cardiovascular events . Besides hyperglycemia, insulin resistance is directly associated with dyslipidemia and hypertension (figure 1). Insulin resistance plays an important role in the development of dyslipidemia by contributing to an increase in the free fatty acid flux released from insulin resistant fat cells . In addition, hypertensive patients in general and salt - sensitive hypertensive patients in particular are often insulin resistant . Given that the vascular system is a direct target for the action of insulin as described above, all the comorbid complications outlined above heavily contribute to increased cardiovascular risk often seen in diabetic, hypertensive, and/or dyslipidemic patients . Endothelial dysfunction is an early event in the development of insulin resistance and contributes to many pathological changes in the vasculature including impairment in the contraction and relaxation of blood vessels and inflammatory and coagulation responses, all of which are closely associated with adverse cardiovascular events . However, sustained hyperinsulinemia or insulin resistance can lead to progressive decline in endothelium - dependent vasodilation [46, 47], as well as impairment of transcapillary insulin transport to skeletal muscle cells in vivo . The interdependence of insulin signaling, glucose metabolism, and endothelial function including the release of endothelium - derived bioactive molecules calls for the development of novel therapeutic strategies to modulate the nos / adma / ddah pathway to treat insulin resistance syndrome and prevent mace . Metformin (glucophage) is the recommended first - line medication for type 2 diabetes . The mechanism by which this drug suppresses hepatic glucose production and stimulates its tissue uptake is through suppression of mitochondrial respiratory chain complex 1 and activation of adenosine monophosphate- (amp-) activated protein kinase (ampk) [49, 50]. Both targeted pathways and the l - arginine - no pathway are mutually restrained in vascular function and homeostasis [51, 52]. For example, in l - arginine treated ecs, coincubation with an ampk inhibitor increased glucose accumulation and blunted the increase in no . Interestingly, the chemical structure of metformin is strikingly similar to that of adma and metformin itself is known to reduce adma and restore no levels in vivo . In addition, metformin has been shown to protect the liver from inflammatory damage through modulation of the ddah / adma pathway (table 1). Thiazolidinediones (tzds or glitazones) are ligands of the nuclear receptor transcription factor peroxisome proliferator - activated receptor gamma (ppar gamma) that have recently been developed as insulin sensitizers to treat patients with type 2 diabetes [55, 56]. Ppar gamma is a member of the ppar family that is mainly expressed in the adipose tissue and is known to masterfully regulate glucose metabolism . Intriguingly, studies have reported that the vasoprotective effect of ppar gamma ligands is directly correlated with no regulation . For example, pioglitazone (actos) has been shown to upregulate ddah gene expression, reduce circulating levels of adma, and enhance no production (figure 2). Although this effect could not be reproduced with other tzds, rosiglitazone (avandia) has been shown to improve endothelial function (table 1) in patients with type 2 diabetes [58, 59]. The effect of no in insulin signaling is demonstrated in multiple animal models that are genetically modified to manipulate the no signaling . For example, loss - of - function studies demonstrated that knockdown of enos gene reduces no and causes vascular dysfunction including insulin resistance . By contrast, the no donor sin-1 has been shown to rescue the insulin resistance phenotype induced by the nos inhibitor n(g)-nitro - l - arginine methyl ester (l - name) [6062]. In addition, exogenous supplementation of the nos substrate l - arginine or the cofactor tetrahydrobiopterin (bh4) is shown to increase insulin sensitivity [63, 64]. These proof - of - principle genetic and pharmacological studies suggest that stimulation of no production by activating enos is an attractive approach to discover and develop novel insulin sensitizers that would also be vasoprotective . Similarly, increased expression or activity of ddah reduces circulating levels of adma and increases nos signaling . Recently, we have demonstrated that upregulation of ddah expression by the bile acid derivative farnesoid x receptor (fxr) agonist int-747 (figure 2) improves insulin sensitivity in an animal model of salt - sensitive hypertension and insulin resistance . A clinical trial on the efficacy of int-747 demonstrated that this compound is able to enhance insulin sensitivity in type 2 diabetics . Meanwhile, we have recently developed a robust high throughput assay to screen for small molecules that modulate ddah enzymatic activity . Unlike transcriptional ddah regulators such as the fxr agonists int-747 and gw4064 that upregulate the gene expression of ddah (table 1), our screening strategy is aimed at identifying compounds that modulate ddah posttranslationally . Using this biochemical assay, we have identified and reported several new chemical entities (nces) and existing drugs that directly modulate ddah activity [6668]. The effect of these agents on insulin signaling and glucose metabolism remains to be seen . For example, we discovered that the antacid drug, proton pump inhibitors (ppis), directly inhibit ddah enzymatic activity and impair endothelium - derived no . Given that ppis are among the most widely sold drug worldwide, it might be important, from drug surveillance perspective, to evaluate their effect on glucose metabolism and insulin sensitivity . Meanwhile, when allosteric ddah activators are developed, it is important to establish their efficacy in glucose metabolism and insulin signaling . In addition to these pharmacological approaches, dietary supplementation with fruits and green vegetables and other life style choices such as cessation of smoking, body weight management, and regular exercise to reduce oxidative stress and maintain or enhance the nos / ddah pathway are possible strategies to prevent and manage cardiovascular disease including diabetes and insulin resistance . Despite the promising efficacy of the glitazones (tzds) in improving insulin sensitivity and management of type 2 diabetes, the serious concern over their cardiovascular safety has urged many countries to remove all approved tzds from the market . In addition, prescription of other antidiabetic drugs such as the sulfonylurea class is increasingly limited due to incidences of associated cardiovascular risk . These unfortunate adversities have intensified the urgency to develop safe and effective insulin sensitizers for diabetic patients . The endothelial nos / ddah pathway is presumed to be a viable target to discover and develop novel insulin sensitizers . Preclinical and clinical studies indicate that downregulation of this pathway is associated with insulin resistance and its upregulation is linked to enhanced insulin sensitivity . In addition, upregulation of nos / ddah is known to increase the vasoprotective molecule nitric oxide and reduce the cardiovascular risk factor adma . Therefore, increased expression and/or activity of endothelial nos or ddah using selective nces is a novel strategy to develop effective insulin sensitizers that are also expected to protect the vascular wall and reduce major adverse cardiovascular events including stroke and heart attack.
How does the care for older people maintain of good quality, according to their needs, while still fundable? Looking at the most vulnerable group of older people with dementia, for which family and friends represent past, present and future, adjustments need to be made . Family, nurses and organization all acknowledge that nursing home care will benefit when these three parties unite their forces as partners in care . Thuis voelen is a guide filled with practical suggestions to accomplish a valuable partnership between family, nurses and organization . Loosely structured conversations between these three parties aim to establish reciprocity and trust and finally to end up with a number of incentives, which concern family as well as employees and range from the moment of intake until the phase of saying goodbye . Advises in the area of architecture, furnishing, public space, human resources and ict are provided as well . A nursing home creates its own worthy care agenda, to accomplish small, practicable changes . Resulting in: a changing (physical) environment; the family enjoys visiting the nursing home and stays longer, nurses enjoy their work environment more and feel relieved. More mutual complicity and responsibility in wanting to improve the quality of life of elderly together . A changing (physical) environment; the family enjoys visiting the nursing home and stays longer, nurses enjoy their work environment more and feel relieved . More mutual complicity and responsibility in wanting to improve the quality of life of elderly together . Thuis voelen is an example of how small scale innovations, new ideas and simple things that matter can establish an improvement of the situation in nursing homes . Within an open - minded organization, any moment an inhabitant and/or his relatives are satisfied and happy, will have a positive effect on the (culture in) organization; time and money will be spared.
Extranodal nhl can occur in any visceral or non - visceral organ, but there are several predominant sites, including the stomach, small intestine, skin, and brain . Nhl in the breast or testis is relatively rare, representing 0.38%-0.7% and 1%-2% of all nhl, respectively . These types of lymphoma are generally high - grade lymphomas with an aggressive clinical course . The main treatment strategy for diffuse large b - cell lymphoma (dlbcl) with involvement of the breast is an anthracycline - containing chemotherapy with or without radiotherapy . The role of central nervous system (cns) prophylaxis remains controversial despite the risk of cns relapse, due to unproven benefit . On the other hand, anthracycline - based chemotherapy combined with locoregional radiotherapy to the contralateral testis and cns prophylaxis the 5-year overall survival rate from previous reports varies from 26% to 71% in breast lymphoma and 56%-87% in testicular lymphoma . Here we report a patient with dlbcl involving both the breast and testis who was successfully treated with complete resection of the involved testis; immunochemotherapy with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone (r - chop); and prophylactic radiotherapy to the contralateral testis . A 76-year - old man visited our hospital (asan medical center, seoul, korea) with a mass on his right breast first being recognized two months ago . He had no b symptoms and an unremarkable medical history, except for hypertension . On physical examination he had a rubbery, non - tender, mobile mass of approximately 4 cm on his right breast . There were no other palpable lymph nodes in the axillary, neck, or inguinal area, but his right testis was slightly harder than the left testis, without a definite mass . Iu / l, and 2 microglobulin was 1.8 g / ml . Screening tests for human immunodeficiency virus, hepatitis c virus, and hepatitis b virus were all negative . On mammography, a heterogeneous, dense, lobulated mass - like lesion was observed in the subareolar portion of the right breast . The pathologic specimen from core needle biopsy revealed diffuse proliferation of large, atypical lymphoid cells (fig . 1b), bcl-6, and irf4/mum1, but was negative for cd10 . In situ hybridization for epstein - barr virus was also negative . Based on these pathologic findings, the patient was diagnosed with dlbcl . A staging work - up including bone marrow biopsy with aspirate, positron emission tomography computed tomography (pet - ct), and abdomen and chest computed tomography (ct) revealed no evidence of other nodal or marrow involvement . However, the pet - ct showed increased metabolism in the right testis, with a maximum standardized uptake value of 8.4 (fig . Pathologic examination revealed a well - defined ovoid mass (3.82.51.6 cm), which was confirmed as dlbcl (fig ., he was finally diagnosed with dlbcl involving both the breast and testis, with an ann arbor clinical stage of 4ea . The patient received a total of six cycles of immunochemotherapy (375 mg / m rituximab on day 1, 750 mg / m cyclophosphamide on day 1, 50 mg / m doxorubicin on day 1, 1.4 mg / m vincristine on day 1, and 50 mg prednisolone bid on days 1 - 5 [r - chop]). Follow - up ct and pet - ct after four cycles and six cycles of r - chop immunochemotherapy confirmed a complete response . After six cycles of r - chop immunochemotherapy, prophylactic radiotherapy of 25 gy with 10 fractions was administered to his left testis . He has been followed at the outpatient clinic regularly without evidence of relapse for 17 months . Extranodal nhl of the breast is uncommon, and its occurrence in male patients is extremely rare . Table 1 provides an overview of previously reported cases of male primary breast lymphoma (pbl) [3 - 13]. None of these cases presented with metastatic disease at first presentation, but three patients relapsed at other sites, including the cns, bone marrow, cervical lymph nodes, and adrenal glands . The prognosis of pbl is somewhat variable, with the 5-year survival rate ranging from 26%-71% . According to some reports, progression to cns is relatively more common in pbl than in other nhl, ranging from 12%-27% . Recent studies, however, suggest that routine cns prophylaxis should not be given despite the high risk of cns relapse in pbl, because there is no survival benefit . In primary testicular lymphoma (ptl), previous studies report a continuous pattern of relapse, even at 10 - 14 years after initial therapy . Similar to pbl, common sites of relapse include extranodal sites such as the cns and contralateral testis . The risk of cns relapse is reported to be 20% and 35% at 5 and 10 years, respectively . Although bilateral involvement is uncommon at diagnosis, up to 35% of ptl patients develop contralateral testicular involvement later in their clinical course, which provides a rationale for prophylactic radiotherapy to the contralateral testis . Anthracycline - containing chemotherapy (i.e., chop) is considered the standard treatment option for early - stage dlbcl . A number of cases benefitted from radiotherapy upon relapse on the contralateral side, which improved overall and progression - free survival in ptl . The role of additional radiotherapy to the involved side of breast, however, is yet to be determined, although it did show positive effects on progression - free survival and the risk of ipsilateral locoregional progression . Here, we present the first reported case of dlbcl with simultaneous breast and testicular involvement, without any nodal presentation . The patient received immunochemotherapy (r - chop) and prophylactic radiotherapy to the contralateral testis because of the high relapse rate of testicular lymphoma . In summary, dlbcl involving both the breast and testis was successfully treated with multimodal strategies and showed a favorable response without evidence of recurrence.
Breast ultrasound has become a standard breast - imaging procedure in addition to mammography, for work - up of patients referred with a palpable mass or with a suspicious lesion detected on the mammogram . Gray - scale breast ultrasound offers the ability to visualize the breast tumor in three dimensions and permits direct measurement of tumor size without magnification . To standardize lesion characterization on ultrasound, the american college of radiology (acr) developed a lexicon of sonographic descriptors of breast masses with attendant assessment categories, i.e. The sonographic breast imaging reporting and data system (bi - rads) lexicon . Technologic advances over the last decade have fueled research in the field of minimal - invasive image - guided ablation techniques for treatment of patients with limited - stage breast cancer . Techniques that have been studied include radiofrequency ablation, cryoablation, focused ultrasound and laser ablation of breast tumors . Different imaging modalities are used to guide the instruments, to monitor the therapeutic procedure and to assess treatment response . Of all imaging modalities, gray - scale breast ultrasound is most often used for breast tumor visualization and real time monitoring of the ablation process . Accurate assessment of breast tumor size is important for planning surgical and minimal - invasive image - guided ablation procedures . Extensive surgical treatment may result in poor cosmetic results, whereas small tumor - free margins may influence the local recurrence rate46 . Several previous studies have assessed the accuracy of gray - scale ultrasound for breast tumor size measurement . Overall, these studies concluded that gray - scale ultrasound of the breast is a reliable method for determining tumor size and favors mammography, but in general true tumor size is underestimated with this technique . In recent years, ultrasound contrast agents have been developed that increase blood echogenicity and improve ultrasound image quality by detection of slow and low - volume blood flow in small tumor vessels (<5 mm). Contrast - enhanced ultrasound (ceus) of the breast has recently been studied for characterization of indeterminate breast lesions . Since breast tumors are strongly vascularized and display neo - angiogenesis in the vital border, it is hypothesized that ceus of the breast may also be a more accurate modality than gray - scale ultrasound for delineation of breast tumor boundaries and tumor size assessment . This prospective feasibility study was designed to assess the accuracy of ceus of the breast for pre - operative tumor size measurement in patients diagnosed with invasive ductal carcinoma of the breast . Seven consecutive female patients, 49 years of age (range 4257 years), with 9 breast lesions were prospectively included in this study . All patients were referred to our department for ultrasound examination and ultrasound (us)-guided large - core needle biopsy of a suspicious breast lesion (bi - rads iv and v) detected on mammography between june 2005 and june 2006 . The diagnosis of invasive ductal breast cancer was confirmed in all patients by us - guided large - core needle biopsy (14 gage). Eligible patients for pre - operative tumor size measurement with contrast - enhanced ultrasound (ceus) of the breast had no history of previous breast surgery . Patients were excluded if use of the ultrasound contrast agent (sonovue, bracco spa, milan, italy) was contra - indicated, due to a history of cardiac failure, right to left shunt, severe pulmonary hypertension, uncontrolled systemic hypertension, adult respiratory disorders and hypersensitivity . Written informed consent was obtained from all patients and the study was performed in accordance with a protocol approved by our institutional panel . All eligible patients prospectively underwent both gray - scale ultrasound and ceus of the breast according to a standardized protocol . High - frequency gray - scale ultrasound examination of the breast was performed first with a philips iu22 scanner (philips medical systems, best, the netherlands) equipped with a 11 mhz linear array transducer . Breast lesion morphology was scored according to the sonographic bi - rads lexicon criteria and classified accordingly . Tumors size (expressed in millimeters) was documented in three dimensions (length, width, and height). For measurements, the tumor edge was defined as the end of the hypoechoic mass before the hyperechoic transition border (so called 1). The maximum dimension on gray - scale ultrasound was finally compared with the maximum histopathologic tumor size . Nonlinear harmonic imaging using a philips iu22 scanner (philips medical systems, best, the netherlands) equipped with a 48 mhz linear array transducer was performed at baseline with a low mechanical index of 0.1, chosen to avoid gas bubble destruction . A suspension of sulfur hexafluoride (sf6) microbubbles was obtained by adding 5 ml saline (0.9% sodium chloride) to 25 mg of the powder, followed by hand agitation . We used a single dose of 2.4 ml sonovue as contrast agent (sf6 volume in a 2.4 ml dose in 0.02 ml), which was intravenously administrated via a 20 gauge canula placed in an arm vein followed by a flushing of 10 ml standard saline . Directly after sonovue administration the microcirculation was studied by recording with clip function for 60 s, without chancing the transducer . Qlab software (philips medical system, best, the netherlands) was used to quantify enhancement on the ceus images in time . By using the region of interest quantification method when enhancement was at peak level (around 10 s) breast tumor size was recorded and measured again in three dimensions . For the measurements, the tumor edge was defined as the end of the hyperechoic mass at the time of maximal contrast enhancement of the lesion (fig . The maximum dimension of the tumor on ceus of the breast was finally compared with the maximum histopathologic tumor size . Both gray - scale and ceus breast ultrasound examination were performed by the same experienced breast radiologist in all cases . Histologic sections of the resected tumors after hematoxylin and eosin (h&e) staining were examined under a light microscope and used for measurement of histopathologic tumor size in millimeters in three dimensions . Margin status was recorded as involved or not involved . For those patients who underwent re - excision for tumor - involved margins, the extent of invasive ductal carcinoma in the re - excision specimen was recorded . To assess the difference between the three methods of tumor size measurement the greatest lesion diameters (mm) obtained by gray - scale breast ultrasound and ceus of the breast were compared with the greatest lesion diameter (mm) on pathology . Tumor size measured in greatest dimension was compared between the groups by using student t - test analysis (p<0.05). Furthermore, the percentage greatest tumor diameter as assessed by gray - scale ultrasound and ceus was within 2 mm of pathologic tumor size, which is considered accurate . The percentage of over- and underestimated tumor size for each modality was calculated using fisher's exact test, with a p - value <0.05 considered to be significant . The mean patient age in this study was 49 years (range 4257 years). Two patients presented with two breast lesions in the same quadrant (multifocal disease), and the remaining five patients presented with a solitary breast lesion . The sonographic characteristics of the lesions according to the sonographic bi - rads criteria are presented in table 1 . Definitive diagnosis on pathology was invasive ductal carcinoma in all cases, with an additional ductal carcinoma in situ (dcis) component in two cases . Gray - scale ultrasound of the breast showed a mean greatest tumor diameter of 15.5 mm (range 10.120.6 mm), compared to 16.5 mm (range 11.518.5 mm) in the ceus group . Mean greatest histopathologic tumor diameter was 15.6 mm (range 9.025.0 mm). Table 2 shows the greatest tumor diameter as measured with each modality compared to pathologic tumor size . Mean greatest tumor diameter as assessed with both ultrasound techniques did not significantly differ (p = 0.23). However, gray - scale ultrasound underestimated tumor size in 6/9 (67%) cases, whereas ceus of the breast underestimated tumor size only in 3/9 (33%) cases . Tumor as measured with gray - scale ultrasound of the breast was within 2 mm of the pathologic tumor size in only 2/9 cases (22%), whereas ceus of the breast accurately assessed tumor size within 2 mm of pathologic tumor size in 6/9 (67%) of the cases (p <0.05). Margin status was recorded as not involved in all nine cases; none of the patients underwent re - excision . To our knowledge this is the first study to assess the accuracy of ceus for pre - operative size assessment of invasive ductal carcinomas of the breast . In this prospective feasibility study ceus of the breast ceus of the breast was significantly more accurate than gray - scale ultrasound of the breast for tumor size measurement . The maximal tumor diameter as assessed by ceus of the breast was within 2 mm of pathologic tumor size in 67% of cases, compared with 22% in the gray - scale ultrasound group (p <0.05). The ability to accurately and reliably measure breast tumor size prior to any surgical treatment or primary medical treatment is essential . As a consequence previous research focused on different methods enabling non - invasive tumor size measurements, including clinical examination (palpation), mammography, gray - scale ultrasound, and magnetic resonance imaging (mri) of the breast . Of these methods palpation proved to have the lowest accuracy, because it is influenced by skin thickening, breast edema, and obesity, and is prone to overestimation of tumor size . Mammography proved to be more accurate than palpation, however it is taken in two standard projections (cranio - caudal and medio - lateral - oblique) not necessarily expressing the largest dimension of the tumor . As a consequence, although all studies showed significant correlation between mammographic and gray - scale ultrasound measurements, the latter technique is considered the most accurate for breast tumor size measurement . Previous studies that compared maximum breast tumor size as measured with gray - scale ultrasound of the breast to tumor size on pathology, reported correlation coefficients in the range of 4084% . However, all concluded that gray - scale ultrasound of the breast still underestimates tumor size in more than half of the patients . Mri of the breast has been reported to be the most accurate imaging modality for non - invasive tumor size assessment . In a prospective study including 111 women with 177 breast lesions, mri was reported to have an accuracy of 85% for evaluation of disease extent . Since, breast mri is expensive, time - consuming and still not available in every institute, we wanted to tackle the problem of tumor size underestimation rate on gray - scale ultrasound, by using contrast - enhanced ultrasound of the breast for tumor size measurement . Ceus of the breast is performed with the second generation contrast agent sonovue, which is made of microbubbles stabilized by phospholipids and containing the inert gas, sf6 . The microbubbles have a high reflectivity and are not extravasated from the vessel lumen, and as a consequence act like true blood pool agents . The effective vessel diameter from which an echo can be detected is in the range of a capillary . It has been postulated that this technique reliably visualizes the neovascularization within and around the tumor, and can potentially be used for tumor boundary identification and lesion characterization . Several studies have proven the potential of ceus of the breast in differentiating malignant from benign breast lesions, with varying sensitivities (6795%) and specificities (5882%) depending on the patient population being studied, the type of equipment, and the criteria used for interpretation of the ceus images . To date, no studies have been performed evaluating the accuracy of ceus of the breast for size measurement of malignant breast tumors . Our study results showed that both gray - scale ultrasound and ceus of the breast underestimated tumor size . Gray - scale ultrasound of the breast underestimated tumor size in 6/9 (67%) cases, which is in agreement with previous literature . However, ceus of the breast underestimated tumor size only in 3/9 (33%) of cases . Although the numbers in this study are small, a trend towards lower tumor size underestimation rate with ceus of the breast was found . More important, the accuracy of ceus of the breast for determining tumor size within 2 mm of pathologic tumor size was 6/9 (67%), compared with 2/9 (22%) for gray - scale ultrasound . This implies that ceus of the breast is a more accurate technique than gray - scale ultrasound of the breast for breast tumor lesion size measurement . A possible limitation of the study is the small number of patients included; as a consequence a well - founded statistical analysis and firm conclusions cannot be made . Furthermore, subgroup analysis reporting accuracy of tumor size assessment for different tumor subtypes could not be made . Second, it is known that ultrasound examination is operator dependent; both the quality of ultrasound and the accuracy of tumor size estimation may depend on the clinician's experience . Since, in our study all the ultrasound measurements were performed by the same breast imaging radiologist, no inter- and intra - observer bias of ceus for lesion size measurement could be calculated . Third, whether to include the echogenic interface (halo) around the hypoechoic lesion for measurement purposes on gray - scale breast ultrasound examination is still a matter of debate in the current literature . In our study the halo was not included for tumor size measurement, which is in agreement with most other previous studies . Finally, currently no standard with regard to lesion size measurement on ceus of the breast exists . Because it is a novel breast imaging modality, it is only rationally assumed that lesion size should be measured at the time of maximal contrast enhancement of the lesion, and that edges are defined as the end of the hyperechoic mass at that time . As a consequence, this feasibility study proves that ceus of the breast is a feasible and safe technique for breast tumor size measurement . In this small group of patients with invasive ductal carcinoma of the breast we showed that ceus of the breast was more accurate than gray - scale ultrasound of the breast for tumor size assessment the maximal tumor diameter as assessed by ceus of the breast was within 2 mm of pathologic tumor size in 67% of cases, compared with 22% in the gray - scale ultrasound group . Gray - scale ultrasound image of an irregular, not parallel oriented, spiculated breast lesion, classified as bi - rads v in the upper outer quadrant of the left breast . For measurements (see lines), the tumor edge was defined as the end of the hypoechoic mass before the hyperechoic transition border (so - called echogenic interface) between tumor and healthy surrounding tissue . Image of the same lesions with ceus of the breast, lesion has become more hyperechoic due to centripetal contrast enhancement . For measurements (see lines) the tumor edge was defined as the end of the hyperechoic mass at time of maximal contrast enhancement of the lesion . Baseline characteristics of 10 breast tumors according to sonographic bi - rads lexicon criteria combined pattern implied both posterior acoustic shadowing and enhancement.
It is currently believed that crsds result from an abnormality of circadian timing system, which regulates the diurnal rhythms of an organism . The core component of this system is the suprachiasmatic nucleus (scn) of the hypothalamus . This internal biological clock has self - generated, endogenous near - clrcadian rhythmlcity, which it conveys through direct and indirect pathways to a widely spread network of subcortical and cortical sites . Thus, many physiological functions, such as hormonal secretion and body temperature, as well as cognitive performance and emotional state, fluctuate according to the time of day . Regulation of the circadian rhythm of sleep - wake cycle involves secretion of the hormone melatonin by the pineal gland, one of the central target sites of the scn . The endogenous biological clock is synchronized or entrained with the environment through time cues, such as light . What abnormality in the complex mechanisms of the circadian timing system gives rise to crsds is still a matter of debate . Among the four disorders of the sleep - wake schedule in addition to sleep, circadian rhythms of melatonin and core body temperature were observed to be delayed in this disorder . Further, the phase angle between sleep timing, core body temperature rhythm, and melatonin rhythm was noted to be altered in patients with dsps . Whereas exposure to bright light at night acutely reduces melatonin concentration in subjects with typical sleep - wake rhythm, this effect is even greater in patients with dsps . Several hypotheses have been proposed to explain how these characteristics produce dsps, none of which has yet been confirmed or refuted . Some findings demonstrate that a genetic origin might be present in crsds . In as many as 44% of patients with crsds, there is evidence that other family members have similar sleep - wake patterns as the patient . In the pedigree of one family with dsps, the trait was found to segregate with either an autosomal dominant mode of herltance with incomplete penetrance or a multifactorial mode of inheritance . Structural polymorphisms on one of the haplotypes of the human period3 gene (hper3) were implicated as contributors to increased susceptibility to dsps . Several pedigrees of familial asps were reported, in which the asps trait segregated as an autosomal - dominant mode of inheritance . Although a mutation of human period2 (hper2) gene was identified in a large family with asps, other findings indicate genetic heterogeneity in this disorder . The exact mechanisms by which mutations in clock genes produce the physiological and behavioral phenotypes of crsds remain to be elaborated . A clinical interview should evaluate the patient's sleep - wake habits and presence of sleep complaints (such as insomnia and daytime sleepiness). Several additional characteristics might be sought for more accurate diagnosis of crsds, such as (i) impairment in different areas of functioning: these patients are frequently unable to keep a steady job, follow a school timetable, and maintain a normal social life; (ii) rigidity of sleep - wake patterns: it is extremely difficult for patients with crsds to adjust to new sleep - wake routines; (iil) hereditary trends: as shown above, other family members, such as parents, siblings, offspring, aunts, and uncles, are likely to have similar sleep - wake schedules to the patient; (iv) history of head injury or brain tumors: previous findings indicate that crsds can emerge as a secondary disorder associated with these conditions; (v) drug intake: as will be described below, crsds can also appear as a side effect of psychoactive medications . If dsps is suspected, it might also be helpful to question the patient about ms or her preferences in regard to mealtimes and hours of alertness . Patients with a delayed sleep - wake schedule usually report lack of appetite in the morning and choose evening hours as the best time for activities involving alertness and concentratlon . The second procedure is the confirmation of information collected in the clinical interview by 7 to 14 days of sleep logs and/or actlgraphic monitoring . The actlgraph is a watch - slzed device worn on the wrist sampling hand motion . A computerized algorithm can provide highly reliable data on sleep and wake periods of the patient . The documentation of sleep - wake cycles requires monitoring for at least several days; therefore, actlgraphy is the most appropriate objective tool for diagnosing crsds, and in most cases polysomnography is not neeessary . Importantly, actlgraphic monitoring must be conducted in free conditions, since sleep - wake schedule obtained under forced conditions can mask the pattern of the schedule, thus misleading the diagnosis . At present, bright - light therapy and melatonin treatment, or a combination of the two, have proved to be the most effective treatment modalities for patients with crsds . These techniques aim to reset the sleep - wake cycle of the patients to match the external 24-h schedule . Bright light is one of the most powerful time cues for the internal circadlan timing system . Light exposure at specific times of the 24-h period can result in a phase - shift in the endogenous circadlan rhythms of a variety of functions, such as melatonin secretion, body temperature, and sleep propensity . These tlme - dependent effects of light were described by phase - response curves (prcs). In general, morning bright - light exposure induces a phase advance, whereas evening bright light exposure induces phase delay . Using the entraining properties of light to synchronize sleep - wake schedule of patients with crsds has become an increasingly popular therapy artificial bright light applied by light devices at the intensities of 2000 to 4000 lux has been successfully used to realign the orcadian phase of patients with dsps and asps, and some evidence supports its effectiveness in treatment of nonentrained type sleep disorders, jet lag, shift work, and dementia . The american academy of sleep medicine has provided the recommended intensities and time 11mits for phototherapy in the treatment of these disorders . Endogenous melatonin secreted by the pineal gland is another potent regulator of the sleep - wake cycle . It is thought that the nighttime increase in melatonin concentration reduces body temperature, which promotes the onset of sleep . Previous findings have demonstrated that pharmacological preparations of melatonin mimic the effects of endogenous melatonin, which are time - dependent: phase advance is produced by melatonin admlnistered in the evening, whereas melatonin administration in the morning induces phase delays . Thus, the prc to melatonin is about 12 h out of phase with the prc to light . Administration of melatonin might be a preferable therapeutic strategy for many patients, who find phototherapy too demanding, leading to decreased compliance . The beneficial effects of 0.5 to 5 mg / day melatonin have been demonstrated in several types of crsds . Importantly, treatment with melatonin not only synchronizes the sleep - wake cycle of patients with crsds, but also significantly and clinically meaningfully improves several dimensions of their daytime functioning . Although some recent well - designed studies indicate that even relatively large doses of melatonin (10 mg / day for a month) have no toxlcological effects, its long - term effects remain to be fully researched and resolved . In patients for whom all of these treatment modalities fail to help, a rehabilitative approach is recommended . The patients should be guided to accept that their condition is permanent, and should be encouraged to consider changes in lifestyle that will be congruent with their sleep - wake cycle . As was described in several recent reviews, chronobiologlcal disturbances may play a crucial role in the pathogenesis of major psychiatric disorders, such as unipolar and bipolar depression, seasonal affective disorder, schizophrenia, and neurodegenerative illnesses . In the present review, we will specifically focus on cases where psychiatric practice might encounter disorders of the sleep - wake schedule . In a large sample of 322 patients with crsds who attended a sleep clinic, 72 patients (22.4%) were diagnosed with personality disorders based on clinical interview . To confirm this preliminary finding a controlled study was conducted, in which the incidence of personality disorders was examined in a group of 50 patients with dsps or freerunning pattern in comparison with 56 healthy controls . Personality disorders in both groups were assessed using the mlllon clinical multiaxlal inventory and personality diagnostic questionnaire - revised . The major finding of this study was that patients with crsds suffer more frequently from personality disorders than do normal controis . No specific pattern or profile of personality disorders could be clearly detected over and above the existence of general personality pathology in a complementary study, the sleep - wake habits of 63 adolescents hospitalized in psychiatric wards were examined . None of the patients had any diagnosed medical disorders, and all received psychoactive medications . The patients suffered from a variety of psychiatric disorders, including schizophrenia and other psychotic disorders; mood disorders; personality disorders; disorders usually first diagnosed in infancy, childhood, and adolescence; anxiety disorders; and substance - related disorders . As predicted, the probability of comorbid dsps among patients with personality disorders was significantly higher than among patients with any other psychiatric dis - order . Further, all of the patients with dsps suffered from disorders characterized by affective lability, namely bipolar disorder, schizoaffective disorder classified as mainly affective, and borderline personality disorder . These findings have led the authors to suggest that there may be an interrelationship between crsds and personality disorders . It is noticeable that both disorders are defined by the diagnostic and statistical manual of mental disorders, fourth edition (dsm - tv) as primarily involvmg a mismatch between the expectations of the society in which the individual lives and his or her own behavioral pattern . It might be that personality disorders are characterized by a deviant sleep - wake pattern as one expression of the general deviation from the expectations of society . On the other hand, peculiarities of the biological clock might lead to emotional, social, and functional difficulties that subsequently escalate into a personality disorder . According to this latter hypothesis, a deviant sleep - wake schedule frequently emerges early in life, possibly harming the mother and the child's mutual ability to adapt to each other . The mother, required to adjust to a biological clock of her infant that differs markedly from her own, becomes tired, frustrated, and angry, causing the infant to respond accordingly the resulting emotional burden, carried by both parties, might jeopardize the attachment processes, thus affecting future prospects of personal and social relationships of the child . At later stages of life, such a child has difficulties following the school timetable of activities, fails to obtain a sufficient amount of sleep at night, loses concentration during the morning and early afternoon hours, and, eventually, falls behind other children in school frequently, the abnormal sleep - wake cycle of individuals with crsds and the accompanying dysfunction at school or work are misattributed by parents, educators, psychologists, and other health care professionals to psychological rather than biological factors, such as laziness and low motivation . This attitude toward individuals with crsds, to which they are subjected since the early childhood or adolescence, adds psychological distress to the practical difficulties of coping with life and contributes to the development of personality disorders . Several cases of disrupted sleep - wake schedule as an latrogenie effect of psychoactive drugs have been documented in the literature . Treatment with a typical neuroleptic, haloperldol, in a patient with chronic schizophrenia was associated with an irregular sleep - wake cycle . Switching treatment to the atypical neuroleptic clozapine established a more organized and stable sleep - wake pattern and improved the clinical state of the patient . To further explore the relationship between type of drug and restactivity patterns, four of these patients received typical neuroleptics (flupentixol or haloperldol) and showed a variety of abnormalities in the daily rest - activity rhythm, eg, delayed circadlan phase syndrome, free - running sleepwake syndrome, and irregular sleep - wake pattern with a clrcabidlan component (approximately 48 h). On the other hand, rest - activity cycles of those patients treated with atypical neuroleptic clozapine (three patients) were highly organized and synchronized with the environmental schedule . Similar effects were observed in a female patient with early - onset alzheimer's disease: when treated with haloperldol, her rest - activity patterns became completely arrhythmic; this was accompanied by marked worsening of the cognitive state . When haloperldol was replaced by clozapine, rapid normalization of the sleep - wake cycle occurred and cognitive functioning improved . Additional evidence arises from a case study describing a male patient with gilles de la tourette syndrome, who was successfully treated with haloperldol prior to haloperldol treatment, the patient reported having a normal sleep - wake schedule . Two years after commencing the treatment, he exhibited an irregular sleep - wake cycle with a dominant 48-h clrcabidlan component . When therapy with haloperldol was changed to atypical neuroleptic risperidone, the timing and duration of sleep episodes became more organized, although his sleep - wake schedule still remained somewhat disturbed . Addition of melatonin as a secondary therapy fully recovered the patient's sleep - wake clrcadian rhythm . This was accompanied by improvement in his quality of life, social interactions, and employment status . These findings support the proposition that whereas atypical neuroleptics like clozapine and risperidone enhance the congruity of the individual's sleep - wake cycle with the environment, typical neuroleptics like haloperldol and flupentixol might alter the circadlan sleep - wake rhythm . Since this effect was evident in several medical disorders, eg, schizophrenia, alzheimer's disease, and tourette syndrome, it was argued that crsds are side effects of typical neuroleptics, rather than an illness - related phenomenon . The exact mechanisms through which typical and atypical neu - roleptics exert their differential effects on sleep - wake cycle remain to be elucidated . Clinical evidence indicates that apart from neuroleptics other psychoactive drugs, such as specific selective serotonin inhibitors (ssris), can trigger the emergence of crsds as a side effect . Hermesh et al described 10 patients with obsessive - compulsive disorder who developed dsps during fluvoxamlne treatment . It was postulated that delayed sleep - wake schedule in this case series was iatrogenic to fluvoxamlne based on the following observations: (i) all patients received no other medications except fluvoxamlne prior to the onset of dsps; (ii) in all patients, dsps first occurred following fluvoxamlne initiation; (iii) when fluvoxamlne was withdrawn or the dose considerably reduced, the sleep - wake cycle returned to normal; and (iv) with reexposure to fluvoxamlne, dsps recurred . Interestingly, emergence of dsps was quite specific to fluvoxamlne; treatment with two other ssris (clomipramine and fluoxetine) has not been associated with any adverse effects on sleepwake cycle of these patients . The authors hypothesized that the alteration of sleep - wake schedule or the lack of it by different ssri agents might depend on the differentlal effects of these drugs on serum melatonin levels . To summarize, the above cases indicate that certain psychoactlve medications might have adverse effects on the circadlan rhythm of the sleep - wake cycle . To date, we have clinical evidence of such effects for haloperldol, flupentixol, and fluvoxamlne . Whether there are additional psychotropics associated with disruptions of the sleepwake schedule, whether the response is doseand timedependent, and what the characteristics are of the particular patients who might develop crsds while on these drugs, remain questions for future research . At this stage, however, sleep - related complaints of patients treated with psychoactive drugs, especially haloperldol, flupentixol, and fluvoxamlne, should not always be regarded as drug - induced insomnia or daytime somnolence . As the findings demonstrate, in some cases crsds individuals with crsds frequently fall to adjust to the activity hours accepted in most social, occupational, and academic settings, due to incompatibility of their internal biological rhythms with the environmental timetable . Consider, for example, a patient with dsps who is expected to arrive at his workplace by 8 or 9 am . In order to fulfill this requirement, this individual is forced to wake up at what might be the middle of his internal night . It is not surprising, therefore, that he will be frequently late and/or absent, a pattern that will most likely subject him to disciplinary actions up to dismissal . If, however, he manages to meet the attendance standards, his performance will be liable to the detrimental effects of sleep loss and time of day . In childhood and adolescence, when crsds usually emerge, the impairment of daytime functioning can be even more remarkable than in adults . Unlike adults, who can at times choose a lifestyle that corresponds to their sleep - wake cycle, the activity hours of persons of younger age are constrained by a strictly predetermined school timetable . The inability to adjust to this timetable, we found that the vast majority of young patients with dsps complained of frequent late arrivals and absences at school, underachievement, and behavioral / social difficulties . Importantly, treatment with melatonin significantly reduced the number of children and adolescents complaining of malfunctioning at school . In some cases, the daytime functional difficulties might be severe enough to be mistakenly interpreted as symptoms of psychiatric disorders . A case of a 14-year - old boy provides a dramatic illustration of such a scenario . During the 4 years prior to his referral to our sleep clinic, the patient suffered from major functioning difficulties, including conflicts with teachers, parents, and peers . At the age of 12, the patient dropped out of school and three months of psychiatric evaluation yielded diagnoses of atypical depressive disorder with possible schizotypal personality disorder . Due to excessive daytime sleepiness, he was referred to our sleep clinic for assessment of a potential sleep disorder . A thorough sleep study revealed that the patient had a 26-h sleepwake schedule and dissociation between oral temperature and salivary melatonin rhythms . Treatment with oral melatonin normalized the sleepwake schedule within a month, and follow - up actigraphy after 6 months of melatonin treatment revealed a full entrainment to a 24-h day . The patient returned to school after a year of absence and succeeded in filling the gaps of missing studies . At the end of the first semester, his parents also reported an improvement in the patient's relationship with his family and peers . In a repeated psychiatric evaluation by licensed psychiatrists, none of the previously described severe diagnoses were present, and the boy showed no evidence of psychopathology, as was previously thought . Over the years of treating patients with crsds, we evidenced a considerable amount of similar case histories, some of which were previously documented . In this context, the association between crsds and attention deficit disorder (add) and attention deflcit / hyperactlvity disorder (adhd) should also be mentioned . A relatively high prevalence (19.3%) of these disorders was reported in a large sample of patients with crsds attending a sleep clinic . In a recent retrospective study of 45 children and adolescents with dsps (aged 6 to 18) who were treated with melatonin, the treatment advanced the sleep - wake cycle of these patients and improved their daytime functioning in educational settings . Interestingly, many of them were able to reduce or discontinue psychotherapy and/or stimulant medication during melatonin therapy . This finding indicates that, at least in some cases, crsd - related dysfunctional behaviors might be erroneously interpreted as symptoms of add / adhd . In a large sample of 322 patients with crsds who attended a sleep clinic, 72 patients (22.4%) were diagnosed with personality disorders based on clinical interview . To confirm this preliminary finding a controlled study was conducted, in which the incidence of personality disorders was examined in a group of 50 patients with dsps or freerunning pattern in comparison with 56 healthy controls . Personality disorders in both groups were assessed using the mlllon clinical multiaxlal inventory and personality diagnostic questionnaire - revised . The major finding of this study was that patients with crsds suffer more frequently from personality disorders than do normal controis . No specific pattern or profile of personality disorders could be clearly detected over and above the existence of general personality pathology in a complementary study, the sleep - wake habits of 63 adolescents hospitalized in psychiatric wards were examined . None of the patients had any diagnosed medical disorders, and all received psychoactive medications . The patients suffered from a variety of psychiatric disorders, including schizophrenia and other psychotic disorders; mood disorders; personality disorders; disorders usually first diagnosed in infancy, childhood, and adolescence; anxiety disorders; and substance - related disorders . As predicted, the probability of comorbid dsps among patients with personality disorders was significantly higher than among patients with any other psychiatric dis - order . Further, all of the patients with dsps suffered from disorders characterized by affective lability, namely bipolar disorder, schizoaffective disorder classified as mainly affective, and borderline personality disorder . These findings have led the authors to suggest that there may be an interrelationship between crsds and personality disorders . It is noticeable that both disorders are defined by the diagnostic and statistical manual of mental disorders, fourth edition (dsm - tv) as primarily involvmg a mismatch between the expectations of the society in which the individual lives and his or her own behavioral pattern . . It might be that personality disorders are characterized by a deviant sleep - wake pattern as one expression of the general deviation from the expectations of society . On the other hand, peculiarities of the biological clock might lead to emotional, social, and functional difficulties that subsequently escalate into a personality disorder . According to this latter hypothesis, a deviant sleep - wake schedule frequently emerges early in life, possibly harming the mother and the child's mutual ability to adapt to each other . The mother, required to adjust to a biological clock of her infant that differs markedly from her own, becomes tired, frustrated, and angry, causing the infant to respond accordingly the resulting emotional burden, carried by both parties, might jeopardize the attachment processes, thus affecting future prospects of personal and social relationships of the child . At later stages of life, such a child has difficulties following the school timetable of activities, fails to obtain a sufficient amount of sleep at night, loses concentration during the morning and early afternoon hours, and, eventually, falls behind other children in school frequently, the abnormal sleep - wake cycle of individuals with crsds and the accompanying dysfunction at school or work are misattributed by parents, educators, psychologists, and other health care professionals to psychological rather than biological factors, such as laziness and low motivation . This attitude toward individuals with crsds, to which they are subjected since the early childhood or adolescence, adds psychological distress to the practical difficulties of coping with life and contributes to the development of personality disorders . Several cases of disrupted sleep - wake schedule as an latrogenie effect of psychoactive drugs have been documented in the literature . Treatment with a typical neuroleptic, haloperldol, in a patient with chronic schizophrenia was associated with an irregular sleep - wake cycle . Switching treatment to the atypical neuroleptic clozapine established a more organized and stable sleep - wake pattern and improved the clinical state of the patient . To further explore the relationship between type of drug and restactivity patterns, four of these patients received typical neuroleptics (flupentixol or haloperldol) and showed a variety of abnormalities in the daily rest - activity rhythm, eg, delayed circadlan phase syndrome, free - running sleepwake syndrome, and irregular sleep - wake pattern with a clrcabidlan component (approximately 48 h). On the other hand, rest - activity cycles of those patients treated with atypical neuroleptic clozapine (three patients) were highly organized and synchronized with the environmental schedule . Similar effects were observed in a female patient with early - onset alzheimer's disease: when treated with haloperldol, her rest - activity patterns became completely arrhythmic; this was accompanied by marked worsening of the cognitive state . When haloperldol was replaced by clozapine, rapid normalization of the sleep - wake cycle occurred and cognitive functioning improved . Additional evidence arises from a case study describing a male patient with gilles de la tourette syndrome, who was successfully treated with haloperldol prior to haloperldol treatment, the patient reported having a normal sleep - wake schedule . Two years after commencing the treatment, he exhibited an irregular sleep - wake cycle with a dominant 48-h clrcabidlan component . When therapy with haloperldol was changed to atypical neuroleptic risperidone, the timing and duration of sleep episodes became more organized, although his sleep - wake schedule still remained somewhat disturbed . Addition of melatonin as a secondary therapy fully recovered the patient's sleep - wake clrcadian rhythm . This was accompanied by improvement in his quality of life, social interactions, and employment status . These findings support the proposition that whereas atypical neuroleptics like clozapine and risperidone enhance the congruity of the individual's sleep - wake cycle with the environment, typical neuroleptics like haloperldol and flupentixol might alter the circadlan sleep - wake rhythm . Since this effect was evident in several medical disorders, eg, schizophrenia, alzheimer's disease, and tourette syndrome, it was argued that crsds are side effects of typical neuroleptics, rather than an illness - related phenomenon . The exact mechanisms through which typical and atypical neu - roleptics exert their differential effects on sleep - wake cycle remain to be elucidated . Clinical evidence indicates that apart from neuroleptics other psychoactive drugs, such as specific selective serotonin inhibitors (ssris), can trigger the emergence of crsds as a side effect . Hermesh et al described 10 patients with obsessive - compulsive disorder who developed dsps during fluvoxamlne treatment . It was postulated that delayed sleep - wake schedule in this case series was iatrogenic to fluvoxamlne based on the following observations: (i) all patients received no other medications except fluvoxamlne prior to the onset of dsps; (ii) in all patients, dsps first occurred following fluvoxamlne initiation; (iii) when fluvoxamlne was withdrawn or the dose considerably reduced, the sleep - wake cycle returned to normal; and (iv) with reexposure to fluvoxamlne, dsps recurred . Interestingly, emergence of dsps was quite specific to fluvoxamlne; treatment with two other ssris (clomipramine and fluoxetine) has not been associated with any adverse effects on sleepwake cycle of these patients . The authors hypothesized that the alteration of sleep - wake schedule or the lack of it by different ssri agents might depend on the differentlal effects of these drugs on serum melatonin levels . To summarize, the above cases indicate that certain psychoactlve medications might have adverse effects on the circadlan rhythm of the sleep - wake cycle . To date, we have clinical evidence of such effects for haloperldol, flupentixol, and fluvoxamlne . Whether there are additional psychotropics associated with disruptions of the sleepwake schedule, whether the response is doseand timedependent, and what the characteristics are of the particular patients who might develop crsds while on these drugs, remain questions for future research . At this stage, however, sleep - related complaints of patients treated with psychoactive drugs, especially haloperldol, flupentixol, and fluvoxamlne, should not always be regarded as drug - induced insomnia or daytime somnolence . As the findings demonstrate, in some cases crsds individuals with crsds frequently fall to adjust to the activity hours accepted in most social, occupational, and academic settings, due to incompatibility of their internal biological rhythms with the environmental timetable . Consider, for example, a patient with dsps who is expected to arrive at his workplace by 8 or 9 am . In order to fulfill this requirement, this individual is forced to wake up at what might be the middle of his internal night . It is not surprising, therefore, that he will be frequently late and/or absent, a pattern that will most likely subject him to disciplinary actions up to dismissal . If, however, he manages to meet the attendance standards, his performance will be liable to the detrimental effects of sleep loss and time of day . In childhood and adolescence, when crsds usually emerge, the impairment of daytime functioning can be even more remarkable than in adults . Unlike adults, who can at times choose a lifestyle that corresponds to their sleep - wake cycle, the activity hours of persons of younger age are constrained by a strictly predetermined school timetable . The inability to adjust to this timetable may be associated with deteriorated school performance . In a recent study, we found that the vast majority of young patients with dsps complained of frequent late arrivals and absences at school, underachievement, and behavioral / social difficulties . Importantly, treatment with melatonin significantly reduced the number of children and adolescents complaining of malfunctioning at school . In some cases, the daytime functional difficulties might be severe enough to be mistakenly interpreted as symptoms of psychiatric disorders . A case of a 14-year - old boy provides a dramatic illustration of such a scenario . During the 4 years prior to his referral to our sleep clinic, the patient suffered from major functioning difficulties, including conflicts with teachers, parents, and peers . At the age of 12, the patient dropped out of school and three months of psychiatric evaluation yielded diagnoses of atypical depressive disorder with possible schizotypal personality disorder . Due to excessive daytime sleepiness, he was referred to our sleep clinic for assessment of a potential sleep disorder . A thorough sleep study revealed that the patient had a 26-h sleepwake schedule and dissociation between oral temperature and salivary melatonin rhythms . Treatment with oral melatonin normalized the sleepwake schedule within a month, and follow - up actigraphy after 6 months of melatonin treatment revealed a full entrainment to a 24-h day . The patient returned to school after a year of absence and succeeded in filling the gaps of missing studies . At the end of the first semester, his school report showed excellent results . His parents also reported an improvement in the patient's relationship with his family and peers . In a repeated psychiatric evaluation by licensed psychiatrists, none of the previously described severe diagnoses were present, and the boy showed no evidence of psychopathology, as was previously thought . Over the years of treating patients with crsds, we evidenced a considerable amount of similar case histories, some of which were previously documented . In this context, the association between crsds and attention deficit disorder (add) and attention deflcit / hyperactlvity disorder (adhd) should also be mentioned . A relatively high prevalence (19.3%) of these disorders was reported in a large sample of patients with crsds attending a sleep clinic . In a recent retrospective study of 45 children and adolescents with dsps (aged 6 to 18) who were treated with melatonin, almost half of the sample had a comorbld diagnosis of the treatment advanced the sleep - wake cycle of these patients and improved their daytime functioning in educational settings . Interestingly, many of them were able to reduce or discontinue psychotherapy and/or stimulant medication during melatonin therapy . This finding indicates that, at least in some cases, crsd - related dysfunctional behaviors might be erroneously interpreted as symptoms of add / adhd . . These disorders can be relatively easily diagnosed and treated with several available treatment modalities . Yet many cases of crsds are underrecognized and misdiagnosed as psychiatric dlsorders or psychophysiological insomnia . Consequently, these patients receive inappropriate treatment, such as hypnotlcs, which can enhance the psychological distress and add to the adjustment difficulties that accompany crsds . It is of great importance to raise the awareness of these disorders on the part of pediatricians, physicians, neurologists, psychiatrists, and psychologists.
Despite decreasing incidence and recent improvements in treatment concepts gastric cancer remains one of the most common cancer entities worldwide with highest incidence rates in eastern asian countries such as korea and japan . However, in order to define prognostic factors or to decide for adjuvant treatment concepts, prediction of postoperative survival is considered to be of utmost importance . The most commonly used tool is the tnm classification, edited by the union international contre le cancer (uicc) and american joint commission on cancer (ajcc). In 2010, as an alternative, nomograms were proposed to provide more accurate survival prediction because not only factors involving tumor extension to locoregional lymph nodes and distant organs but also predictive factors such as number of dissected lymph nodes, tumor size, lymphatic vessel infiltration (lvi), and tumor location represent prognostic factors in gastric cancer surgery . One of the first nomograms was developed in the us and cross - validated in 2 european institutions . Among them was a cohort from the department of surgery of the technische universitaet muenchen (tum). In contrast, the nomogram did not prove to be applicable in eastern asian (korean) patients due to possibly different treatment strategies, tumor location, or ethnic differences . In consequence further general applicability of the korean nomograms was considered questionable due to the high specialization of the reporting centers, which developed the nomograms . Therefore, eom et al developed a nomogram based on multicentric data (8 institutions) with a korean cross - validation study . This nomogram was constructed using a multivariate cox proportion hazard regression model of potential survival - related factors . Here, a line is drawn according to any of the clinicopathologic factors to an axis indicating specific score values . After adding the respective values, the sum value displays the probability of 5-year survival by connecting the score value with the 5-year survival axis . It was demonstrated that survival prediction was accurate not only in specialized centers but also in general hospitals . However, the authors concluded that validation in western patients is deemed to be useful considering general applicability of the tool . In order to obtain comparable results, we report of a validation study for the multicenter korean nomogram in gastric cancer patients having undergone primary curative oncologic surgery at the department of surgery of the tum . The prospectively documented gastric cancer database was reviewed for patients having undergone gastric cancer surgery with curative intent between 1982 and 2008 at the surgical department of the tum . Data were obtained from the medical records and transferred to the institutional database as soon as the patients were discharged from inpatient hospital care . Exclusion criteria were: metastatic disease, neoadjuvant / perioperative chemotherapy, extension to the distal esophagus, gastric stump cancer, hospital mortality within 30 days, loss of follow - up within a 60-month period and residual cancer after surgery (r1/r2). The dataset consisted of patients age, sex, tumor size, location (upper, middle, lower third), histological type (differentiated type: papillary, well - differentiated, and moderately differentiated tubular adenocarcinoma; undifferentiated type: poorly differentiated tubular adenocarcinoma, signet ring cell carcinoma, mucinous adenocarcinoma, and undifferentiated adenocarcinomas), lymphovascular invasion, pt-, pn-, and uicc - stage, extent of lymph node dissection and follow - up period with survival status . Lymph node dissection was categorized as d1 plus or d2 as defined by the japanese treatment guidelines . T- and n - stages were evaluated according to the 7th ajcc / uicc tumor - node - metastasis (tnm) classification . Patients were followed up after surgery regularly according to institutional guidelines including physical examinations, laboratory tests (cea), endoscopy, and computed tomography (ct). The examinations were scheduled every 6 months for the first 3 years and annually for the next 2 years . Follow - up time was determined from the day of surgery to the last follow - up date . The control group consisted of the patients having been included in the multicenter korean nomogram development set published before . Intergroup comparisons were analyzed by -testing, continuous variables are presented as mean standard deviation . T tests or wilcoxon tests were used whenever appropriate . Risk calculation according to the proposed nomogram (figure 1) was performed for every patient . The hazard ratio and corresponding 95% confidence interval [ci]) for each of the potential risk factors was estimated by a cox proportional hazard regression model . The korean patient group was used as the development set, and the german patient group as the validation set . All of the statistical analyses with respect to the model's performance were performed in the german patients cohort . The previously developed model was validated in the german cohort with respect to its discrimination ability using c - statistics and its calibration ability using hosmer the discriminative power depends on the ability of a model to correctly distinguish between nonevents and events, which can be quantified by calculating the c - statistic developed for the respective survival model . The c - statistic is a measure that is analogous to the receiver operating characteristic (roc) curve, which indicates the probability that a model produces higher risks for those who develop events compared with those who do not develop events . H - l chi - square statistic measures how closely the predicted probabilities agree numerically with the actual outcomes . This chi - square statistic was calculated by categorizing the data into 5 groups (quintiles) based on the predicted probabilities that are generated by the model in ascending order . For each quintile, the average predicted probabilities were compared to the event rate estimated by the kaplan meier method . The statistical analysis was carried out in analogy to the korean validation cohort in the eom paper . All of the results were interpreted by a specialist in biostatistics (bh nam). This analysis was approved by the local institutional review board (ethikkommission der fakultaet fr medizin der technischen universitaet muenchen). A total of 2771 patients underwent surgery for gastric cancer at tum between 1982 and 2008 . Among these patients, 659 patients with neoadjuvant / perioperative chemotherapy, 33 with neoadjuvant chemoradiation, 84 with r1-resection, 257 with r2-resection, 291 patients with metastatic disease, 307 with adenocarcinoma of the esophagogastric junction (aeg), 19 carcinomas of the gastric remnant, 42 patients with in - hospital mortality, 75 patients without resection (open&close), and 96 patients with a follow - up of <60 months were excluded from the analysis . Finally 908 patients were enrolled in this validation analysis . The control cohort consisted of 1579 patients and apart from ajcc / uicc stages, the tum cohort was different in almost all analyzed parameters compared to korean patients . Tum patients were significantly older (p <0.0001) and the amount of female patients was considerably higher (p <0.001). Tumor size tended to be smaller in german patients (p <0.0001) and tumors tended to be located in the more proximal parts of the stomach (p <0.0001). Tumors were more undifferentiated compared to korean patients and lvi was less frequent in german patients (p <0.0001, respectively). T - stages were more advanced in the tum cohort (p <0.0001), whereas lymph node metastases were diagnosed more frequently in koreans (p <0.0001). The extent of lymph node dissection was significantly different: almost all the korean patients received a d2 dissection whereas in contrast d1 + dissection was performed more frequently in the german cohort (p <0.0001). Therefore, the amount of patients with <15 retrieved lymph nodes is significantly higher compared to the korean - multicenter cohort (p <0.0001). Demographic and clinicopathological characteristics of the knds and tum cohorts median overall follow - up was 64.7 months (1218), 74 (8214 months) for survivors and 25.9 (1218) months for deceased patients in the german cohort . Median follow - up was significantly longer compared to the korean cohort (52 [1105] months overall, 57 [1105] months for survivors, 18 [1100] months for nonsurvivors, p <0.0001). During the follow - up period 398 patients (43.8%) died (351/1579 [23.4%] in the korean cohort, p <0.0001). In the univariate model age (older than 70 years), tumor size (above 5 cm), location in the upper third and tumor - involvement of the whole stomach, undifferentiated histology, lvi, pt-, pn - stage, and d1 + dissection were significantly related to survival . Multivariate regression analysis revealed age (older than 70 years), tumor extension to the complete stomach, presence of lvi, pt- and pn - stage to be significantly related to overall survival . The prognostic factors were almost comparable in the korean cohort . In korean patients tumor size> 10 cm was related to worse overall survival exclusively and pn1-stage was not predictive for os in contrast to the tum cohort . Risk factors for overall survival comparing korean and german patients (cox proportional hazards regression model) the validation of the korean nomogram in the tum cohort was performed by evaluating the performance of the model with respect to its discrimination and calibration abilities . The h - l chi - square statistic was 2.16, and the calibration plot (predicted and actual events [deaths]) according to the respective quintiles is presented in figure 2 (p = 0.989). The ratios between expected and observed risks for each quintile range from 84% to 107% of the predicted value . Each stage was significantly different from each other (group 1 vs group 2: p <0.0001; group 2 vs group 3: p = 0.0018; group 3 vs group 4: p = 0.015; group 4 vs group 5: p <0.0001). Calibration plot of predicted and actual events (deaths) according to the quintiles in the hosmer lemeshow statistic . H - l type = 2.162 (p = 0.989). C - index = 0.761 (95% ci 0.7350.787). The observed and expected event probabilities and their respective ratios (o / e) meier survival plots according to the respective quintiles in the german validation cohort (p <0.0001). Group 1 vs group 2: p <0.0001; group 2 vs group 3: p = 0.0018; group 3 vs group 4: p = 0.015; group 4 vs group 5: p <0.0001 . A total of 2771 patients underwent surgery for gastric cancer at tum between 1982 and 2008 . Among these patients, 659 patients with neoadjuvant / perioperative chemotherapy, 33 with neoadjuvant chemoradiation, 84 with r1-resection, 257 with r2-resection, 291 patients with metastatic disease, 307 with adenocarcinoma of the esophagogastric junction (aeg), 19 carcinomas of the gastric remnant, 42 patients with in - hospital mortality, 75 patients without resection (open&close), and 96 patients with a follow - up of <60 months were excluded from the analysis . Finally 908 patients were enrolled in this validation analysis . The control cohort consisted of 1579 patients and apart from ajcc / uicc stages, the tum cohort was different in almost all analyzed parameters compared to korean patients . Tum patients were significantly older (p <0.0001) and the amount of female patients was considerably higher (p <0.001). Tumor size tended to be smaller in german patients (p <0.0001) and tumors tended to be located in the more proximal parts of the stomach (p <0.0001). Tumors were more undifferentiated compared to korean patients and lvi was less frequent in german patients (p <0.0001, respectively). T - stages were more advanced in the tum cohort (p <0.0001), whereas lymph node metastases were diagnosed more frequently in koreans (p <0.0001). The extent of lymph node dissection was significantly different: almost all the korean patients received a d2 dissection whereas in contrast d1 + dissection was performed more frequently in the german cohort (p <0.0001). Therefore, the amount of patients with <15 retrieved lymph nodes is significantly higher compared to the korean - multicenter cohort (p <0.0001). Demographic and clinicopathological characteristics of the knds and tum cohorts median overall follow - up was 64.7 months (1218), 74 (8214 months) for survivors and 25.9 (1218) months for deceased patients in the german cohort . Median follow - up was significantly longer compared to the korean cohort (52 [1105] months overall, 57 [1105] months for survivors, 18 [1100] months for nonsurvivors, p <0.0001). During the follow - up period 398 patients (43.8%) died (351/1579 [23.4%] in the korean cohort, p <0.0001). In the univariate model age (older than 70 years), tumor size (above 5 cm), location in the upper third and tumor - involvement of the whole stomach, undifferentiated histology, lvi, pt-, pn - stage, and d1 + dissection were significantly related to survival . Multivariate regression analysis revealed age (older than 70 years), tumor extension to the complete stomach, presence of lvi, pt- and pn - stage to be significantly related to overall survival . The prognostic factors were almost comparable in the korean cohort . In korean patients tumor size> 10 cm was related to worse overall survival exclusively and pn1-stage was not predictive for os in contrast to the tum cohort . Risk factors for overall survival comparing korean and german patients (cox proportional hazards regression model) the validation of the korean nomogram in the tum cohort was performed by evaluating the performance of the model with respect to its discrimination and calibration abilities . The h - l chi - square statistic was 2.16, and the calibration plot (predicted and actual events [deaths]) according to the respective quintiles is presented in figure 2 (p = 0.989). The ratios between expected and observed risks for each quintile range from 84% to 107% of the predicted value . Each stage was significantly different from each other (group 1 vs group 2: p <0.0001; group 2 vs group 3: p = 0.0018; group 3 vs group 4: p = 0.015; group 4 vs group 5: p <0.0001). Calibration plot of predicted and actual events (deaths) according to the quintiles in the hosmer lemeshow statistic . H - l type = 2.162 (p = 0.989). C - index = 0.761 (95% ci 0.7350.787). The observed and expected event probabilities and their respective ratios (o / e) meier survival plots according to the respective quintiles in the german validation cohort (p <0.0001). Group 1 vs group 2: p <0.0001; group 2 vs group 3: p = 0.0018; group 3 vs group 4: p = 0.015; group 4 vs group 5: p <0.0001 . Despite decreasing incidence gastric cancer remains 1 of the most common malignancies worldwide with a special focus on eastern asia (korea, japan, and china). Therefore, uniform staging methods are of utmost importance to compare treatment outcomes and treatment results from randomized controlled trials between different parts of the world . The most commonly used staging system is the tnm system proposed by the uicc / ajcc . However, several authors demonstrated before that survival probabilities may vary within the respective uicc / ajcc stages and that survival predictability has not necessarily improved after revision of the sixth edition . Furthermore predictive factors such as tumor location, tumor size, extent of lymph node dissection, and presence of lvi are not covered by classical tnm staging . Therefore, more uniform staging methods are required omitting those possible drawbacks . A conceivable option to resolve this issue the best known nomogram was developed at the memorial sloan kettering cancer center in the united states and validated in cohorts across the world with different outcomes . Validation studies in germany and the netherlands revealed its applicability in the western hemisphere whereas the mskcc - nomogram failed to adequately predict survival in korean patients . The snuh - nomogram was externally validated not only in korea itself but also in japan, whereas the seoul - st mary's nomogram was only validated internally . They only incorporated patients having undergone open surgery and d2-lymphadenctomy by very specialized surgeons with high caseload . The nomograms were criticized not to be applicable to stage adopted lymph node dissection (d1 +) in early gastric cancer patients concluding that previous nomograms are not suited for more than half of the gastric cancer patients in korea . Due to that criticism the korean multicenter data incorporated data from 8 different institutions with different case load and also reduced lymphadenectomy cases (d1 + gastrectomy due to egc). The authors concluded that their multicenter - nomogram was suitable for survival prediction in specialized and local hospitals in korea . Similar to the developers of the snuh - nomogram the authors concluded that despite successful external validation within korea and japan, a confirmation of the results in a western patient cohort was required in order to obtain general applicability . Comparison between the korean and the german group revealed marked differences in baseline characteristics except for the distribution of uicc stages . It is well known that korean patients are significantly younger at the time of diagnosis and treatment compared to western patients . The amount of patients older than 70 years was twice as high in the german cohort . Additionally a preponderance of proximal tumor locations was noted and is congruent with other studies . These facts may basically be related to and explained by the existence of a national screening program in korea, which is accessible from the age of 40 and inexistent in germany, where egd is only performed in case of symptoms or positive family history . However, distribution of the ajcc / uicc stages was not different between the 2 groups . Nonsurprisingly the amount of patients having undergone d2-dissection is significantly higher in the korean cohort and represented by the significantly higher number of harvested lymph nodes . D2-dissection has only been implemented in europe as a standard of care since publication of the long - term results of the dutch trial . Nonetheless the authors of the nomogram emphasized the applicability of the nomogram also for patients not having undergone d2-dissection due to more limited disease stages . Interestingly the type of lymph - node dissection did not have any significant correlation to overall survival in both of the analyzed cohorts . Analysis of prognostic factors revealed the same predictors of overall survival as in the korean cohort with the following exceptions: tumor size> 10 cm was not related to os in the german cohort . Tumor involvement of the whole stomach was exclusively related to os in the tum patients which, however, implies a tumor size> 10 cm . Interestingly, limited metastasis to 1 or 2 lymph nodes was an independent predictor of os only in the german patient cohort . This may be explained by a more aggressive tumor phenotype which may be represented by more advanced pt - stages . Finally, comparative analysis of the predictive abilities of the korean nomogram in the german patient cohort demonstrated its validity . C - statistics were used as goodness - of - fit measure to discriminate expected and actual events . C - values of 0.7 to 0.8 generally indicate moderately good discrimination and excellent discriminative ability is indicated by values over 0.8 . The c - index of 0.76 may be interpreted as an acceptable value for predictability of the nomogram in western patients . The nomogram is considered to be more accurate the higher the value of the c - index is . The lower value in the german cohort thus means a decreased predictability compared to the korean validation set . This may be represented by ethnic and biologic differences, which are not considered in the statistical model . However, goodness - of - fit analysis by h - l chi - square statistic revealed no statistical difference in the german validation . This result indicates that the predictive ability of the nomogram does not significantly differ from the observed os . Further, stage distribution according to the predicted quintiles revealed a homogenous distribution of the survival curves with statistically significant differences over all stages . This staging system might be an additional tool to the conventional tnm - staging for survival prediction . This stands in marked contrast to the popular mskcc - nomogram, which failed to predict survival in comparison between korean and us patients . Reasons for this may be that the mskcc - nomogram incorporated slightly different parameters like lauren histotype and a differentiation between positive and negative lymph nodes . Not only the lauren histotype but also stage distribution was significantly different between korean and us patients . Further the amount of patients with a number of <15 retrieved lymph nodes was 22% in the mskcc cohort compared with 6% in this analysis . However, the construction- and validation - datasets are based on a single - center cohort with patients having undergone d2-dissection only . This does not reflect surgical reality in these days as more stage - adopted resection is performed in korea (ie, d1 + dissection for early gastric cancer). Finally the snuh - nomogram was only validated in a specialized japanese single - center cohort . Despite its similarities with the snuh - nomogram this multicenter nomogram is not only applicable in specialized korean centers but also in general hospitals and maybe in western (european) collectives . The predictability of the korean multicenter - nomogram in the tum cohort also reflects the necessity to treat patients in specialized institutions, especially in western countries . This again stirs up the debate if centralization to specialized hospitals for specific types of cancers improves oncologic outcomes . From the authors point of view further, patients having undergone neoadjuvant / perioperative chemotherapy were excluded from this analysis which may have led to a selection bias . The reason for excluding these patients was that neoadjuvant regimens in the reported period (19822008) were not standardized and applied on an irregular basis . Chemotherapy at that time was applied to technically irresectable patients who would not have met the inclusion criteria for this analysis . Since neoadjuvant chemotherapy has become a standard of treatment in europe these results will have to be reanalyzed in the future . Further the general applicability of the nomogram in german community hospitals may not be guaranteed due to the specialization of the tum in gastric cancer treatment and a centralization effect . Conclusively this validation analysis of a korean multicenter nomogram for survival - prediction after curative gastric cancer surgery demonstrated its applicability in a specialized western treatment center for gastric cancer . Further study is needed to obtain data on generalized applicability in western gastric cancer patients.
The calcifying odontogenic cyst (coc) was first described by gorlin and his colleagues (1962), as a separate entity of odontogenic origin . As all lesions are not cystic and the biological behavior is often not compatible with a cyst, there is a controversy as to whether coc is a cyst or a tumor . Based on the dualistic concept, some authors consider that coc contains two entities: a cyst and a neoplasm . However, others regard coc as a tumor with a tendency for cyst formation . Based on this monistic concept the world health organization (who) has classified all cocs as neoplasms . The cystic lesions are termed as calcifying cystic odontogenic tumors (ccot) and the neoplastic entity as a dentinogenic ghost cell tumor(dgct). Microscopically, it consists of ameloblastomatous epithelial islands, with areas of ghost cell formation and varying amount of dentinoid material . The purpose of this article is to report a case of dentinogenic ghost cell tumor in a 21-year - male, in the posterior region of the mandible, which is at a comparatively younger age and at an infrequent site . A 21-year - old male patient reported to the department of oral pathology and microbiology, government dental college and hospital, aurangabad, with the complaint of swelling in the left posterior region of the lower jaw, of one - month duration . Extraorally a diffuse swelling was seen over the left mandibular angle region [figure 1]. Diffuse swelling over mandibular angle region on left side intraoral examination revealed the presence of a bony hard swelling, extending from 37 to the retromolar region [figure 2]. The color of the lesional area was the same as that of the adjacent mucosa . It showed a smooth surface all over, except a small growth distal to 37, which subsided after antibiotic treatment . Bony hard swelling extending from 37 to retro - molar region with a small 1 1 cm reddish growth, distal to 37 radiographs revealed a well - defined lesion extending from 34 to the ramus of the mandible with scalloped borders . An occlusal radiograph showed buccal cortical expansion in the retromolar region on the left side [figure 4]. Root resorption of 36 and 37 buccal cortical expansion in the left retro - molar region histopathological examination revealed a tumor mass composed of islands of odontogenic epithelial cells with hyperchromatic nuclei in a mature connective tissue stroma . Ameloblastoma - like islands with peripheral columnar, polarized basal cells, and central stellate reticulum - like cells were also noted . Numerous ghost cells with faint eosinophillic cytoplasm and a shadow of nuclear outlines were noted within the epithelial islands as well as in the connective tissue [figure 5]. Many foreign body - type multinucleated giant cells were observed surrounding the ghost cells in the connective tissue stroma [figure 6]. Some ghost cells exhibited calcification . Masses of dentinoid - like material were present in close proximity to the epithelial islands; in which some showed calcification [figure 7]. Ameloblastomatous epithelial island with ghost cells and dentinoid material (h and e, 40) giant cells surrounding ghost cells in the connective tissue stroma (h and e, 100) calcifying ghost cells and dysplastic dentin (h and e, 100) tumor cells infiltrating adjacent bone (h and e, 400) calcifying odontogenic cyst constitutes 1 to 2% of all odontogenic tumors in which 88.5% are cystic and the remaining 11.5% are solid tumors . (1981), termed these solid tumors as dentinogenic ghost cell tumors as they are made up of an ameloblastomatous epithelium with areas of ghost cell formation and varying amounts of dentinoid . Fejerskov and krogh (1972), interpreted it as a tumor or hamartoma with a marked tendency for cystic degeneration . They stated that there is no reason to assume that epithelial change can develop only in the pre - existing cyst wall, but rather cystic degeneration can take place in the of the proliferating epithelial island . These authors also noted that the lesion is not invariably cystic and suggested the term calcifying ghost cell odontogenic tumor . As it appeared to be one variety of ameloblastoma and could recur after conservative surgical treatment, shear (1983), preferred the term dentinoameloblastoma . Ellis and shmookler (1986), preferred the term epithelial odontogenic ghost cell tumor as epithelial cells appearing like ghost cells were the most distinctive feature of this neoplasm . (1990), suggested the term odontogenic ghost cell tumor for the same neoplastic form of coc . The average age for the presentation of this lesion is 50 years, (range 17 72 years) with slight male predilection . Tumor occurs in the maxilla and the mandible with equal frequency, with canine to first molar region the most often the affected site . Patients are usually without symptoms, although with a few complain of pain or discomfort . The present lesion was seen in a 21-year - old male, in the posterior region of the mandible on the left side, at a comparatively younger age, and contrary to the common anterior region of the jaw . Root resorption or an impacted tooth in relation to the tumor mass is also noted in some cases . Microscopic features show islands of odontogenic epithelial cells with hyperchromatic nuclei in a mature connective tissue . Loosely arranged stellate reticulum - like cells may be seen enclosed by the odontogenic epithelium . The epithelium may show ameloblastomatous proliferation with a well - defined basal layer of columnar or cuboidal cells and hyperchromatic nuclei, which are polarized away from the basement membrane . On account of the multipotentiality of odontogenic epithelium, the histological presence of areas similar to different odontogenic tumors, such as, ameloblastoma, odonto - ameloblastoma, ameloblastic fibro - odontoma, odontoma, adenomatoid odontogenic tumor, and cementoma may be seen . Two characteristic features of dgct distinguish it from ameloblastoma and other odontogenic tumors, numerous ghost cells, and masses of dentinoid material . Ghost cells are characterized by the loss of nuclei, preservation of basic cellular outlines, and resistance to degradation . Although cellular outlines are usually well defined, they may be blurred, and as a result the groups of ghost cells appear fused . Dystrophic calcification may occur in some of the ghost cells, initially seen as fine basophilic granules and later as small spherical bodies . Sometimes ghost cells break through the basement membrane and come in contact with the connective tissue, where they evoke a foreign body reaction with the formation of multinucleated giant cells . Most authors interpreted the changes in the ghost cells as aberrant or incomplete keratinizations or even as true keratinizations . Abram and howell (1968), presented the concept that the keratin appeared to originate from the degeneration of squamous cells . Levy (1973), investigated the ghost cells in odontomas and suggested that they represented squamous metaplasia with subsequent calcification caused by ischemia . Sedano and pindborg (1975), thought that ghost cells represented different stages of normal and aberrant keratin formation and that they were derived from the metaplastic transformation of odontogenic epithelium . Other investigators were of the opinion that ghost cells might be the product of abortive enamel matrix in the odontogenic epithelium, however, the morphology of ghost cells seems to be different from that of the enamel matrix . Another characteristic feature is the formation of dentinoid or osteoid material, which represents an inflammatory response of the body to the presence of ghost cells . Abrams and howell (1968), stated that masses of ghost cells induce granulation tissue to lay down the juxtaepithelial osteoid, which may calcify . Sauk (1972), sapp and gardner (1977), and nagao et al . (1982), stated that the juxtaepithelial osteoid or dentinoid are frequently found in areas free of either granulation tissue or ghost cells and postulated that it might be an inductive phenomenon rather than an inflammatory response . To date, it is not clear whether this material represents a true inductive effect or merely a metaplastic change in the connective tissue . Similar histopathological features are seen in the present case, and hence it has been diagnosed as dentinogenic ghost cell tumor . In the immunohistochemical evaluation of a case of dgct, by piatelli a et al . The epithelial cells were positive for cytokeratins, characterizing the presence of an odontogenic epithelium, while the calcified bodies and ghost cells were devoid of any immunoreactivity, representing that they were derived from the metaplastic transformation of the odontogenic epithelium or were a product of the coagulative necrosis of the odontogenic epithelium . There was also a strong positivity of the odontogenic epithelium for bcl-2 and mib-1, whereas, only a rare positivity for p-53 . The ghost cells, giant cells, and dentinoid material were completely negative . It was concluded that the cells that expressed bcl-2 and mib-1 probably represented the portion of the tumor that proliferated and that could undergo malignant transformation . Initially enucleation was the primary treatment for central dgct, but local recurrence was noted . The patient has been under observation since nine months and no recurrence has been noted till now . Coc has been seen to be of extensive diversity in its clinical and histopathological features as well as in its biological behavior . The present case of 21-year - old male was diagnosed as dgct, a tumorous form of coc, due to its characteristic histological features; numerous ghost cells and dentinoid material.
However, toothbrushes are rare in many third - world countries, where locally available chewing sticks are commonly used . The most common type of chewing stick, miswak, is derived from salvadora persica, a small tree or shrub with a spongy stem and root, which is easy to crush between the teeth . Miswak is a chewing stick used in many developing countries as a traditional toothbrush for oral hygiene . Reports on the oral health of miswak users are contradictory; several studies have claimed that chewing sticks are effective in reducing plaque and gingival inflammation. [58] when used properly, the miswak is reported to be as effective as a toothbrush . It was found that the chewing stick removed plaque from interproximal sites to virtually the same extent as from other more accessible sites . The conventional tooth brushing has been reported to be relatively ineffective for the removal of interproximal plaque . The use of miswak has also been reported to inhibit the formation of dental plaque chemically and exert antimicrobial effect against many oral bacteria . However, some studies have reported more plaque formation and gingival bleeding in individuals who used chewing sticks in comparison with toothbrush users . In few studies, it has been found that the toothbrush was more efficient as an oral hygiene aid than the miswak . Surprisingly, despite the widespread use of miswak since ancient times, relatively little scientific attention has been paid to its oral health beneficial effects . In 1987, world health organization encouraged the developing nations to use miswak for oral hygiene because of tradition, availability, and low cost . Recently, various authors have concluded that chewing sticks or its extract has therapeutic effect on gingival diseases . Thus, a study was designed to clinically evaluate the effect of miswak stick as an adjunct to tooth brushing on plaque levels and gingival health in subjects diagnosed with mild to moderate chronic generalized marginal gingivitis in comparison with those of toothbrush users . Thirty subjects diagnosed with mild to moderate chronic generalized marginal gingivitis were selected randomly from the out patient department (opd) presenting to jss dental college and hospital, mysore . The inclusion criteria included subjects to be within the age group of 18 - 35 years; subjects who were having full set of teeth; subjects who were diagnosed with mild to moderate chronic generalized marginal gingivitis; subject having a probing pocket depth less than or equal to 3 mm; and subjects showing gingival index (loe and silness) more than 1, and plaque index (turesky modified quigley - hein plaque index) more than 1 . Exclusion criteria were the subjects who were systemically compromised; pregnant and lactating mothers; subjects having orthodontic appliance; subjects who have grossly decayed teeth, mal - positioned teeth or crowded teeth, overhanging restorations, crowns, and fixed partial dentures; patients who used antibiotics in the previous three months; and patients with xerostomia and on antihistamines were also excluded . The study was performed according to a randomized, single - blind (clinical investigator), parallel - armed design . Before the start of the study, during the first visit oral hygiene habits were recorded by a structured interview based on a prepared questionnaire . The questionnaire included education level, smoking habit, dental visit, usage of chewing stick and/or toothbrush and its frequency . The subjects were randomly assigned to one of the three groups consisting of 10 subjects in each group . Group a- subjects were asked to brush with the provided toothbrush without toothpaste during morning, mid day, and evening.group b- subjects were asked to brush with the provided toothbrush without toothpaste thrice daily, and in addition, miswak sticks were given to use during morning, mid day, and evening.group c- subjects were asked to use miswak sticks during morning, mid day, and evening . Group a- subjects were asked to brush with the provided toothbrush without toothpaste during morning, mid day, and evening . Group b- subjects were asked to brush with the provided toothbrush without toothpaste thrice daily, and in addition, miswak sticks were given to use during morning, mid day, and evening . Group c- subjects were asked to use miswak sticks during morning, mid day, and evening . Subjects were trained and instructed on a model to use miswak or toothbrush (without toothpaste) or both, depending on the respective assigned group . Sufficient numbers of commercially available standard sized (20 cm in length and 1 cm in diameter) miswak branch - lets / twigs (miswak haleemi, haleemi natural products, karachi, pakistan, 75700) [figure 1] were purchased from local market of mysore, india, and were stored in the refrigerator until implemented into the study . Demonstrating a miswak stick and a toothbrush used in the study subjects falling under group c were given fresh sticks of standard sized miswak sticks . Subjects falling under group a were given a new straight handled, medium soft toothbrush without toothpaste (ajay quest toothbrush, raghav lifestyle products, new delhi, india) [figure 1]. Subjects falling under group b were given both miswak sticks and tooth brushes without toothpaste . Subjects using miswak (group b and c) were advised to use freshly prepared miswak stick at each indicated time interval and store in refrigerator packed in provided paper when not in use . Recording of clinical indices and photographs were taken at baseline, 2 4, 6, and 8 week time intervals . The clinical parameters measured were: a) gingival index, according to loe and silness and b) plaque index, according to turesky modified quigley - hein plaque index . Both gingival inflammation and plaque were registered at four sites per tooth (buccal, mesial, distal, and lingual) for all teeth, except the third molar, and were analyzed according to respective indexing system . Photograph of stained plaque on anterior dentition was taken by using a digital camera (kodak c713, eastman kodak company, rochester, ny 14650), keeping the camera approximately perpendicular (90) and at a distance of 25 cm from the labial surface of upper central incisor . Photographic images were analyzed by computer software (uth scsa image tool [it version 2]) for the percentage of plaque coverage . The total labial teeth surfaces and the area covered by plaque all images were coded and the analysis of the images was done in a blinded way . The analysis of the images was done at baseline, 2 4, 6, and 8 week time intervals . Recorded data were analyzed using software program (statistical package version 17, statistical package for the social sciences (spss), chicago, il). All parametric variables were analyzed using repeated measures of analysis of variance (anova) for examining the mean differences from baseline to 8 week and student's t test (independent samples) for comparison of mean differences between groups at specific time intervals . The inclusion criteria included subjects to be within the age group of 18 - 35 years; subjects who were having full set of teeth; subjects who were diagnosed with mild to moderate chronic generalized marginal gingivitis; subject having a probing pocket depth less than or equal to 3 mm; and subjects showing gingival index (loe and silness) more than 1, and plaque index (turesky modified quigley - hein plaque index) more than 1 . Exclusion criteria were the subjects who were systemically compromised; pregnant and lactating mothers; subjects having orthodontic appliance; subjects who have grossly decayed teeth, mal - positioned teeth or crowded teeth, overhanging restorations, crowns, and fixed partial dentures; patients who used antibiotics in the previous three months; and patients with xerostomia and on antihistamines were also excluded . The study was performed according to a randomized, single - blind (clinical investigator), parallel - armed design . Before the start of the study, during the first visit, intraoral examination was performed . Oral hygiene habits were recorded by a structured interview based on a prepared questionnaire . The questionnaire included education level, smoking habit, dental visit, usage of chewing stick and/or toothbrush and its frequency . The subjects were randomly assigned to one of the three groups consisting of 10 subjects in each group . Group a- subjects were asked to brush with the provided toothbrush without toothpaste during morning, mid day, and evening.group b- subjects were asked to brush with the provided toothbrush without toothpaste thrice daily, and in addition, miswak sticks were given to use during morning, mid day, and evening.group c- subjects were asked to use miswak sticks during morning, mid day, and evening . Group a- subjects were asked to brush with the provided toothbrush without toothpaste during morning, mid day, and evening . Group b- subjects were asked to brush with the provided toothbrush without toothpaste thrice daily, and in addition, miswak sticks were given to use during morning, mid day, and evening . Group c- subjects were asked to use miswak sticks during morning, mid day, and evening . Subjects were trained and instructed on a model to use miswak or toothbrush (without toothpaste) or both, depending on the respective assigned group . Sufficient numbers of commercially available standard sized (20 cm in length and 1 cm in diameter) miswak branch - lets / twigs (miswak haleemi, haleemi natural products, karachi, pakistan, 75700) [figure 1] were purchased from local market of mysore, india, and were stored in the refrigerator until implemented into the study . Demonstrating a miswak stick and a toothbrush used in the study subjects falling under group c were given fresh sticks of standard sized miswak sticks . Subjects falling under group a were given a new straight handled, medium soft toothbrush without toothpaste (ajay quest toothbrush, raghav lifestyle products, new delhi, india) [figure 1]. Subjects falling under group b were given both miswak sticks and tooth brushes without toothpaste . Subjects using miswak (group b and c) were advised to use freshly prepared miswak stick at each indicated time interval and store in refrigerator packed in provided paper when not in use . Recording of clinical indices and photographs were taken at baseline, 2 4, 6, and 8 week time intervals . The clinical parameters measured were: a) gingival index, according to loe and silness and b) plaque index, according to turesky modified quigley - hein plaque index . Both gingival inflammation and plaque were registered at four sites per tooth (buccal, mesial, distal, and lingual) for all teeth, except the third molar, and were analyzed according to respective indexing system . Photograph of stained plaque on anterior dentition was taken by using a digital camera (kodak c713, eastman kodak company, rochester, ny 14650), keeping the camera approximately perpendicular (90) and at a distance of 25 cm from the labial surface of upper central incisor . Photographic images were analyzed by computer software (uth scsa image tool [it version 2]) for the percentage of plaque coverage . The total labial teeth surfaces and the area covered by plaque were directly counted in pixels . The plaque area all images were coded and the analysis of the images was done in a blinded way . The analysis of the images was done at baseline, 2 4, 6, and 8 week time intervals . Recorded data were analyzed using software program (statistical package version 17, statistical package for the social sciences (spss), chicago, il). All parametric variables were analyzed using repeated measures of analysis of variance (anova) for examining the mean differences from baseline to 8 week and student's t test (independent samples) for comparison of mean differences between groups at specific time intervals . None of the participants complained of discomfort or showed any signs of adverse reactions with use of prescribed chewing sticks . Data analyzed by repeated measure of anova for time and cleaning method interactions showed a significant reduction in plaque score (f=20.612; p<0.0001) from baseline to 8 week in all the groups . Student's t test done for detecting a difference at specific time intervals showed statistically significant (p<0.0001) decrease in plaque score in group b compared to group a and group c, respectively, from 2 to 8 week . Whereas, there was no statistical significant difference found when group a was compared with group c (p>0.05) from 2 to 4 week . At the 6 and 8 weeks, there was significant difference (p<0.05) between group a and group c [table 1]. Comparison of plaque scores between the groups at various times intervals result obtained from repeated measure of anova showed a highly significant decrease (f=11.981; p<0.0001) in mean gingival score from baseline to 8 week in all the groups . Student's t test showed statistically significant difference (p<0.05), when group b was compared with groups, a and c respectively . The difference was not significant when group a was compared with group c (p>0.05) [table 2]. Comparison of gingival scores between the groups at various times intervals analysis done by results repeated measure of anova showed that there was highly significant reduction (f=4.942; p<0.0001) in stained plaque area from baseline to 8 week in all the groups . Student's t test showed statistically significant (p<0.05) reduction of gingival score in group b, when compared with group a and group c, respectively . The difference was not significant, when group a was compared with group c (p>0.05) until 4 week . Further at the 6 and 8 week, the difference between the groups, a and c, was significant (p<0.05) [table 3]. Data analyzed by repeated measure of anova for time and cleaning method interactions showed a significant reduction in plaque score (f=20.612; p<0.0001) from baseline to 8 week in all the groups . Student's t test done for detecting a difference at specific time intervals showed statistically significant (p<0.0001) decrease in plaque score in group b compared to group a and group c, respectively, from 2 to 8 week . Whereas, there was no statistical significant difference found when group a was compared with group c (p>0.05) from 2 to 4 week . At the 6 and 8 weeks, there was significant difference (p<0.05) between group a and group c [table 1]. Result obtained from repeated measure of anova showed a highly significant decrease (f=11.981; p<0.0001) in mean gingival score from baseline to 8 week in all the groups . Student's t test showed statistically significant difference (p<0.05), when group b was compared with groups, a and c respectively . The difference was not significant when group a was compared with group c (p>0.05) [table 2]. Analysis done by results repeated measure of anova showed that there was highly significant reduction (f=4.942; p<0.0001) in stained plaque area from baseline to 8 week in all the groups . Student's t test showed statistically significant (p<0.05) reduction of gingival score in group b, when compared with group a and group c, respectively . The difference was not significant, when group a was compared with group c (p>0.05) until 4 week . Further at the 6 and 8 week, the difference between the groups, a and c, was significant (p<0.05) [table 3]. Current study was designed to investigate the clinical therapeutic effect of miswak as an adjunct to tooth brushing method in the treatment of chronic gingivitis . The selection of miswak from the salvadora persica tree for the present study was based on number of factors . The use of miswak is most common in the middle east region and in the indian subcontinent, its taste is acceptable, it is inexpensive, and has been reported to have anti - plaque and many therapeutic pharmacological properties. [58] there are studies[48] on the comparison of toothbrushes and miswak for plaque removal . Reports on the oral health of miswak users are contradictory and published literatures suggest that its therapeutic role in oral health needs to be verified before implementing into general practice of dentistry . Current dental literature[1416] describes several clinical indices and methods for measuring plaque and gingival inflammation, both quantitatively and qualitatively . The indices used to measure the accumulation of dental plaque and gingival inflammation are usually based on subjective estimations of the plaque - covered areas of the tooth surface and inflammatory changes of gingiva . However, these indices on an ordinal scale are visual determinations resulting in data of less sensitivity . Photogrammetric techniques giving measurements in an interval scale have therefore been used to obtain more accurate measurements of the plaque area . The results of the present study showed that there was a significant reduction in plaque score and improvement in gingival health after the use of miswak as an adjunct to tooth brushing method . The miswak stick when not used as adjunct to tooth brush was not effective as the conventional toothbrush in reducing plaque until 4 week . Later at the 6 and 8 week, there was significant difference in plaque score in toothbrush user compared to miswak user . This initial reduction in the plaque score among the miswak users could be due to hawthorne effect . The hawthorne effect is a form of reactivity, whereby subjects improve or modify an aspect of their behavior being experimentally measured simply in response to the fact that they are being studied, but not in response to any particular experimental manipulation . The effect might have resulted from subject's initial excitement of using a new product (miswak) and trying to get a good result out of their use . After some time, as the weeks progressed, there could be a probability that the subjects lost their interest in using the miswak effectively . On the other hand, toothbrush is a part of routine oral hygiene procedure and is being practiced by individuals since the eruption of first dentition in the oral cavity . Previous studies have revealed that higher plaque and gingival bleeding in chewing stick users as compared with toothbrush users . Studies have also revealed that poor oral hygiene with those using chewing sticks may be a reflection of poor techniques . Furthermore, the difference in topographic design of handles and bristles of miswak stick might be other reason of poor plaque control in miswak users . Studies have revealed that miswak bristles are not similar to the toothbrush bristles that are used to remove plaque from the tooth surfaces mechanically . Unlike a conventional toothbrush, the angulations in the toothbrush enable it to adapt more easily to the distal tooth surfaces, particularly on the posterior teeth . However, the techniques employed for removing plaque mechanically from outer surface and interproximal sites are similar with the toothbrush and the chewing stick, e.g., vertical and horizontal brushing . These techniques primarily depend upon the people's attitudes, knowledge, and manual dexterity . These mechanical differences were evident in our result also, and miswak bristles were not as effective as tooth brush bristles on outer surface area as well as interproximal area . These findings suggest that mechanically, miswak when not used in conjunctions to tooth brushes is not as effective as toothbrushes in cleaning the tooth surfaces . In the present study, a standardized design was developed to minimize the effects of other variables, which could affect plaque control, including type of toothbrush and/or chewing stick (miswak), frequency of tooth brushing and/or miswak, and the technique of tooth brushing and/or miswak . The study condition was standardized by instructing all subjects on how to use the identical conventional toothbrushes and chewing sticks under direct supervision of investigator . Furthermore, all participants were issued with identical conventional toothbrushes and fresh chewing sticks of fairly uniform length . Studies have revealed that in anterior region, the strokes and bristle angulations remain the same in both toothbrush and miswak stick and differ to great extent in posterior dentition . Thus, an attempt was made to measure the covered plaque area from matched mechanical cleansing procedure in anterior dentition, which was not possible in posterior dentition . Our result showed a beneficial effect of miswak on gingival health when used as an adjunct to tooth brushing method . This change could be attributed to the effect of different therapeutic components reported in the extract of miswak . Mentioned therapeutic components have been shown to beneficially influence the gingival health and plaque inhibition in various in vitro and in vivo studies. [72228] silica in miswak acts as an abrasive material to remove plaques and stains . Tannins (tannic acid) exert an astringent effect on the mucous membrane, thus reducing the clinically detectable gingivitis . Sodium bicarbonate (baking soda), nahco3, has mild abrasive properties and has a mild germicidal action. [2527] high concentrations of chloride inhibit calculus formation . These therapeutic findings of previous studies could be the reason for improved gingival health in the present study . In the present study, subjects were advised to use freshly prepared miswak stick at indicated time interval . Previous studies have revealed that freshly prepared miswak sticks have no cytotoxic effect on periodontal tissue . However, the same plant used even after 24 hours does contain harmful cytotoxic components and has potential to damage periodontal cells . Based on these findings, we recommended cutting the used portion of the miswak after it has been used for one day and preparing a fresh part for the next use . In the present study, miswak stick was used instead of miswak extract in mouthwash or toothpaste form assuming that miswak stick will provide both the mechanical and chemical effect supported by previous published literatures. [1928] our results showed beneficial chemical therapeutic action of miswak stick juice on gingival health, which was extracted on chewing miswak stick . Our results are in agreement with a controlled study, where it was reported that powdered miswak if used with a mechanically proper device i.e. Toothbrush, will give better results than miswak sticks alone or commercial toothpowder on gingival health . Presently, many commercial preparations are available in market as toothpaste, but no preparation presently exists in the form of mouthwash or in - office therapeutic medicament in the market . Thus, in future, well - controlled studies are required to evaluate miswak based therapeutic medicament, which can be used in treatment of mild to moderate gingival diseases . Our results showed significant improvement in plaque score and gingival health when miswak was used as an adjunct to tooth brushing . Our findings clearly indicate that miswak cannot replace the toothbrush, but can be used an adjunct to toothbrush, utilizing the mechanical efficacy of toothbrush and chemical effects of miswak . Henceforth, in future, a well - designed study is required to ascertain the efficacy of miswak or miswak extract in the treatment of gingival diseases.
When making any agrochemical spray application, the primary concerns are ensuring maximum biological efficacy while minimizing any off - target movement and associated adverse environmental impact or other non - target biological harm . One of the principal factors to consider when setting up any sprayer, prior to an application, is droplet size, which has long been recognized as one of the primary parameters influencing overall spray deposition, efficacy, and drift . While there are a number of other factors that potentially impact spray deposition and drift, droplet size is one of the easiest to change to fit the needs of a given application scenario . Droplet size from any agricultural spray nozzle is influenced by a number of factors including, but not limited to, nozzle type, nozzle orifice size, spray pressure and spray solution physical properties . With aerial applications, the additional influence of air shear resulting from the airspeed of the aircraft and the nozzle's orientation relative to that airshear, causes secondary breakup of the sprays leaving the nozzles . With all of these factors, applicators are faced with the difficult task of making proper nozzle selection and operational setup decisions that insure that all pesticide products labels are met and that the resulting spray droplet size is such that on - target deposition and biological efficacy are maintained while minimizing off - target movement . The goal of this method is to provide clear, concise information on the droplet size resulting from the various combinations influencing factors to support an applicator's operational decisions . While there are a number of instruments available for measuring droplet size from sprays, measurements from agrochemical spray nozzles are typically either laser diffraction, imagery, or phase doppler based . The imagery and phase doppler based methods are single particle counter methods, meaning that smaller areas within the spray cloud are focused on, with individual particles being measured . Whereas laser diffraction methods take an ensemble measurement, meaning the distribution of a group of particles is rapidly measured . While these methods differ in principle, with proper setup and use, comparable results can be obtained . Laser diffraction methods have been widely adopted by the agricultural application community due to the ease of use, ability to rapidly measurement high number density sprays and the large dynamic measurement range . As an ensemble measurement is made, a single traverse of a spray plume through the line of measurement is all that is required for a composite droplet size of the entire spray . This allows for efficient evaluations of droplet size from a large number of spray nozzles and operational parameter combinations . By comparison, the single particle counter methods necessarily focus on much smaller areas within a spray cloud in order to capture individual particles, meaning that multiple measurement locations must be evaluated and combined to return a composite result . This requires significantly more time, effort and spray solution to evaluate a single spray plume than laser diffraction based methods . The increased spray volume required can present a significant problem if actual pesticide products are being tested as a result of increased costs of the material used and the disposal costs . However, the single particle counter methods offer the advantage of providing a temporal sample, in that they measure the number of droplets per unit time passing through a sample volume, whereas laser diffraction provides a spatial sample as the measurement is proportional the number of droplets within a given volume . Were all droplet velocities within a given spray the same however, for most spray systems the droplet velocities are correlated to droplet size, resulting in a bias with spatial sampling methods . Overcoming this spatial bias from laser diffraction measurements through appropriate testing methodology the spatial bias is reduced when testing nozzles in a concurrent airstream of 13 m / sec and with the measurement location located an appropriate distance from the nozzle, as the combination of these two parameters results in homogeneous droplet velocities throughout the spray cloud . Further, the spatial bias is small (5% or less) for aerial nozzle testing due to the high concurrent airspeeds evaluated . To determine the optimal test method to reduce the spatial bias with our current low and high speed wind tunnel facilities, the series of reference nozzles used to determine agricultural spray size classifications were evaluated for droplet size using both laser diffraction and imaging methods . Sizing evaluations were conducted under multiple combinations of concurrent air velocity and measurement distance (distance from the nozzle exit to the point of measurement), representative of the operational range of the existing facilities . Laser diffraction measurements were compared to imagery results to determine the potential spatial bias and the optimal combination of measurement distance and concurrent airspeed was selected as the standard operational procedure . A measurement distance of 30.5 cm and a concurrent airspeed of 6.7 m / sec for evaluation of ground spray nozzles in the low speed wind tunnel reduced spatial bias to 5% or less . Spatial biases of 3% or less were obtained for aerial nozzle evaluations in the high speed tunnel, for all airspeeds tested, with a measurement distance of 45.7 cm . Using these standard methods, the authors were also able to demonstrate that lab to lab variability could be minimized, providing for consistent interlaboratory droplet size data . All droplet size testing demonstrated as part of this work was conducted at the usda - ars - aerial application technology research unit's spray atomization research facility . A laser diffraction system was positioned downstream of the nozzle at the distances specified in the protocol section . For ground nozzle testing, the laser diffraction system was configured, following the manufacturer's instructions, to have a dynamic size range of 18 - 3,500 m across 31 bins . Likewise for the aerial nozzle testing the system was configured with a dynamic size range of 9 to 1,750 m, also across 31 bins . Aerial based spray nozzle evaluations were conducted in high speed air to simulate aerial application conditions . Ground sprayer nozzles were tested in a larger wind tunnel section with a single concurrent airspeed to minimize the spatial bias from laser diffraction . Nozzles being tested were positioned upstream of the laser diffraction system at the distances given in the protocol section . Nozzles were mounted on a linear traverse allowing for the spray plume to be traversed vertically through the measurement zone during a given measurement cycle . The protocol for ground nozzle testing describes an experiment examining three typical nozzles at two spray pressures while the aerial nozzle testing describes an experiment examining two typical spray nozzles at two spray pressures and three airspeeds . Both testing scenarios use an " active blank " spray solution, rather than water only, to mimic the effects of real world spray solutions . Prior to any testing, align the laser diffraction system components following the guidelines provided by the manufacturer to ensure proper system functionality and data quality . Follow proper safety precautions associated with the use of a class iiia laser avoiding direct eye exposure . Use proper personal protection equipment if active ingredient chemical spray solutions are being used . Prepare the " active blank " by adding 47.5 ml (reflects a mix rate of 0.25% v / v) of a 90% non - ionic surfactant to 19 l of water and mixing well using a stir rod in a cordless drill . Depending on the amount of testing to be done, larger volumes of active blank may be required . Pour the " active blank " spray mixture into the stainless steel pressure tanks, seal the tank and attach the input air pressure hose and the outgoing liquid hose feeding the spray nozzle . Confirm that the distance between the nozzle outlet and the measurement zone is 30.5 cm (12 in) using a tape measure . If it is, continue . If not, adjust by moving either the laser diffraction system or the nozzle . Install a standard 110 degree flat fan nozzle with a #05 orifice (noted as an xrc11005 nozzle) in the nozzle body attached to the traverse system . Adjust the nozzle orientation such that the long axis of the flat fan nozzle is oriented vertically in the tunnel but either rotating the nozzle within the mounting ring on the check valve or by changing the position of the check valve if the nozzle cannot be rotated to the correct position . Turn on the wind tunnel and set the airspeed to 6.7 m / sec by adjusting the fan speed and confirming the airspeed in the tunnel using a hot wire anemometer . Set the spray pressure to 276 kpa (40 psi) by adjusting the incoming air pressure using an inline pressure regulator . Confirm the pressure using an electronic pressure gauge installed immediately upstream of the spray nozzle . Position the nozzle at the top of the tunnel by activating and running the linear traverse to the top - most position prior to initiating the measurement process . Are properly recorded in the laser diffraction system data recording software by confirming that the parameters recorded on the user parameters interface window match the testing conditions . Note: this data parameter recording screen may vary by laser diffraction instrument . Initiate a reference measurement by selecting the reference measurement icon in the operating software to account for any dust or background particles . Initiate start of the measurement cycle . Depending on the laser diffraction system being used, a few seconds is typically required to focus the sensor prior to initiating the measurement process . Once the system indicates it is ready to start the measurement process, activate the spray by opening the liquid feed valve on the pressure tank . Once the spray is started, lower the nozzle through the laser beam using the traverse mechanism until the entire spray plume has passed through the measurement zone . Note: on the laser diffraction system used by the authors, the actual measurement process does not initiate until the spray passing through the measurement zone achieves an optical concentration of 0.5%, and continues until an elapsed time of 10 - 12 sec has elapsed . Determine if additional replicates are required by computing the mean and standard deviation for the dv0.1, dv0.5, and dv0.9 of the three replicates and ensuring that standard deviation is 10%, or less, of the mean . Set the spray pressure to 414 kpa (60 psi) and repeat steps 2.7 - 2.12 . Export and save droplet size data using the method provided within the operating software . Prepare the " active blank " by adding 47.5 ml of a 90% non - ionic surfactant to 19 l of water and mixing well using a stir rod in a cordless drill . Note: depending on the amount of testing to be done, larger volumes of active blank may be required . Pour the " active blank " spray mixture into the stainless steel pressure tanks, seal the tank and attach the input air pressure hose and the outgoing liquid hose feeding the spray nozzle . Confirm that the distance between the nozzle outlet and the measurement zone is 45.7 cm (18 in) using a tape measure . If it is, continue . If not, adjust by moving the laser diffraction system the required distance from the nozzle . Install a standard 20 degree flat fan nozzle with a #15 orifice (noted as a 2015 nozzle) in a check valve and nozzle body onto the boom traverse section at the wind tunnel outlet . Ensure that the nozzle is correctly positioned with the nozzle body oriented horizontally and parallel to the airstream . Set the airspeed at the tunnel outlet to 53.6 m / sec (120 mph) and confirm speed using pitot tube attached to an airspeed indicator . Set the spray pressure to 207 kpa (30 psi) by adjusting the incoming air pressure using an inline pressure regulator . Position the nozzle at the top position of the traverse prior to initiating the measurement process . Are properly recorded in the laser diffraction system data recording software by confirming that the parameters recorded on the user parameters interface window match the testing conditions . Note: this data parameter recording screen may vary by laser diffraction instrument . Initiate a reference measurement by selecting the reference measurement icon in the operating software to account for any dust or background particles . Initiate start of the measurement cycle . Depending on the laser diffraction system being used, a few seconds is typically required to focus the sensor prior to initiating the measurement process . Once the system indicates it is ready to start the measurement process, activate the spray by opening the liquid feed valve on the pressure tank . Once the spray is started, lower the nozzle through the laser beam using the traverse mechanism until the entire spray plume has passed through the measurement zone . Note: on the laser diffraction system used by the authors, the actual measurement process does not initiate until the spray passing through the measurement zone achieves an optical concentration of 0.5%, and continues until an elapsed time of 5 - 7 sec has elapsed . Determine if additional replicates are required by computing the mean and standard deviation for the dv0.1, dv0.5, and dv0.9 of the three replicates and ensuring that standard deviation is 10%, or less, of the mean . Repeat steps 3.4 3.12 for each additional nozzle, pressure, nozzle orientation and airspeed combination of interest . Prior to any testing, align the laser diffraction system components following the guidelines provided by the manufacturer to ensure proper system functionality and data quality . Follow proper safety precautions associated with the use of a class iiia laser avoiding direct eye exposure . Use proper personal protection equipment if active ingredient chemical spray solutions are being used . Prepare the " active blank " by adding 47.5 ml (reflects a mix rate of 0.25% v / v) of a 90% non - ionic surfactant to 19 l of water and mixing well using a stir rod in a cordless drill . Depending on the amount of testing to be done, larger volumes of active blank may be required . Pour the " active blank " spray mixture into the stainless steel pressure tanks, seal the tank and attach the input air pressure hose and the outgoing liquid hose feeding the spray nozzle . Confirm that the distance between the nozzle outlet and the measurement zone is 30.5 cm (12 in) using a tape measure . If it is, continue . If not, adjust by moving either the laser diffraction system or the nozzle . Install a standard 110 degree flat fan nozzle with a #05 orifice (noted as an xrc11005 nozzle) in the nozzle body attached to the traverse system . Adjust the nozzle orientation such that the long axis of the flat fan nozzle is oriented vertically in the tunnel but either rotating the nozzle within the mounting ring on the check valve or by changing the position of the check valve if the nozzle cannot be rotated to the correct position . Turn on the wind tunnel and set the airspeed to 6.7 m / sec by adjusting the fan speed and confirming the airspeed in the tunnel using a hot wire anemometer . Set the spray pressure to 276 kpa (40 psi) by adjusting the incoming air pressure using an inline pressure regulator . Confirm the pressure using an electronic pressure gauge installed immediately upstream of the spray nozzle . Position the nozzle at the top of the tunnel by activating and running the linear traverse to the top - most position prior to initiating the measurement process . Are properly recorded in the laser diffraction system data recording software by confirming that the parameters recorded on the user parameters interface window match the testing conditions . Note: this data parameter recording screen may vary by laser diffraction instrument . Initiate a reference measurement by selecting the reference measurement icon in the operating software to account for any dust or background particles . Initiate start of the measurement cycle . Depending on the laser diffraction system being used, a few seconds is typically required to focus the sensor prior to initiating the measurement process . Once the system indicates it is ready to start the measurement process, activate the spray by opening the liquid feed valve on the pressure tank . Once the spray is started, lower the nozzle through the laser beam using the traverse mechanism until the entire spray plume has passed through the measurement zone . Note: on the laser diffraction system used by the authors, the actual measurement process does not initiate until the spray passing through the measurement zone achieves an optical concentration of 0.5%, and continues until an elapsed time of 10 - 12 sec has elapsed . Determine if additional replicates are required by computing the mean and standard deviation for the dv0.1, dv0.5, and dv0.9 of the three replicates and ensuring that standard deviation is 10%, or less, of the mean . Set the spray pressure to 414 kpa (60 psi) and repeat steps 2.7 - 2.12 . Prepare the " active blank " by adding 47.5 ml of a 90% non - ionic surfactant to 19 l of water and mixing well using a stir rod in a cordless drill . Note: depending on the amount of testing to be done, larger volumes of active blank may be required . Pour the " active blank " spray mixture into the stainless steel pressure tanks, seal the tank and attach the input air pressure hose and the outgoing liquid hose feeding the spray nozzle . Confirm that the distance between the nozzle outlet and the measurement zone is 45.7 cm (18 in) using a tape measure . If it is, continue . If not, adjust by moving the laser diffraction system the required distance from the nozzle . Install a standard 20 degree flat fan nozzle with a #15 orifice (noted as a 2015 nozzle) in a check valve and nozzle body onto the boom traverse section at the wind tunnel outlet . Ensure that the nozzle is correctly positioned with the nozzle body oriented horizontally and parallel to the airstream . Turn on the wind tunnel blower and set the airspeed at the tunnel outlet to 53.6 m / sec (120 mph) and confirm speed using pitot tube attached to an airspeed indicator . Set the spray pressure to 207 kpa (30 psi) by adjusting the incoming air pressure using an inline pressure regulator . Position the nozzle at the top position of the traverse prior to initiating the measurement process . Are properly recorded in the laser diffraction system data recording software by confirming that the parameters recorded on the user parameters interface window match the testing conditions . Note: this data parameter recording screen may vary by laser diffraction instrument . Initiate a reference measurement by selecting the reference measurement icon in the operating software to account for any dust or background particles . Initiate start of the measurement cycle . Depending on the laser diffraction system being used, a few seconds is typically required to focus the sensor prior to initiating the measurement process . Once the system indicates it is ready to start the measurement process, activate the spray by opening the liquid feed valve on the pressure tank . Once the spray is started, lower the nozzle through the laser beam using the traverse mechanism until the entire spray plume has passed through the measurement zone . Note: on the laser diffraction system used by the authors, the actual measurement process does not initiate until the spray passing through the measurement zone achieves an optical concentration of 0.5%, and continues until an elapsed time of 5 - 7 sec has elapsed . Determine if additional replicates are required by computing the mean and standard deviation for the dv0.1, dv0.5, and dv0.9 of the three replicates and ensuring that standard deviation is 10%, or less, of the mean . Repeat steps 3.4 3.12 for each additional nozzle, pressure, nozzle orientation and airspeed combination of interest . The resulting data from this method can be expressed in a variety of formats, depending on the user's preference and the operational capabilities of the laser diffraction system . Typically this data is presented as a plot of the volume weighted droplet size distribution (figures 1 and 2) or as descriptive droplet size metrics (tables 1 and 2). These results can then be used to examine the impact that changes in nozzle or operational parameters have on the resulting spray droplet size . We examined two different aerial spray nozzles, both with the same orifice size but with different spray fan angles . With these two aerial nozzles, we also examined the effects of spray pressure and airspeed on droplet size . Examining the 2015 nozzle operated at a spray pressure of 207 kpa and comparing the volume weighted distributions resulting from the same nozzle being operated in 53.6 m / sec versus 71.5 m / sec airspeed, it is immediately obvious that the higher airspeeds results in a dramatic shift in the incremental and cumulative distributions toward smaller droplet diameters (figures 1 and 2) which is the result of increased breakup of spray droplets at the higher airspeed . While the graphical representation of the results provide a very visual representation of the results, quantitative values derived from these distributions are more practical for larger data sets . Typical droplet size metrics used in agricultural spray research include the dv0.1, dv0.5 and dv0.9 values, which correspond to the droplet diameters such that 10, 50 and 90% (respectively) of the spray volume is contained in droplets of equal or lesser diameter . These data are the same as those shown in the graphical distributions, but provide a more convenient format of expressing the data . Comparing the data for both the 2015 and 4015 spray nozzles at both pressures and all three airspeeds, general trends can be observed (table 1). The 4015 flat fan nozzle results in smaller droplet sizes than the 2015 at the same pressure and airspeed, as indicated by the smaller volume weighted diameters (dv0.1, dv0.5, and dv0.9) and the increase in the total volume of the spray comprised of droplet of 100 m or less . Dv0.1, dv0.5, and dv0.9 are the droplet diameters such that 10, 50 and 90%, respectively, of the total spray volume is comprised of droplets of equal or lesser diameter . This is the result of the increase spray fan angle seeing greater breakup at the outer edges of the liquid fan angle . Within the same nozzle type and spray pressure, all droplet size metrics decrease with increasing airspeeds, again as a result of increasing breakup of droplets at the higher airspeeds . An interesting phenomenon with the aerial spray nozzles is seen when looking at the effects of spray pressure within each nozzle and airspeed combination . This is caused by a decrease in the relative velocity difference between the liquid exiting the nozzle and the surrounding airstream, as the liquid exit velocity increases as pressure increases (table 1). Looking at the results from the ground nozzles and spray pressures tested, the effect of nozzle type on droplet size is significant with the tti11003 resulting in droplet sizes that are more than double that the xrc11003 and the ai11003 droplet sizes falling in the middle of the other two (table 2). Within each nozzle type, the effects of pressure figure 1.incremental droplet size distribution for a 20 degree flat fan aerial spray nozzle with a #15 orifice operated at 207 kpa and in an airspeed of 53.6 m / sec . The blue curve represents the incremental volume weighted distribution which provides the percentage of the total spray volume contained in droplets falling with the range of each measurement bin as measured by the laser diffraction system . The cumulative data allows for the volume - weighted diameters specific to a certain percentage of total spray volume to be determined . As illustrated in the figure, to obtained the dv0.5 volume diameter, locating the 50% point on the cumulative curve and the associated droplet diameter shows that 50% of the total spray volume is contained in spray droplets of diameter 551 m or smaller . Figure 2.incremental droplet size distribution for a 40 degree flat fan aerial spray nozzle with a #15 orifice operated at 207 kpa and in an airspeed of 71.5 m / sec . As in figure 1, the blue curve represents the incremental volume weighted distribution and the red curve is the cumulative distribution . Compared to the results shown in figure 1, the incremental distribution shows a significant shift toward smaller droplet diameters as a result of the increased airspeed and therefore secondary droplet breakup . Determining the dv0.5 volume diameter shows that 50% of this spray volume is contained in droplets of diameter 350 m or smaller . The secondary, smaller peak on the right, toward the larger end of the droplet size scale is typically the result of either vibrations or other noise in the system or the presence of ligaments associated with incomplete atomization within the spray cloud . As droplet size distributions for typical agricultural spray nozzles and solutions are typically log - normally distributed, the presence of a secondary peak in the distribution may be a valid result from an atypical spray solution and/or nozzle combination, but is more likely an indicator of some confounding issue in the measurement process . Volume weighted diameters (averages standard deviations across three replicate measurements) for 2015 and 4015 flat fan aerial spray nozzles operated at spray pressures of 207 and 414 kpa and in airspeeds of 53.6, 62.6 and 71.5 m / sec . Volume weighted diameters (averages standard deviations across three replicate measurements) for three ground sprayer nozzles (xrc11005, ai11005 and tti11005) operated at spray pressures of 276 and 414 kpa . There are a number of critical steps that should be followed when applying this method . With both aerial and ground nozzle evaluations, the distance from the exit of the nozzle to the line of measurement similarly, the concurrent airspeed used in ground nozzle testing should be verified and adjusted to the 6.7 m / sec recommended . Differences in airspeed from that recommended will significantly influence the results due to sampling bias issues at lower airspeeds, and potentially increase secondary breakup at higher airspeeds . Also, proper alignment of the laser diffraction system components is critical in order to ensure the system is operating at the accuracy and precision specification certified by the manufacturer . Proper setup and alignment of the nozzles relative to the concurrent airflow is critical to ensuring quality data, as even slight misalignments of a few degrees in the nozzles positioning can result in significant impacts on the resulting droplet size data . The methods presented can be applied to any spray nozzle configuration or spray solution for both ground and aerial system . With ground sprayers, changes in spray droplet size are typically a function of nozzle type and size, spray pressure and spray solution type . With aerial sprayer the additional role of changes in airspeed and the orientation of the nozzle to surrounding airstream are critical to the resulting droplet size . This method can be used to evaluate the combined effect of these factors on the final droplet size . However there are rare instances when some modifications to the recommended methods are required . Specifically, spray solutions or nozzles that require further distances from the nozzle for complete breakup of spray into discrete particles will require adjusting the distance between nozzle and measurement point . To date, the only nozzle / spray solution treatments that have required this sort of adjustment have been straight stream nozzles at all operational settings and narrow angle flat fan nozzles with spray additives that increase the solutions viscosity, when measured under aerial application testing conditions . The laser diffraction system will still return droplet size data in the event of incomplete breakup of the spray cloud, but the resulting data will typically be biased toward much larger droplet sizes as a result of spray ligaments being measured by the system . While these ligaments are not readily apparent to the naked eye, their presence will typically show up visually in the distribution plot as a secondary peak at the larger end of the droplet size scale (figure 3). Though caution is advised in assuming that this secondary peak is the result of the presence of ligaments, as external vibrations or other interference with the laser diffraction system may cause a similar response . As a user's experience level increases, making the distinction between the two based on errors becomes easier . In the case where spray atomization is incomplete, we have found that extending the sampling distance to 1.8 m (for aerial spray nozzles) resolves the issue and returns quality data . This 1.8 m distance is in fact the standard distance at which our group evaluates all straight stream nozzles under aerial application conditions . When working with ground sprayer nozzles, there are a class of nozzle designs that use a twin, flat fan orifice outlet the may require modification to the nozzle mounting setup to insure the entire spray plume passes through the sampling area without fouling the laser diffraction system's lenses . While this method is designed to minimize the sampling bias due to spatial biases associated with laser diffraction systems, it does not completely eliminate them, meaning that the droplet size values return cannot be taken as " absolute " . Laser diffraction does not provide a means to measure, and adjust, the resulting droplet size data for the non - homogeneous droplet velocities amongst the different droplet sizes in the composite spray cloud . This becomes critical when inter - laboratory data sets are compared, particularly with respect to ground spray nozzles . The method currently accepted to standardize the results and allow comparisons between laboratories uses a series of highly calibrated reference spray nozzles, whose droplet size data are used to establish a set of classification categories . Further details on the nozzles and classification definitions can be found in the american society of agricultural and biological engineers (asabe) " spray nozzle classification by droplet spectra " international standard (asae / ansi, 2009). As discussed in the introduction, there are other droplet sizing systems besides laser diffraction . Where laser diffraction provides a composite measure of droplet size across the entire spray plume, these other methods focus in on a small area with the spray cloud, sampling only a small portion of the overall spray cloud . Obtaining a representative sample of the entire plume with these others methods requires a much more rigorous, and time consuming, multi - chordal traverse of the spray plume's cross section area, resulting in a large number of sub - samples that must be combined to generate a composite result . Once this method has been successfully integrated into a research program and the techniques mastered by the users, the next challenge is conducting well - structured experiments aimed at understanding the role each of the influence factors play with respect to the formation of droplet size . This is a bigger challenge than it seems given the seemingly endless combination of nozzle type, nozzle setup and operational factors, airspeed and nozzle position (aerial spraying) and real - world tank mixes used by the agricultural application industry . Even more of a challenge is finding a way the makes this information available to the applicators in a format that is easily useable . One option our group has used with great success is a class of experimental designs called response surfaces that allow for the development of droplet size prediction models based on a limited number of experimental treatments allowing for an extremely efficient evaluation of multiple spray nozzles and solutions . This structured design method has been used to develop a series of droplet size models for the most commonly used aerial and ground nozzles used by agricultural applicators.
The role of axillary surgery in breast cancer is to stage the axilla and in those with lymph node metastases to treat the axilla with axillary clearance . Adequate axillary dissection is important in node - positive patients both to ensure removal of all involved nodes to optimise local control and to obtain the maximum prognostic information . When staging the axilla, an additional goal, particularly in node - negative patients, is to minimise morbidity . Various strategies for doing so have been developed, the most recent being dual localisation sentinel lymph node biopsy (slnb). This has been recommended by the united kingdom national institute for health and clinical excellence as the preferred technique for staging the axilla in radiologically or cytologically (where tested) node - negative patients . If slnb analysis demonstrates metastasis to the axilla, it is recommended that patients undergo axillary clearance . Traditionally, sentinel lymph nodes are analysed histologically, and patients who are found to have metastases in these nodes often undergo a delayed completion axillary dissection (dalnd) after a delay when the histological result is available . In two large united kingdom datasets of slnb, the almanac trial and the new start training programme, approximately 30% of patients were slnb positive and therefore required further axillary surgery alternative analysis techniques have been developed for use in conjunction with slnb to enable rapid intraoperative analysis of sentinel lymph nodes including touch imprint cytology, frozen section analysis, and molecular analysis such as osna and the veridex polymerase chain reaction (pcr) assay as used in our institution . These diagnostic techniques enable node - positive patients to undergo immediate axillary clearance under the same general anaesthetic (ga) without the need for a second operation and another ga . Anecdotally, dalnd is often more technically challenging than primary axillary clearance or immediate completion axillary dissection (ialnd) following intraoperative slnb analysis ., who have shown that a dalnd requires a greater operative time than ialnd . With increased difficulty associated with delayed axillary clearance, it is important to establish whether optimal oncological results are maintained using this approach . It is also important to establish oncological equivalence between primary axillary clearance and slnb with ialnd, as although there is no scarring to contend with, the axillary contents are fragmented by the latter approach . Have shown no difference in median nodal yields from axillary clearance with or without slnb, in either the immediate or delayed setting . Earlier studies support these findings, but only compared two of the three possible patient groups [7, 9]. We determined to assess the nodal harvest from the axilla in these various contexts, within a single centre, with the same surgeons and pathologists all working in accordance with the same standardised protocols . Our hypothesis was that dalnd might yield a lower number of lymph nodes due to increased technical difficulty and scarring compared to ialnd . Sentinel node biopsy using dual localisation with radioisotope and patent blue v dye was introduced into portsmouth breast unit for staging the axilla in breast cancer in 1999 as part of the almanac trial . After the initial trial results in 2004, slnb was adopted as the standard of care for all patients with clinical or radiological t1 cancers . Axillary clearance without sentinel node biopsy remained the standard approach for those with more advanced tumours . During 2006, the application of slnb was extended to tumours up to 3 cm in diameter . From december 2007, after the introduction of intra - operative assessment using a pcr - based assay, slnb became the index standard for axillary assessment of all breast cancer patients irrespective of t - stage, unless there was clinical or radiological evidence of nodal involvement . All operations were performed or supervised by consultant surgeons, unless a dedicated breast surgical trainee had completed accreditation with the slnb technique . Sentinel nodes were sectioned into 2 mm slices and alternate sections analysed by pcr and conventional histology . Portsmouth breast unit maintains a prospective database of all patients undergoing surgical treatment of breast cancer . From this, 50 consecutive histologically node - positive patients were retrospectively identified in each of the three groups . The sample size was determined by the size of our smallest group (those undergoing ialnd), with the other patient groups matched in number . Group 1 consisted of consecutive node - positive patients who had undergone primary axillary clearance prior to november 2007; group 2 consisted of patients who had undergone slnb and proceeded to dalnd following histological node analysis following the introduction of slnb but prior to the adoption of intra - operative analysis (between november 2007 and november 2009); group 3 consisted of consecutive patients who had undergone slnb and ialnd following positive intra - operative slnb pcr analysis (after november 2009). Data was collected from electronic histopathological and clinic letter databases and analysed using spss v14 (spss inc ., data collected included age, sex, tumour type, grade, size (t score), hormone receptor status (oestrogen and progesterone), her-2 receptor status, and the nature of surgery performed on the breast primary . Histological invasive tumour size was documented in all cases, and total tumour size was noted in the histology report . Statistical analysis was performed to test the null hypothesis that all patients were taken from the same population group . Age was analysed using analysis of variance (anova), and all other parameters were assessed using pearson's chi - squared test . The primary outcome was the total number of lymph nodes harvested from the axilla (combining axillary dissection with sentinel node harvest where performed). The secondary outcome was the total number of histologically positive lymph nodes harvested . Where not explicitly stated, nodal metastases were assumed to be macrometastases without extracapsular spread . Where lymph nodes were pcr positive but histologically negative, histological specimens were sent for immunohistochemistry . Our three groups were statistically similar in all respects, except the nature of surgery performed on the primary breast tumour (see table 1). Our patient groups were similar in terms of sex, with only one male among 150 patients . Tumour size, as reflected by t score, was similar across patient groups, with the majority of tumours less than 5 cm in diameter (t1 and t2). There were similar numbers of multifocal tumours in the three groups (range 7 to 11). Tumour grades were similar, with the majority of tumours being graded 2 or 3, and most tumours were of ductal type . Oestrogen and progesterone receptors positivity dominated, and few tumours tested positive for her-2 receptors . Patients in group 1 were more likely to have their primary tumours treated with mastectomy than breast conserving treatment, in contrast with groups 2 and 3 . This difference was most marked in group 2, where only 2 patients underwent mastectomy . These differences in nature of surgery performed on the breast were strongly statistically significant (p <0.001). All three patient groups were statistically similar regarding both outcome measures total number of nodes harvested from the axilla and the total number of positive nodes harvested (table 2 and figures 1 and 2). The mean total number of nodes harvested ranged from 14.6 to 15.4 with clearly overlapping 95% confidence intervals as illustrated in figure 1 . The mean number of positive nodes was higher in group 1 at 5.1, compared with 3.2 and 3.52 in groups 2 and 3, respectively, but 95% confidence intervals overlap, and no statistically significant difference was found (figure 2). Consistent with this the median total number of nodes harvested was 14 in all three groups . Here, we demonstrate that both the total number of lymph nodes harvested from the axilla and the number of positive nodes are unaffected, whether axillary clearance is performed as a primary procedure, as a delayed procedure following slnb, or as an immediate procedure following intra - operative analysis of sentinel lymph nodes (sln). The mean number of positive nodes harvested was higher, although not significantly so, in patients undergoing primary axillary clearance compared to those having an axillary clearance following slnb . This tendency may reflect bias due to preoperative selection of patients with clinically or radiologically positive nodes in the primary axillary clearance cohort . Our patient groups were comparable over all parameters except the nature of surgery performed on the primary breast tumour . This demonstrates that our results are valid, as although our patients were selected by being consecutive rather than through a rigorous case matching process, the groups were statistically similar in all but one measured characteristic . The difference between groups in the nature of surgery performed is likely to be the result of previous local policy, prior to the introduction of intra - operative sln analysis, if only offering slnb to patients with smaller breast tumours undergoing breast conserving surgery . With the introduction of intra - operative sln analysis, it is documented that nodal harvest is determined not only by surgical technique, but also by the degree to which nodes are pursued at histopathological analysis . In this single - centre study, we believe that variations in lymph node count due to individual surgeon or pathologist - specific technique is likely to have been minimised by standardised surgical and pathological protocols . We conclude from this study that there is no statistically significant difference in the number of lymph nodes or number of positive nodes harvested from the axilla, regardless of timing of axillary clearance or the use of slnb . The goal of axillary clearance is to remove all potentially involved lymph nodes and fatty tissue, and using the number of nodes as a measure of adequacy of tissue retrieval, we have shown equivalence between each technique in our unit . Therefore, even in more surgically challenging circumstances, such as the scarred axilla of a previous slnb, surgical technique is robust in ensuring adequate clearance of the axilla.
This disease is a genetically heterogeneous eye disorder, which affects the central nucleus of the eye and frequently results in visual impairment or even blindness in children . The prevalence of congenital cataract is estimated between 1 and 15 in 10,000 newborns (1). It has been reported that the rate of congenital / infantile cataract in males was 10% higher than females (2 - 4). To date, surgery has been the first line of treatment for congenital cataract, but it has many complications such as strabismus, nystagmus, amblyopia, posterior capsular opacity, and glaucoma . So, despite dramatic improvements in the treatment of congenital cataracts, still a large proportion (75%) of childhood blindness in developing countries is due to congenital cataract (2 - 6). Although the aforementioned disease can be either bilateral or unilateral, and also with or without genetic basis, it has been shown that the unilateral cataract is rarely inherited, whereas approximately 50% of bilateral cases have a genetic background . This trait can be transmitted in different patterns such as autosomal dominant, autosomal recessive and x - linked; however, the autosomal dominant pattern seems to be the most common inheritance pattern (1, 7, 8). Identification of the genetic variations underlying hereditary cataract and subsequent functional studies will improve our insight into normal and abnormal lens development and the mechanisms of cataract pathogenesis . Although identification of genetic variations in congenital cataract has not yet led to establish a better and newer treatment, it may be possible to cure congenital cataracts before birth or in early childhood with advances in genetic science . So far, several chromosomal regions are suggested to be involved in the molecular etiology of the disease . In addition, several genes such as crystallin genes (cryaa, cryab, crybb1, crybb2, cryba1/a3, cryga, crygb, crygc, crygd, and crygs), gja3, gja8, mip and aqp0) have been reported to be associated with the inherited cataract (1, 7 - 12). Although more than 90% of soluble lens proteins consist of crystallin proteins, connexin proteins play an important role in maintenance of lens structure and transparency by controlling water and ion balance in the lens . The mutations identified in the connexin genes that encode these correspon - ding proteins have been associated with inheritance of cataracts in human (9 - 12). In the present study, we conducted a mutation screening of the entire coding sequences of gja8 gene; the gene which encodes connexin proteins, among ten iranian families with adcc . This study was approved by the institutional review board committees (irb) at tehran university of medical sciences (tums, iran). A written informed consent was obtained from parents or guardians before mutation analysis . In this study, 20 patients from 10 unrelated iranian families with adcc were diagnosed and enrolled based on the following criteria: (1) bilateral congenital cataracts that had been approved by detailed ophthalmologist s examination; (2) no other ocular or systemic disease; (3) no other congenital and syndrome related malformation; (4) no history of any teratogenic drug use during pregnancy; (5) compatible family pedigree with an autosomal dominant pattern of the disease . The exclusion criteria were the various diseases, infections, or trauma that mimics inherited cataracts as well as individuals requiring sedation for study procedures . Genomic dna was isolated from five milliliters whole blood using a qiaamp dna mini kit (qiagen, hilden, germany). The pcr amplification was typically carried out using specific primer pairs of coding regions (http://simgene.com/primer3) and exon - intron boundaries of gja8 gene (table 1), 0.2u taq dna polymerase (roche, mannheim, germany), 10 pmole of each primer, 200 m of each dntps, 0.67 l of 50 mm mgcl2, 60 ng dna and 2.5 l of pcr buffer in 25 l of pcr reactions . The pcr conditions included an initial denaturation step for 3 min at 95 c, 30 sec at 95 c, 45 sec at 64 c with a 1 c decrease every second cycle down to 55 c, then 55 c for 14 cycles, 1 min at 72 c for extension, and finally 10 min at 72 c (13 - 15). Pcr products were separated on 2% agarose gels and visualized with ethidium bromide, as described previously (15 - 18). The primer sequences used in this study subsequently, to determine any mutation the pcr product was subjected to direct sequencing (gene fanavaran, iran). Sequence data searches were performed in non - redundant nucleic and protein databases blast (http://www.ncbi.nlm.nih.gov/blast). In order to investigate whether the identified mutation could affect the biological function of the altered protein, we analyzed the mutated form of protein using polyphen2 program (http://genetics .- bwh.harvard.edu / pph2/). This study was approved by the institutional review board committees (irb) at tehran university of medical sciences (tums, iran). A written informed consent was obtained from parents or guardians before mutation analysis . In this study, 20 patients from 10 unrelated iranian families with adcc were diagnosed and enrolled based on the following criteria: (1) bilateral congenital cataracts that had been approved by detailed ophthalmologist s examination; (2) no other ocular or systemic disease; (3) no other congenital and syndrome related malformation; (4) no history of any teratogenic drug use during pregnancy; (5) compatible family pedigree with an autosomal dominant pattern of the disease . The exclusion criteria were the various diseases, infections, or trauma that mimics inherited cataracts as well as individuals requiring sedation for study procedures . Genomic dna was isolated from five milliliters whole blood using a qiaamp dna mini kit (qiagen, hilden, germany). The pcr amplification was typically carried out using specific primer pairs of coding regions (http://simgene.com/primer3) and exon - intron boundaries of gja8 gene (table 1), 0.2u taq dna polymerase (roche, mannheim, germany), 10 pmole of each primer, 200 m of each dntps, 0.67 l of 50 mm mgcl2, 60 ng dna and 2.5 l of pcr buffer in 25 l of pcr reactions . The pcr conditions included an initial denaturation step for 3 min at 95 c, 30 sec at 95 c, 45 sec at 64 c with a 1 c decrease every second cycle down to 55 c, then 55 c for 14 cycles, 1 min at 72 c for extension, and finally 10 min at 72 c (13 - 15). Pcr products were separated on 2% agarose gels and visualized with ethidium bromide, as described previously (15 - 18). The primer sequences used in this study subsequently, to determine any mutation the pcr product was subjected to direct sequencing (gene fanavaran, iran). Sequence data searches were performed in non - redundant nucleic and protein databases blast (http://www.ncbi.nlm.nih.gov/blast). In order to investigate whether the identified mutation could affect the biological function of the altered protein, we analyzed the mutated form of protein using polyphen2 program (http://genetics .- bwh.harvard.edu / pph2/). According to studied medical records, interviewing the patients, and medical examinations, probands from ten families with adcc were enrolled in this study . All probands and their families were clinically examined by an ophthalmologist to identify adcc affected individuals . Genetic analysis of these probands revealed three missense mutations including c.130g> a (p.v44 m), c.301g> t (p.r101l) and c.134g> t (p.w45l) in gja8 gene . In family 1, the proband was a 3-year - old girl from a family with 12 adcc affected individuals . Molecular genetic studies revealed a novel mutation c.301g> t (p.r101l) in the gja8 gene . The aforementioned mutation also was detected in gja8 gene of her mother who suffered from adcc (figure 1). This mutation was not observed in any of the unaffected family members or in the 100 healthy control individuals . Ophthalmological evaluation, pedigree analysis and molecular study of family 1 . A: slit - lamp photographs of eyes from probavd revealed congenital proband . B: the pedigree of family 1 shows 12 affected patients (arrow indicates the proband) and co - segregation of c.301g> t (p.r101l) through the family . Filled symbols represent autosomal dominant congenital cataracts (adcc) patient and open symbols show individuals without clinical adcc . C: dna chromatogram showed a heterozygous missense mutation in the codon 101 gja8 in which g> t (arrow indicates the position of nucleotide substitution) in family 2, the proband was a 4-year - old girl from a family with 4 affected individuals with adcc . Ocular examination using slit- lamp confirmed posterior polar cataract . Through bidirectional sequencing of entire coding regions and intron - exon boundaries of the gja8 gene, a missense mutation, c.130 g> a (p.v44 m), was identified . In the affected members of family 2, the known mutation, c.130 g> a (p.v44 m), b: the pedigree of family 2 shows 4 affected patients (arrow indicates the proband). Filled symbols represent autosomal dominant congenital cataracts (adcc) patient and open symbols show individuals without clinical adcc . C: dna sequencing revealed a heterozygous mutation in codon 44 for amino acid valin to methionine c.130g> a (p.v44 m). Arrow indicates the position of nucleotide substitution in family 3, the proband was a 4-year old boy from a family with 11 adcc affected individuals . Mutation analysis of gja8 using bidirectional sequencing as described above revealed a missense mutation, c.134 g> t (p.w45l). This mutation was also detected in the gja8 gene of other affected family members (figure 3). Filled symbols represent autosomal dominant congenital cataracts (adcc) patients and open symbols show individuals without clinical adcc . C: heterozygous mutation in coding region of gja8 gene showed in dna chromatogram c.134g> t (p.w45l) in order to predict the potential biological effect of the novel missense mutation (r101l) within coding region of gja8 gene, we utilized the polyphen 2 program . In order to predict the potential biological effect of the novel missense mutation (r101l) within coding region of gja8 gene, we utilized the polyphen 2 program . So far, along with the development of molecular genetics, various loci and more than 20 genes such as cryaa, cryab, cryba1/a3, and gja8 have been reported to be associated with congenital cataracts (7, 9 - 12). In this study, we conducted mutation detection within gja8 gene among ten iranian families with adcc . Three missense mutations, including c.130g> a (p.v44 m), c.301g> t (p.r101l) and c.134g> t (p.w45l) in the coding region of gja8 (cx50) gene were detected in the patients with adcc . Gap junction proteins, are a unique family of transmembrane proteins that form specific channels between cells and are encoded by a gene family with at least 14 members . These proteins connect the cytoplasm of neighboring cells and play an important role in intercellular communication in the lens . The gja8 gene encodes 433 amino acid residues of gap junction protein alpha 8 or connexin 50 . The encoded protein contains four transmembrane domains that are joined by two extracellular and one cytoplasmic loop and flanked by cytoplasmic n- and c - termini . This protein creates gap junction channels by forming hexamers, or hemi - channels, that can dock between adjacent cells . The normal function of these channels have been shown to be involved in the trafficking of ions and small molecular metabolites (19 - 24). Mutations identified in iranian patients with congenital cataract studies have reported that gja8 mutations are responsible for a significant proportion of adcc (1, 7, 25, 26). Up to now, more than 20 mutations in gja8 gene have been reported in hereditary congenital cataract (1, 7, 8, 19, 23, 25 - 27). Even though our results are the first report that demonstrated the genetic alterations of gja8 gene in iranian population, various studies reported the association of gja8 mutations with adcc in different ethnic groups (25 - 27). Two out of three identified mutations, c.130g> a (p.v44 m) and c.134g> t (p.w45l) (v44 m and w45l), through the present work, are located in the extracellular loop 1 near the tm1/e1 border . It has been well documented that this part of gja8 gene is supposed to be a hot - spot sequence . In line with our suggestion, several studies reported a high incidence rate of mutation in this region among congenital cataract (18 - 20, 22 - 28). Additionally, different studies have shown that mutations in this region impair the normal functions of cx50 and are introduced as disease - causing mutation in adcc (29, 30). Moreover, our in silico study of c.130g> a (p.v44 m) and c.134g> t (p.w45l) mutations, which are located on the first exon of gja8 gene, predicted that these mutations are probably damaging to the structure and function of the protein (with a specificity of 0.99 and 1.00, respectively). Then, this is quite understandable that these mutations likely lead to the disruption of the gap junction formation and altered protein trafficking or channel assembly of heteromeric connexins consisting of gja8 mutant and wild - type subunits . The third identified genetic variation c.301g> t (p.r101l) seems to be a novel mutation . Bioinformatic studies using polyphen program suggested that the r101las is probably damaging with a score of 0.997 and specificity of 0.98 . Arginine has a basic polarity and positively charged r group, and hydropathy index of -4.5, which is totally different from leucine with a non - polar, neutral r group, and hydropathy index of + 3.8 . Therefore, this base substitution might completely change the 3d structure, characteristics, and normal function of the resulting protein . Further studies are required to reveal the possible implication of this genetic alteration with adcc . We identified a novel heterozygous c301g> t (p.r101l) genetic variation and two known mutations in gja8 in the three iranian families with adcc . Our data also showed that the mutation in gja8 gene accounts for ~30% of adcc in iranian population . The in silico and experimental studies suggested that these genetic alterations might be vital in hemichannel formation . However, further investigations are essential to define the exact molecular mechanisms, which are implicated by these mutations in the abnormal function as well as their pathogenic effects on the development of adcc.
Cellular transport such as the movement of molecules and macromolecules from one location to another within cells and the formation of complex molecular structures make the properties of the network more intricate . However, all of this apparent complexity can be systematically illustrated as a simple interaction network, particularly through an understanding of protein protein interaction (ppi) networks . The collection of all protein interactions in an organism is typically referred to as an interactome (1). Ppi provides useful information of functional linkage between interacting partnerships within cells (3). Therefore, ppi can help to reveal signal transductions (4,5), post - translational modifications and developmental processes (6). In addition, it can serve to aid in the identification of novel regulatory components and pathways, and provide a valuable approach to understand functional specificities at the molecular level . Sequence - based annotation efforts have led to the identification of a number of cellular components, which can be regarded as a one - dimensional annotation . Accumulated information regarding interactions, and advancement of various high - throughput technologies make it possible to generate systematical, or two - dimensional annotations (7), such as interaction maps . The past several years have witnessed an exponential increase in the amount of biological data, mainly due to the development and application of high - throughput technologies, including gene expression microarrays and mass spectrometry to characterize dna, rna and proteins . Currently, interactomes have been created for several model organisms, such as saccharomyces cerevisiae (8,9), drosophila melanogaster (10), caenorhabditis elegans (11) and homo sapiens, among others (12). In the plant kingdom, arabidopsis thaliana has been widely employed as a model organism to elucidate important biological principles . In fact, several years ago the entire sequence of the arabidopsis genome was reported, and most of its genes annotated (13). However, there are still 30% of these gene products yet to be characterized because sequence homology was not effective at assigning gene function . Only a few interactions of specific protein families in a. thaliana have been reported (14,15), however, an enhanced understanding of ppis can suggest important future directions for researchers to study gene / protein relationships and functions . Meanwhile, many experimental procedures have been developed to analyze ppis, including biochemical methods [e.g. Protein affinity chromatography (1618), affinity blotting, coimmunoprecipitation and cross - linking (1922)]; molecular biological methods [e.g. Protein probing, the two - hybrid system (2325) and phage display (26)] and genetic methods [e.g. The isolation of extragenic suppressors and synthetic mutants (27)]. High - throughput experimental techniques have enabled us to study ppis at the proteome scale . This is achieved via systematic identification of physical interactions among all proteins in an organism . The ever - increasing volume of ppi data is becoming the foundation for new biological discoveries . However, these data are distributed in numerous sources and it has been confirmed that some data are noisy, data quality varies significantly, and data often cannot be verified against each other . Bioinformatics and computational approaches have been used to assess the reliability of high - throughput results and to gain confidence in published data (28). The methodology can also integrate raw data into useful information and provide experimentally testable hypotheses, thereby expanding our knowledge about new mechanisms in biological processes (2932). Other computational prediction methods based on known protein structural interactions can also be useful to analyze large - scale ppi rules . This prediction methodology evaluates interaction rules among complete genomes using protein structural interactome maps (33). Consequently, numerous researches using computational methods have been carried out to investigate gene and protein functions, ppis and gene regulation relationships(3439). These approaches have been applied to interactomes of h. (40), s. cerevisiae (41), c. elegans (37), plasmodium falciparum (42,43), among other organisms . However, rapidly increasing amounts of biological data generated from genome - wide and proteome technologies on modern biochemistry and molecular biology need to be well stored, compararable, organized and accessible . An appropriate repository and maintenance system for atpid was developed using a. thaliana as the model system for a comprehensive resource of ppis . This database contains 28 062 interaction pairs, of which 3866 involve 1875 proteins obtained from the literature and 800 pairs were from enzyme complexes in kegg . In addition, bioinformatics predictions or literature surveys provided 5562 proteins with subcellular location information . Intuitive and user - friendly query interfaces have made all the features of atpid easily accessible . This database provides invaluable resources for researchers to study ppis and protein functions in arabidopsis, data can also be used to address questions regarding gene functions and biological processes in other taxa . Atpid is a non - commercial public access database (http://atpid.biosino.org/) that provides data download services for standalone analyses or data mining, including protein interaction properties and other areas of interest in plant biology . The power and expressivity of any network lies largely in the data model used to represent molecular interactions . From a computational perspective, we applied uniform systematic benchmarks and statistical approaches to specifically train our ppi network for arabidopsis . In addition, to assure data quality, we treated each resource separately as weighted features and reconstructed the ppi network through the proper integration of various protein interaction datasets according to the nave bayesian network theory . In this way, protein interaction data are generated in the following ways: experimental results are obtained from related papers in pubmed and other available databases; and data are made accessible from bioinformatics predictions . Manually collected protein interactions were extracted from not only thousands of published articles, but also intact (44), bind (45) and tair databases (13). We deposited protein interaction data possessing physical evidence or experimental references related to the association between two proteins into atpid . First, we mapped ppi collected from the literature lacking agi locus identifiers to ipi (46) and removed symbols without a match . Additionally, protein pairs in enzyme complexes were also inferred as a part of gsp based on the assumption that subunits in an enzyme complex have high functional association and potential physical interactions . Enzyme complexes from kegg (47) were obtained to extract the intersection of interactions from text mining and complexes of enzymes directly garnered from the kegg database . We subsequently used the decision tree to determine how many proteins belonging to an enzyme complex resulted in a less false positive and higher accuracy . Because many subunits or components of an enzyme complex are mapped from sequence similarity with other species or orthologs, we compared true protein interaction data to reduce noise and redundant information . Eventually, 800 unique pairs were obtained through enzyme complex after excluding the redundancies from the 3866 pairs via text mining . Consequently, a total of 4666 interaction pairs involving 2285 proteins were generated (table 1). Such protein interaction resources, called gsp (golden standard positive) are stored in atpid and used to score the interaction network that assigns each predictive interaction pair with quantized measures . Table 1.overview of gsp resourcesppi resourcesnumber of.ppi pairsnumber of proteins in ppi pairsgsp ppi literatures from pubmed1259740 inact1528677 bind1475538 tair107369838661875protein complexes kegg (enzyme complex)1700856total46662285 manually collected protein interactions are extracted directly from thousands of published articles in pubmed . Inact provides a freely available, open source database system for protein interaction data in embl - ebi . Bind is a new resource to perform cross - database searches of available sequence, interaction, complex and pathway information . It integrates a range of component databases including genbank and bind, the biomolecular interaction network database . Kegg, a reference knowledge base linking genomes to biological systems and environments, provides resourceful enzyme complex information . After mapping various symbols to agi, we found 3866 ppi pairs involving 1875 proteins with literature supports . Combined with enzyme complexes from kegg, the total number of gsp is up to 4666 involving with 2285 proteins . Overview of gsp resources manually collected protein interactions are extracted directly from thousands of published articles in pubmed . Inact provides a freely available, open source database system for protein interaction data in embl - ebi . Bind is a new resource to perform cross - database searches of available sequence, interaction, complex and pathway information . It integrates a range of component databases including genbank and bind, the biomolecular interaction network database . Kegg, a reference knowledge base linking genomes to biological systems and environments, provides resourceful enzyme complex information . After mapping various symbols to agi, we found 3866 ppi pairs involving 1875 proteins with literature supports . Combined with enzyme complexes from kegg, the total number of gsp is up to 4666 involving with 2285 proteins . For predicting ppis in atpid, we applied computational approaches, including conserved protein interactions (i.e. Interologs) (48), gene expression data (49,50), genomic context (i.e. Gene neighbor algorithm) (51,52), gene fusion (rosetta stone method) (53,54), phylogenetic profiles (55,56) and go annotation . The optimized phylogenetic profiles were constructed and assessed using the method of sun et al . (57). Ortholog map files in inparanoid (58) and dip interaction data for other organisms were also collected (59). To infer arabidopsis protein interactions, we mapped several model organismal (e.g. S. cerevisiae, d. melanogaster, c. elegans and h. sapiens) protein interaction data and orthologs to arabidopsis . In addition, we used the atlas of arabidopsis development microarray data (acc.no: me00319) from the tair database (13) to identify co - expressed genes . These data were used to calculate the shared smallest biological processes (ssbp) value of each pair for all proteins employing go annotation methods (40). Therefore, proteins involved in the same process are more likely to interact than proteins in distinct processes . Furthermore, proteins exhibiting high functional specificity are more likely to interact than proteins functioning in more comprehensive processes . Based on this assumption, we first identified all biological process terms shared by two proteins . Subsequently, we counted how many other proteins are assigned to each of the common terms and produced the shared biological process term with the smallest count (ssbp). In general, the smaller the ssbp count, the more specific the biological process term, and the greater the functional similarity between two proteins . In this way, we used ssbp to predict ppis . We also investigated the assumption that some of the operons contained within a particular organism may be conserved across other organisms based on the gene neighbors method . The conservation of an operon's structure provides additional evidence that genes within an operon are functionally coupled and are perhaps components of a protein complex or pathway . The underlying assumption of the method is that if a composite protein is uniquely similar to two component proteins in another species, the component proteins are most likely to interact (53). Gene - fusion events were identified in complete genomes, based solely on sequence comparisons . The predictive datasets from such individual methods were integrated employing nave bayesian networks (40). The bayesian networks approach was used to integrate more than seven predictive data sources and to subsequently build a model to infer novel ppis for arabidopsis . The essence of the approach is to provide a mathematical rule, given some predictive evidence, to explain how to adjust the odds that a pair of proteins interacts, either in a true interaction instance (gsp) or correspondingly, in negative protein interactions, known as gsn (golden standard negative). However, protein localization data provides indirect evidence, given we assume that proteins in different cellular compartments do not interact (60). Hence, gsn values were constructed based on this assumption using subcellular localization data from the suba database (61). Individual likelihood ratios were easily calculated by counting the number of protein pairs with values that overlap with the gsp and gsn sets in the predictive dataset . The confidence scores (lr) for each inferred ppi pair were the sum of the logarithmic form of all seven individual likelihood ratios from corresponding methods . The atpid querying results page depicts the lr score from each method with open, partially or completely filled circles that indicate positive correlations with the confidence level of the interaction relationship . The detailed number of each predictive dataset is shown in table 2 and all predictive datasets can be downloaded from the atpid website . Table 2.overview of the number of individual predictive datasetnumber of predictive ppi pairsnumber of proteins in the ppi pairso: ortholog interaction datasets30451359 g: shared biological function: go ontology553523e: co - expression14 8378024f: gene fusion method65705671n: gene neighbors method20081637p: phylogenetic profile method15 7238751d: enriched domain pair21821288atpid28 062 (putative ppi with gsp)12 50623 396 (putative ppi without gsp)11 706through integration by nave bays network, atpid achieved 28 062 ppi pairs with 23 396 pairs from prediction methods . There are seven individual datasets from various approaches, identified by o, g, e, f, n, p and d. the details of each method can be browsed on atpid faq . Overview of the number of individual predictive dataset through integration by nave bays network, atpid achieved 28 062 ppi pairs with 23 396 pairs from prediction methods . There are seven individual datasets from various approaches, identified by o, g, e, f, n, p and d. the details of each method can be browsed on atpid faq . The power and expressivity of any network lies largely in the data model used to represent molecular interactions . From a computational perspective, we applied uniform systematic benchmarks and statistical approaches to specifically train our ppi network for arabidopsis . In addition, to assure data quality, we treated each resource separately as weighted features and reconstructed the ppi network through the proper integration of various protein interaction datasets according to the nave bayesian network theory . In this way, protein interaction data are generated in the following ways: experimental results are obtained from related papers in pubmed and other available databases; and data are made accessible from bioinformatics predictions . Manually collected protein interactions were extracted from not only thousands of published articles, but also intact (44), bind (45) and tair databases (13). We deposited protein interaction data possessing physical evidence or experimental references related to the association between two proteins into atpid . First, we mapped ppi collected from the literature lacking agi locus identifiers to ipi (46) and removed symbols without a match . Additionally, protein pairs in enzyme complexes were also inferred as a part of gsp based on the assumption that subunits in an enzyme complex have high functional association and potential physical interactions . Enzyme complexes from kegg (47) were obtained to extract the intersection of interactions from text mining and complexes of enzymes directly garnered from the kegg database . We subsequently used the decision tree to determine how many proteins belonging to an enzyme complex resulted in a less false positive and higher accuracy . Because many subunits or components of an enzyme complex are mapped from sequence similarity with other species or orthologs, we compared true protein interaction data to reduce noise and redundant information . Eventually, 800 unique pairs were obtained through enzyme complex after excluding the redundancies from the 3866 pairs via text mining . Consequently, a total of 4666 interaction pairs involving 2285 proteins were generated (table 1). Such protein interaction resources, called gsp (golden standard positive) are stored in atpid and used to score the interaction network that assigns each predictive interaction pair with quantized measures . Table 1.overview of gsp resourcesppi resourcesnumber of.ppi pairsnumber of proteins in ppi pairsgsp ppi literatures from pubmed1259740 inact1528677 bind1475538 tair107369838661875protein complexes kegg (enzyme complex)1700856total46662285 manually collected protein interactions are extracted directly from thousands of published articles in pubmed . Inact provides a freely available, open source database system for protein interaction data in embl - ebi . Bind is a new resource to perform cross - database searches of available sequence, interaction, complex and pathway information . It integrates a range of component databases including genbank and bind, the biomolecular interaction network database . Kegg, a reference knowledge base linking genomes to biological systems and environments, provides resourceful enzyme complex information . After mapping various symbols to agi, we found 3866 ppi pairs involving 1875 proteins with literature supports . Combined with enzyme complexes from kegg, the total number of gsp is up to 4666 involving with 2285 proteins . Overview of gsp resources manually collected protein interactions are extracted directly from thousands of published articles in pubmed . Inact provides a freely available, open source database system for protein interaction data in embl - ebi . Bind is a new resource to perform cross - database searches of available sequence, interaction, complex and pathway information . It integrates a range of component databases including genbank and bind, the biomolecular interaction network database . Kegg, a reference knowledge base linking genomes to biological systems and environments, provides resourceful enzyme complex information . After mapping various symbols to agi, we found 3866 ppi pairs involving 1875 proteins with literature supports . Combined with enzyme complexes from kegg, the total number of gsp is up to 4666 involving with 2285 proteins . For predicting ppis in atpid, we applied computational approaches, including conserved protein interactions (i.e. Interologs) (48), gene expression data (49,50), genomic context (i.e. Gene neighbor algorithm) (51,52), gene fusion (rosetta stone method) (53,54), phylogenetic profiles (55,56) and go annotation . The optimized phylogenetic profiles were constructed and assessed using the method of sun et al . (57). Ortholog map files in inparanoid (58) and dip interaction data for other organisms were also collected (59). To infer arabidopsis protein interactions, we mapped several model organismal (e.g. S. cerevisiae, d. melanogaster, c. elegans and h. sapiens) protein interaction data and orthologs to arabidopsis . In addition, we used the atlas of arabidopsis development microarray data (acc.no: me00319) from the tair database (13) to identify co - expressed genes . These data were used to calculate the shared smallest biological processes (ssbp) value of each pair for all proteins employing go annotation methods (40). Therefore, proteins involved in the same process are more likely to interact than proteins in distinct processes . Furthermore, proteins exhibiting high functional specificity are more likely to interact than proteins functioning in more comprehensive processes . Based on this assumption, we first identified all biological process terms shared by two proteins . Subsequently, we counted how many other proteins are assigned to each of the common terms and produced the shared biological process term with the smallest count (ssbp). In general, the smaller the ssbp count, the more specific the biological process term, and the greater the functional similarity between two proteins . In this way, we used ssbp to predict ppis . We also investigated the assumption that some of the operons contained within a particular organism may be conserved across other organisms based on the gene neighbors method . The conservation of an operon's structure provides additional evidence that genes within an operon are functionally coupled and are perhaps components of a protein complex or pathway . The underlying assumption of the method is that if a composite protein is uniquely similar to two component proteins in another species, the component proteins are most likely to interact (53). Gene - fusion events were identified in complete genomes, based solely on sequence comparisons . The predictive datasets from such individual methods were integrated employing nave bayesian networks (40). The bayesian networks approach was used to integrate more than seven predictive data sources and to subsequently build a model to infer novel ppis for arabidopsis . The essence of the approach is to provide a mathematical rule, given some predictive evidence, to explain how to adjust the odds that a pair of proteins interacts, either in a true interaction instance (gsp) or correspondingly, in negative protein interactions, known as gsn (golden standard negative). However, protein localization data provides indirect evidence, given we assume that proteins in different cellular compartments do not interact (60). Hence, gsn values were constructed based on this assumption using subcellular localization data from the suba database (61). Individual likelihood ratios were easily calculated by counting the number of protein pairs with values that overlap with the gsp and gsn sets in the predictive dataset . The confidence scores (lr) for each inferred ppi pair were the sum of the logarithmic form of all seven individual likelihood ratios from corresponding methods . The atpid querying results page depicts the lr score from each method with open, partially or completely filled circles that indicate positive correlations with the confidence level of the interaction relationship . The detailed number of each predictive dataset is shown in table 2 and all predictive datasets can be downloaded from the atpid website . Table 2.overview of the number of individual predictive datasetnumber of predictive ppi pairsnumber of proteins in the ppi pairso: ortholog interaction datasets30451359 g: shared biological function: go ontology553523e: co - expression14 8378024f: gene fusion method65705671n: gene neighbors method20081637p: phylogenetic profile method15 7238751d: enriched domain pair21821288atpid28 062 (putative ppi with gsp)12 50623 396 (putative ppi without gsp)11 706through integration by nave bays network, atpid achieved 28 062 ppi pairs with 23 396 pairs from prediction methods . There are seven individual datasets from various approaches, identified by o, g, e, f, n, p and d. the details of each method can be browsed on atpid faq . Overview of the number of individual predictive dataset through integration by nave bays network, atpid achieved 28 062 ppi pairs with 23 396 pairs from prediction methods . There are seven individual datasets from various approaches, identified by o, g, e, f, n, p and d. the details of each method can be browsed on atpid faq . The process of creating a release atpid database begins with extracting the published and other relevant information from various databases (figure 1). Automated and manual quality assurance procedures are administered to verify data completeness and consistency . If necessary, material in the development database is revised and a new database version is generated . Ortholog maps, domain attributes and network displays are developed with crosslinks to other relevant external resources (62). Following the final testing of data and the web server, the latest release (14 july 2007) contains 28 062 ppi pairs involving 12 506 proteins . Of the ppi pairs, 23 396 pairs were inferred by the integration of several methods, while the other 3866 pairs involving 1875 proteins were manually curated from the literature and other 800 pairs were determined from enzyme complexes from kegg . In addition to protein interaction data, we added subcellular location annotations to nearly 5000 proteins from swissprot and suba databases (61,63) and popular prediction tools, including targetp (64), predotar (65) and mitoprotii (66), which can promote subproteome and protein function research . Atpid spans roughly 41% of the estimated 30 480 peptides with interaction annotations in the arabidopsis genome and reflects the labor - intensive nature of manual curation . Our future plans are to manually mine thousands of protein interactions to acquire information through bulk importation of data from other sources or experimental results . Thus increasing ppi information and power as a resource . Ppi will also provide enhanced resource training data for reconstructing interaction networks with higher accuracy and larger coverage of the arabidopsis genome . In turn, the database can aid users in querying more detailed information about interaction pathways or maps comprising of interesting protein attributes . Quick start main interaction querying box with links to each of the seven method theories used in atpid, the atpid database statistics, and announcements regarding the function of the website . Ppi query is the main function of atpid, which makes available manually collected ppi data and predicted ppi through integrated data resources . Query flow is illustrated in figure 2 and demonstrates how querying a protein name or protein pair on the query page accesses ppi information (http://atpid.biosino.org/query.php). Atpid allows several types of query keywords used by other databases, including uniprotkb / swiss - prot i d, tair agi, entrez gene name, refseq provisional i d (ncbi) or international protein index (ipi) symbols . This search is appropriate when the user would like to know which other protein(s) have the highest probability of interacting with the protein of interest . Pair search allows the user to submit a protein pair to ascertain if an interaction between two proteins has been documented . For example, the user is interested in the hap3a protein, which encodes a subunit of the ccaat - binding complex and binds to the ccaat box motif present in some plant promoter sequences . Search results show a summary of the protein attributes in the first table, including the locus, gene / protein symbol, the number of interactions (six from gsp and seven by inference), function description and database cross - references to entrez, tair, ipi, uniprotkb / trembl, uniparc and kegg (figure 3). Figure 3.the atpid interface of ppi simple search results for the queried protein, hap3a . There are generally three tables: (1) protein attributes with gene model descriptions and ppi summary, showing how many protein pairs can be predicted and the inferred interactions overlapped with gsp . (2) the second table represents interactants of the queried protein, hap3a . Users can view what the experimental evidence is to support this consequence and link to corresponding literature(s). (3) the third table represents potential interactants of the queried protein, hap3a, by nave bays integration across the seven approaches . Each approach is graphically represented by circles . When the cursor is over a circle, it will display the corresponding score (lr) for the method . There are generally three tables: (1) protein attributes with gene model descriptions and ppi summary, showing how many protein pairs can be predicted and the inferred interactions overlapped with gsp . (2) the second table represents interactants of the queried protein, hap3a . Users can view what the experimental evidence is to support this consequence and link to corresponding literature(s). (3) the third table represents potential interactants of the queried protein, hap3a, by nave bays integration across the seven approaches . When the cursor is over a circle, it will display the corresponding score (lr) for the method . The second table of the search results presents inferred ppi pairs belonging to gsp listed with supporting evidence, including literature references from pubmed and experimental detection methods from text mining . Each of the seven prediction approaches is depicted by a letter acronym within a circle . When the user places the cursor over each circle, it displays the full name of the method . The circle under each method indicates the confidence strength for the predicted method and the related protein . The more the circle is filled, the more likely the pair of proteins is to interact . The corresponding score for the specific method is displayed when the cursor is held over the circle . Network display above the information table provides the link to a new window that displays the interaction network about hap3a (figure 4). In the network display page, the query protein is represented as a triangle; the functional partners of the queried protein are represented by a circle, derived from the first level displayed in the ppi network that directly linked to the query protein . The associated functional partners of the queried protein are shown as squares, derived from the second level in the network . A red node represents a protein with known function (i.e. Annotated), whereas a gray node represents an unknown functional protein (i.e. Without annotation). The line between each protein indicates the functional relationship; a red line infers the interaction from text mining, and a blue line indicates the predictive function relationship . By holding the cursor over each protein, the related annotation for the protein is displayed and allows the user to navigate the network and easily check a proteins relationship . Figure 4.atpid interface of the (network display) window for the queried protein, hap3 . In the (network display) window, we can view the interaction network involved with queried protein(s) and the neighbor component(s) intuitively . Atpid interface of the (network display) window for the queried protein, hap3 . In the (network display) window, we can view the interaction network involved with queried protein(s) and the neighbor component(s) intuitively . (text format output) can export the interaction pair information for further analysis . In the pair search results window when users submit potential interaction pairs, the domain attribute of one partner protein (e.g. At1g09030) each module is linked to pfam and when the user places the cursor over the module, details of the domain will also be displayed . Thus, atpid provides comprehensive knowledge through a friendly and convenient interface that should be easy to use by biologists . The database server is located at sibs (shanghai institute of biological science) data service platform . The atpid development environment is apache+php+mysql, which allows for more efficient calculation rate performances and augmentation of the program . Quick start main interaction querying box with links to each of the seven method theories used in atpid, the atpid database statistics, and announcements regarding the function of the website . Ppi query is the main function of atpid, which makes available manually collected ppi data and predicted ppi through integrated data resources . Query flow is illustrated in figure 2 and demonstrates how querying a protein name or protein pair on the query page accesses ppi information (http://atpid.biosino.org/query.php). Atpid allows several types of query keywords used by other databases, including uniprotkb / swiss - prot i d, tair agi, entrez gene name, refseq provisional i d (ncbi) or international protein index (ipi) symbols . This search is appropriate when the user would like to know which other protein(s) have the highest probability of interacting with the protein of interest . Pair search allows the user to submit a protein pair to ascertain if an interaction between two proteins has been documented . For example, the user is interested in the hap3a protein, which encodes a subunit of the ccaat - binding complex and binds to the ccaat box motif present in some plant promoter sequences . Search results show a summary of the protein attributes in the first table, including the locus, gene / protein symbol, the number of interactions (six from gsp and seven by inference), function description and database cross - references to entrez, tair, ipi, uniprotkb / trembl, uniparc and kegg (figure 3). Figure 3.the atpid interface of ppi simple search results for the queried protein, hap3a . There are generally three tables: (1) protein attributes with gene model descriptions and ppi summary, showing how many protein pairs can be predicted and the inferred interactions overlapped with gsp . (2) the second table represents interactants of the queried protein, hap3a . Users can view what the experimental evidence is to support this consequence and link to corresponding literature(s). (3) the third table represents potential interactants of the queried protein, hap3a, by nave bays integration across the seven approaches . When the cursor is over a circle, it will display the corresponding score (lr) for the method . There are generally three tables: (1) protein attributes with gene model descriptions and ppi summary, showing how many protein pairs can be predicted and the inferred interactions overlapped with gsp . Users can view what the experimental evidence is to support this consequence and link to corresponding literature(s). (3) the third table represents potential interactants of the queried protein, hap3a, by nave bays integration across the seven approaches . Each approach is graphically represented by circles . When the cursor is over a circle, it will display the corresponding score (lr) for the method . Search results presents inferred ppi pairs belonging to gsp listed with supporting evidence, including literature references from pubmed and experimental detection methods from text mining . Each interactant can be linked to a new pair search results window . Each of the seven prediction approaches is depicted by a letter acronym within a circle . When the user places the cursor over each circle, it displays the full name of the method . The circle under each method indicates the confidence strength for the predicted method and the related protein . The more the circle is filled, the more likely the pair of proteins is to interact . The corresponding score for the specific method is displayed when the cursor is held over the circle . Network display above the information table provides the link to a new window that displays the interaction network about hap3a (figure 4). In the network display page, the query protein is represented as a triangle; the functional partners of the queried protein are represented by a circle, derived from the first level displayed in the ppi network that directly linked to the query protein . The associated functional partners of the queried protein are shown as squares, derived from the second level in the network . A red node represents a protein with known function (i.e. Annotated), whereas a gray node represents an unknown functional protein (i.e. Without annotation). The line between each protein indicates the functional relationship; a red line infers the interaction from text mining, and a blue line indicates the predictive function relationship . By holding the cursor over each protein, the related annotation for the protein is displayed and allows the user to navigate the network and easily check a proteins relationship . Figure 4.atpid interface of the (network display) window for the queried protein, hap3 . In the (network display) window, we can view the interaction network involved with queried protein(s) and the neighbor component(s) intuitively . Atpid interface of the (network display) window for the queried protein, hap3 . In the (network display) window, we can view the interaction network involved with queried protein(s) and the neighbor component(s) intuitively . (text format output) can export the interaction pair information for further analysis . In the pair search results window when users submit potential interaction pairs, the domain attribute of one partner protein (e.g. At1g09030) can be viewed graphically (figure 5). Each module is linked to pfam and when the user places the cursor over the module, details of the domain will also be displayed . Thus, atpid provides comprehensive knowledge through a friendly and convenient interface that should be easy to use by biologists . The database server is located at sibs (shanghai institute of biological science) data service platform . The atpid development environment is apache+php+mysql, which allows for more efficient calculation rate performances and augmentation of the program . Atpid serves as a major reference site for ppis using arabidopsis as a model plant system . A number of new features and applications are currently under construction, such as a gene regulation and dynamic ppi network that function under different conditions with increased gene expression and proteomics data . Currently, the subcellular localization predictions of a. thaliana are available for both the chloroplast and mitochondrion and the predictive organellar proteins have been added into atpid . In addition, we plan to conduct further assessments of proteins to other cellular and/or subcellular locations, including nuclear, cytoplasmic and extracellular proteins . Another important field of research is to elucidate the relationships between phenotype and genotype . For example, we plan to collect data relevant to mutants and their respective phenotypes . These highly varied types of data will be available through atpid in the near future.
Multiple myeloma (mm) is a haematological malignancy characterised by a malignant proliferation of monoclonal plasma cells in the bone marrow producing monoclonal immunoglobulins and by the formation of focal osteolytic lesions in the skeleton . The disorder might evolve from a common pre - malignant condition called monoclonal gammopathy of undetermined significance (mgus). The clinical manifestations of mm include bone pains caused by lytic bone lesions, anaemia, hypercalcaemia, immunodefi - ciency and renal failure . The incidence of the disease is about 3 to 4 per 100,000 in the netherlands and mortality rates are only slightly lower . Although the disorder is not curable in most cases, the overall survival varies depending on the age of onset and other prognostic features . However, several families have been described with multiple cases of mm, suggesting there may be a genetic predisposition [5 - 11]. Since no causative germline gene mutations have been identified, diagnostic dna testing of families with an inherited type of mm and presymptomatic genetic screening for unaffected relatives are unavailable . Here we report on two families with familial clustering of mm, we review the literature on familial mm, and we discuss the options for screening healthy relatives for mm in these familial cases . Two unrelated families were referred to our clinic asking about the possible heritability of mm in their family and the options for screening of healthy relatives . Family 1 had three first - degree relatives affected by mm (figure 1). The index patient (iii-2) was a 65-year old female who was diagnosed with mm after presenting with pain in the shoulder region and fatigue . Cytogenetic findings in bone marrow corresponded with this diagnosis (46, xx, 54 -x, 1q+, + 1p-, + 3, + 5, + 6q-, + 9, 11q+, -13, 14q+, + 15, + 16, + 19, + 20). Her father (ii-3) had been diagnosed with mm at 71 years of age after the discovery of a lytic lesion in the spine and an igg paraproteinaemia . The brother of the index patient (iii-1) had been diagnosed with mm at 45 years of age . He had several lytic bone lesions in his spine and pelvis and an igg paraproteinaemia of 140 pedigree of family 1 with multiple myeloma . Diagnosis and age of diagnosis are given, as well as age of death . Solid blocks represent patients with multiple myeloma; open blocks represent patients without multiple myeloma . Mm = multiple myeloma; lu = lung cancer; l = leukaemia; d = deceased; number in parenthesis = age of onset, current age, or age of death . The mother of the index patient died of leukaemia (type unknown) at 75 years of age . Two of the patient's paternal uncles died of lung cancer at an old age (smoking habits unknown). There was no known shared or individual exposure to toxic chemicals or radiation in the patients . Family 2 had two first - degree relatives affected by mm (figure 2). The index patient is a 38-year old male who was diagnosed with mm after discovery of a lytic lesion in the hip . Pedigree of family 2 with multiple myeloma (see figure 1 for explanation of symbols). His father was diagnosed with mm at the age of 60 years after having a pathological fracture of the upper arm . Cytogenetic findings in bone marrow corresponded with this diagnosis (46, xy, 58, xy, + 3, + 5, + 6, t(8; 22)(q24; q11), + 9, + 11, + 15, + 18, + 19, + 21). The patterns of other types of cancer seen in these two families did not suggest any known hereditary cancer syndrome . It usually occurs incidentally within a family, but several familial cases have been reported in the literature [5 - 11]. The frequency of familial mm seems to be approximately 3.2 per 1000 cases of mm, leading to an occurrence of familial mm in approximately 1 per 10 million persons per year, making it a rare event . Have been implicated, for example radiation and pesticide exposure were reported to have led to a higher incidence of mm . However, alexander et al . Concluded that no environmental risk factors could consistently be established when they compared multiple epidemiologic studies on environmental influences . Chance alone might have caused the clustering within families; however, several families have shown an inheritance pattern that is very suggestive of an underlying genetic factor . A number of authors have proposed an autosomal dominant mode of inheritance, with reduced penetrance, to explain the occurrence of multiple cases of mm within a family [6 - 11,15]. . Postulated an autosomal recessive inheritance with low penetrance based on their findings of a high prevalence of b - cell diseases (mm or mgus monoclonal gammopathy of unknown origin) among siblings of mm patients . However, they only screened siblings and showed no data on paternal or offspring disease state . Both our families would fit the hypothesis of an autosomal dominant pattern of inheritance, although environmental agents, low penetrance predisposing genes, or clustering by chance or combinations of these could be alternative explanations for the familial pattern . Unfortunately, because of the small number of informative meioses and the lack of dna samples from some of the affected relatives, linkage analysis cannot be expected to solve the puzzle in these two families . In the first family all three affected persons had an igg paraproteinaemia, which might suggest a common genetic cause, but in the second family the type of monoclonal immunoglobulin differed between the two patients . A discrepancy in type of immunoglobulin produced within a family was also shown by lynch et al . In an mm / mgus family and also by ogmundsdottir . Some studies suggest that germline gene mutations and polymorphisms may be associated with a risk of mm; however, results have not been consistent and no causative inherited genetic factor has been detected so far . It has been well established that mm is characterised by extensive genomic abnormalities in tumour cells . The abnormalities consist of numerical and gross structural changes, deletions, duplications and translocations . Only two of the patients in our families showed characteristic complex chromosomal changes, refuting a relation between specific somatic chromosomal abnormalities and constitutional genetic factors . Small structural chromosomal changes (shared by relatives) could have been overlooked due to the limited spatial resolution of the technique . Because no genetic factor or exact mode of inheritance has been determined for mm, the risk for healthy relatives of mm patients can only be estimated from large case - control or cohort studies . These cases showed an sir (standardised incidence ratio) of mm in offspring from an affected parent of 2.45 . Landgren et al . Conducted a case - control study to evaluate the risk of developing mm for someone with first - degree relatives with mm . They found a significantly increased risk for mm in relatives of cases (rr (relative risk) 1.67). Among female relatives, hemminki found an sir of 4.25 for mm among offspring with a parent presenting with mm . Eriksson et al . Calculated an rr of 5.64 for first - degree relatives of mm patients . Brown et al . Found an almost four - fold excess risk for mm in a large case - control study in the us among subjects with a first - degree relative with mm . From the icelandic cancer registry, ogmundsdottir et al . Calculated a significantly increased risk of developing mm for first - degree relatives (rr 2.33), with the risk being largest for female relatives (rr 3.23). Overall, the risk of mm in first - degree relatives of cases with mm seems to be between two- and four - fold . Similar familial risks have been reported for cancers with a known, highly penetrant, predisposition gene, like breast cancer, colon cancer and ovarian cancer . To the best of our knowledge, there are no reports estimating the risk for subjects with more than one (first - or second - degree) relative suffering from mm, as seen in our families, but we presume the risks are higher for relatives of familial cases of mm than for those with only a single family member suffering from mm . The risk of developing other types of cancer seems to be slightly higher for relatives of mm patients . An increased risk for other lymphoproliferative disorders in the relatives of mm patients has been shown, especially for cll (chronic lymphatic leukaemia) and nhl (non - hodgkin lymphoma) [9 - 11,23]. Significant associations were found between mm in a patient and rectal, stomach, cervical, prostate, bladder, endocrine gland and connective tissue malignancies in family members . It has been shown that familial cases of mm occur at an earlier age than sporadic cases, which is a hallmark of heritable cancer . Anticipation, or the tendency in a multi - generational family for cancer to occur earlier in subsequent generations, was observed in some studies for mm [5,7 - 10], but not in all . In general, anticipation could be explained by ascertainment bias (due to screening of relatives or to them being more aware of the disease, thus leading to earlier discovery) or changes of incidence over time, but it might also point towards a genetic cause of the disease . In both our families there seems to be anticipation . The cancer occurred on average 16 years earlier in the first family, and 23 years earlier in the second family . Patients from families like the ones reported here often have questions regarding the possibly hereditary nature of mm in their family and ask if there is a need to screen healthy relatives . There are no primary preventive measures for mm as yet and no consensus guidelines for screening healthy relatives in mm families ., who suggested screening using blood and urine protein electrophoresis in first - degree relatives of familial mm patients and follow - up surveillance of individuals with mgus, but they did not specify the frequency of screening or the preferred age for starting screening . A population with a high risk of mm in which screening is recommended is the group of patients with mgus (monoclonal gammopathy of unknown origin). Mgus can be seen as a pre - malignant precursor of mm and it is defined by the presence of a monoclonal protein, but the absence of a large amount of plasma cells in the bone marrow and end - organ damage characteristic for mm (renal insufficiency, lytic bone lesions, anaemia, hypercalcaemia) [9 - 14]. Mgus progresses into mm in about 1% of cases per year and progression cannot be prevented . Periodic screening is therefore advised for patients with mgus in order to detect progression into mm and to prevent or delay serious complications such as renal failure or pathologic fractures . The quality of life of patients can be improved by averting these events and the cost of long - term dialysis or surgical intervention for skeletal complications would be reduced . Since there are no known predictors to indicate which mgus patients will progress to mm, screening is recommended for all patients . These cases were either found by collecting familial cases or by direct screening of relatives . After screening 19 relatives using electrophoresis of urine and blood, they detected two siblings with mgus . These individuals were advised about long - term follow - up because of their increased risk for mm . When considering the usefulness of a screening program the wilson and jungner criteria are often used . These indicate that there should be an important health problem in order to start screening and that it should only be offered if there is a certain degree of increased risk . The previously calculated relative risks (rrs) do not translate into a high lifetime risk because of the low risk for mm in the general population, and they therefore do not carry the same weight as similar rrs that have been calculated for relatives of patients with common types of cancer like colorectal cancer or breast cancer . However, although familial mm is very rare, screening should be offered to selected families for whom the health problem is significant . Calculations did not take more than one affected relative into account and may therefore have significantly underestimated the risk for relatives in the rare families with multiple cases of mm . Indeed, several observations have suggested the existence of hereditary mm, and germline gene mutations with significant penetrance may therefore underlie familial mm . In the absence of known risk estimates for the relatives in these families, we conclude for practical purposes that a significant risk cannot be ruled out at the present time and the notion of screening in these families cannot simply be dismissed . According to the wilson and jungner criteria, there should be a suitable and acceptable diagnostic test for the disease and good treatment options . Screening for mm can be done by blood and urine protein electrophoresis, which is minimally invasive and relatively easy to perform . On detecting a monoclonal protein, a distinction between mgus or mm should be made by further study, possibly including a bone marrow biopsy . Treatment options for mm have improved over the last few years with respect to survival and quality of life . The goal of cancer screening in general is to detect a pre - malignant, treatable lesion (as in the case of screening for adenomatous colorectal polyps) or to detect a cancer in an early, treatable stage (as in screening for breast cancer), and early detection should positively influence the natural cause of the disease . Even though mm is an incurable disease in most cases and preventing the benign precursor mgus from progressing is impossible, screening might nevertheless be beneficial because detection of mgus or early - state mm is advantageous for morbidity and delays mortality from the disease . The screening test for mm is cheap, and early detection could prevent costly complications . The disadvantages of screening for mm are the possible distress for relatives in whom mgus or mm is detected, since mgus and mm are not curable and progression from mgus to mm can neither be predicted nor prevented . However, weighing the advantages and disadvantages for screening of healthy relatives in mm families, we conclude that screening might be beneficial for this distinct group . We suggest that screening should comprise annual protein electrophoresis of blood and urine and that it should be restricted to individuals with more than one first - degree affected relative or to those with one first - degree and at least one second - degree relative with mm . Since mm is rarely diagnosed before age 40, we propose that relatives should be screened from this age . If families present with mm at a younger age, then screening could be started five years earlier than the youngest age at diagnosis (with the five years being an arbitrary period). Counselling the families on the limitations and possible advantages of screening should of course precede any screening . We suggest that this screening should be performed in a research setting where the outcome can be closely monitored to determine its clinical value and the psychological burden . If, in the future, germline gene mutations are identified as the cause of mm, more accurate risk estimates will become available . If lifetime risks for mutation carriers should indicate the need for screening, then presymptomatic dna testing can help in targeting this surveillance . In summary, although risk estimates for asymptomatic relatives in these families are not yet available, a clinically significant risk of developing mm cannot be excluded . In a research setting, these families could be offered screening for mm . - more than one first - degree relative diagnosed with multiple myeloma, or - one first - degree and at least one second - degree relative with multiple myeloma annual protein electrophoresis to test immunoglobulins in blood and urine, starting at age 40 years . If families present with mm at a younger age, then screening could be started at say five years earlier than the youngest age at diagnosis in those families . Because no genetic factor or exact mode of inheritance has been determined for mm, the risk for healthy relatives of mm patients can only be estimated from large case - control or cohort studies . These cases showed an sir (standardised incidence ratio) of mm in offspring from an affected parent of 2.45 . Landgren et al . Conducted a case - control study to evaluate the risk of developing mm for someone with first - degree relatives with mm . They found a significantly increased risk for mm in relatives of cases (rr (relative risk) 1.67). Among female relatives, hemminki found an sir of 4.25 for mm among offspring with a parent presenting with mm . Eriksson et al . Calculated an rr of 5.64 for first - degree relatives of mm patients . Brown et al . Found an almost four - fold excess risk for mm in a large case - control study in the us among subjects with a first - degree relative with mm . From the icelandic cancer registry, ogmundsdottir et al . Calculated a significantly increased risk of developing mm for first - degree relatives (rr 2.33), with the risk being largest for female relatives (rr 3.23). Overall, the risk of mm in first - degree relatives of cases with mm seems to be between two- and four - fold . Similar familial risks have been reported for cancers with a known, highly penetrant, predisposition gene, like breast cancer, colon cancer and ovarian cancer . To the best of our knowledge, there are no reports estimating the risk for subjects with more than one (first - or second - degree) relative suffering from mm, as seen in our families, but we presume the risks are higher for relatives of familial cases of mm than for those with only a single family member suffering from mm . The risk of developing other types of cancer seems to be slightly higher for relatives of mm patients . An increased risk for other lymphoproliferative disorders in the relatives of mm patients has been shown, especially for cll (chronic lymphatic leukaemia) and nhl (non - hodgkin lymphoma) [9 - 11,23]. Significant associations were found between mm in a patient and rectal, stomach, cervical, prostate, bladder, endocrine gland and connective tissue malignancies in family members . It has been shown that familial cases of mm occur at an earlier age than sporadic cases, which is a hallmark of heritable cancer . Anticipation, or the tendency in a multi - generational family for cancer to occur earlier in subsequent generations, was observed in some studies for mm [5,7 - 10], but not in all . In general, anticipation could be explained by ascertainment bias (due to screening of relatives or to them being more aware of the disease, thus leading to earlier discovery) or changes of incidence over time, but it might also point towards a genetic cause of the disease . In both our families there seems to be anticipation . The cancer occurred on average 16 years earlier in the first family, and 23 years earlier in the second family . Patients from families like the ones reported here often have questions regarding the possibly hereditary nature of mm in their family and ask if there is a need to screen healthy relatives . There are no primary preventive measures for mm as yet and no consensus guidelines for screening healthy relatives in mm families . The only recommendation we could find in the literature is from hodgson et al ., who suggested screening using blood and urine protein electrophoresis in first - degree relatives of familial mm patients and follow - up surveillance of individuals with mgus, but they did not specify the frequency of screening or the preferred age for starting screening . A population with a high risk of mm in which screening is recommended is the group of patients with mgus (monoclonal gammopathy of unknown origin). Mgus can be seen as a pre - malignant precursor of mm and it is defined by the presence of a monoclonal protein, but the absence of a large amount of plasma cells in the bone marrow and end - organ damage characteristic for mm (renal insufficiency, lytic bone lesions, anaemia, hypercalcaemia) [9 - 14]. Mgus progresses into mm in about 1% of cases per year and progression cannot be prevented . Periodic screening is therefore advised for patients with mgus in order to detect progression into mm and to prevent or delay serious complications such as renal failure or pathologic fractures . The quality of life of patients can be improved by averting these events and the cost of long - term dialysis or surgical intervention for skeletal complications would be reduced . Since there are no known predictors to indicate which mgus patients will progress to mm, screening is recommended for all patients . These cases were either found by collecting familial cases or by direct screening of relatives . After screening 19 relatives using electrophoresis of urine and blood, they detected two siblings with mgus . These individuals were advised about long - term follow - up because of their increased risk for mm . When considering the usefulness of a screening program the wilson and jungner criteria are often used . These indicate that there should be an important health problem in order to start screening and that it should only be offered if there is a certain degree of increased risk . The previously calculated relative risks (rrs) do not translate into a high lifetime risk because of the low risk for mm in the general population, and they therefore do not carry the same weight as similar rrs that have been calculated for relatives of patients with common types of cancer like colorectal cancer or breast cancer . However, although familial mm is very rare, screening should be offered to selected families for whom the health problem is significant . Calculations did not take more than one affected relative into account and may therefore have significantly underestimated the risk for relatives in the rare families with multiple cases of mm . Indeed, several observations have suggested the existence of hereditary mm, and germline gene mutations with significant penetrance may therefore underlie familial mm . In the absence of known risk estimates for the relatives in these families, we conclude for practical purposes that a significant risk cannot be ruled out at the present time and the notion of screening in these families cannot simply be dismissed . According to the wilson and jungner criteria, there should be a suitable and acceptable diagnostic test for the disease and good treatment options . Screening for mm can be done by blood and urine protein electrophoresis, which is minimally invasive and relatively easy to perform . On detecting a monoclonal protein, a distinction between mgus or mm should be made by further study, possibly including a bone marrow biopsy . Treatment options for mm have improved over the last few years with respect to survival and quality of life . The goal of cancer screening in general is to detect a pre - malignant, treatable lesion (as in the case of screening for adenomatous colorectal polyps) or to detect a cancer in an early, treatable stage (as in screening for breast cancer), and early detection should positively influence the natural cause of the disease . Even though mm is an incurable disease in most cases and preventing the benign precursor mgus from progressing is impossible, screening might nevertheless be beneficial because detection of mgus or early - state mm is advantageous for morbidity and delays mortality from the disease . The screening test for mm is cheap, and early detection could prevent costly complications . The disadvantages of screening for mm are the possible distress for relatives in whom mgus or mm is detected, since mgus and mm are not curable and progression from mgus to mm can neither be predicted nor prevented . However, weighing the advantages and disadvantages for screening of healthy relatives in mm families, we conclude that screening might be beneficial for this distinct group . We suggest that screening should comprise annual protein electrophoresis of blood and urine and that it should be restricted to individuals with more than one first - degree affected relative or to those with one first - degree and at least one second - degree relative with mm . Since mm is rarely diagnosed before age 40, we propose that relatives should be screened from this age . If families present with mm at a younger age, then screening could be started five years earlier than the youngest age at diagnosis (with the five years being an arbitrary period). Counselling the families on the limitations and possible advantages of screening should of course precede any screening . We suggest that this screening should be performed in a research setting where the outcome can be closely monitored to determine its clinical value and the psychological burden . If, in the future, germline gene mutations are identified as the cause of mm, more accurate risk estimates will become available . If lifetime risks for mutation carriers should indicate the need for screening, then presymptomatic dna testing can help in targeting this surveillance . In summary, although risk estimates for asymptomatic relatives in these families are not yet available, a clinically significant risk of developing mm cannot be excluded . In a research - more than one first - degree relative diagnosed with multiple myeloma, or - one first - degree and at least one second - degree relative with multiple myeloma annual protein electrophoresis to test immunoglobulins in blood and urine, starting at age 40 years . If families present with mm at a younger age, then screening could be started at say five years earlier than the youngest age at diagnosis in those families.
Image denoising is commonly used in medical imaging in order to help medical doctors to see abnormalities in the images . Image denoising was first applied to 2d images [13] and then extended to 3d data [46], 3d data can either be collected as several 2d images over time or as one 3d volume . A number of medical imaging modalities (e.g., computed tomography (ct), ultrasound (us) and magnetic resonance imaging (mri)) now provide the possibility to collect 4d data, that is, time - resolved volume data . This makes it possible to, for example, examine what parts of the brain that are active during a certain task (functional magnetic resonance imaging (fmri)). While 4d ct data makes it possible to see the heart beat in 3d, the drawback is that a lower amount of x - ray exposure has to be used for 4d ct data collection, compared to 3d ct data collection, in order to not harm the patient . When the amount of exposure is decreased, the amount of noise in the data increases significantly . Three - dimensional image denoising has previously been applied to several time points independently, but there has not been much work done on true 4d image denoising where the algorithm considers several volumes at the same time (and not a single volume at a time). Applied 4d anisotropic diffusion filtering to ultrasound volumes and jahanian et al . Applied 4d wavelet denoising to diffusion tensor mri data . For ct data, it can be extra beneficial to use the time dimension in the denoising, as some of the reconstruction artefacts vary with time . It is thereby possible to remove these artefacts by taking full advantage of the 4d data . While true 4d image denoising is very powerful, the drawback is that the processing time increases exponentially with respect to dimensionality . The rapid development of graphics processing units (gpus) has resulted in that many algorithms in the medical imaging domain have been implemented on the gpu, in order to save time and to be able to apply more advanced analysis . To give an example of the rapid gpu development, . Some examples of fields in medical imaging that have taken advantage of the computational power of the gpu are image registration [913], image segmentation [1416] and fmri analysis [1720]. In the area of image denoising, howison made a comparison between different gpu implementations of anisotropic diffusion and bilateral filtering for 3d data . Su and xu in 2010 proposed how to accelerate wavelet - based image denoising by using the gpu . Describe gpu - based image manipulation and enhancement techniques for dynamic volumetric medical image visualization, but enhancement in this case refers to enhancement of the visualization, and not of the 4d data . Recently, the gpu has been used for real - time image denoising . In 2007, chen et al . Used bilateral filtering on the gpu for real - time edge - aware image processing . Fontes et al . In 2011 used the gpu for real - time denoising of ultrasound data and goossens et al . In 2010 managed to run the commonly used nonlocal means algorithm in real time . To our knowledge, there has not been any work done about true 4d image denoising on the gpu . In this work we therefore present a novel algorithm, based on local adaptive filtering, for 4d denoising and describe how to implement it on the gpu, in order to decrease the processing time and thereby significantly increase the clinical value . In this section, the algorithm that is used for true 4d image denoising will be described . To show how a higher number of dimensions, the power of dimensionality, can improve the denoising result, the size of the 4d data is 127 127 9 9, but there is no signal variation in the last two dimensions . The data contains a large step, a thin line, and a shading from the top left corner to the bottom right corner . The denoising was done on one 127 127 image, for the 3d case, the denoising was done on one 127 127 9 volume and for the 4d case all the data was used . A single anisotropic lowpass filter was used for the denoising, and the filter had the same dimensionality as the data and was oriented along the structures . The original test data, the test data with noise and the denoising results are given in figure 1 . It was introduced for 2d by knutsson et al . In 1983 and then extended to 3d in 1992 . In this work, the main idea is to first estimate the local structure tensor (by using a first set of filters) in each neighbourhood of the data and then let the tensor control the reconstruction filters (a second set of filters). The term reconstruction should in this paper not be confused with the reconstruction of the ct data . The local structure tensor t is in 4d a 4 4 symmetric matrix in each time voxel, (1)t=(t1t2t3t4t2t5t6t7t3t6t8t9t4t7t9t10)=(xxxyxzxtxyyyyzytxzyzzzztxtytzttt), and contains information about the local structure in the data, that can be used to control the weights of the reconstruction filters . The result of the adaptive filtering is that smoothing is done along structures (such as lines and edges in 2d), but not perpendicular to them . Compared to more recently developed methods for image denoising (e.g., nonlocal means, anisotropic diffusion and bilateral filtering), adaptive filtering is in our case used for 4d image denoising for three reasons . Nonlocal means can give very good results, but the algorithm can be extremely time consuming (even if gpus are used). Bilateral filtering does not only require a multiplication for each filter coefficient and each data value, but also an evaluation of the intensity range function (e.g., an exponential) which is much more expensive to perform than a multiplication . Second, the tuning of the parameters is for our denoising algorithm rather easy to understand and to explore . When a first denoising result has been obtained, it is often obvious how to change the parameters to improve the result . Third, the adaptive filtering approach has been proven to be very robust (it is extremely seldom that a strange result is obtained). Adaptive filtering has been used for 2d image denoising in commercial clinical software for over 20 years and a recent 3d study proves its potential, robustness, and clinical acceptance . The nonlocal means algorithm only works if the data contains several neighbourhoods with similar properties . The local structure tensor can, for example, be estimated by using quadrature filters [5, 30]. Quadrature filters q are zero in one half of the frequency domain (defined by the direction of the filter) and can be expressed as two polar separable functions, one radial function r and one directional function d, (2)q(u)=r(\|\|u\|\|)d(u), where u is the frequency variable . The radial function is a lognormal function (3)r(\|\|u\|\|)=exp (c ln 2(\|\|u\|\|u0)), c=4b2ln (2), where u0 is the centre frequency of the filter and b is the bandwidth (in octaves). The directional function depends on the angle between the filter direction vector n^ and the normalized frequency coordinate vector u as cos (), (4)d(u)={(utn^)2,utn^>0,0,otherwise . Quadrature filters are cartesian nonseparable and complex valued in the spatial domain, the real part is even and in 2d acts as a line detector, while the imaginary part is odd and in 2d acts as an edge detector . In 3d, the even and odd filters correspond to a plane detector and a 3d edge detector . In 4d, the complex - valued filter response q is an estimate of a bandpass filtered version of the analytical signal with magnitude a and phase, (5)q = a(cos ()+isin ())=aei. The tensor is calculated by multiplying the magnitude of the quadrature filter response qk with the outer product of the filter direction vector n^k and then summing the result over all filters k, (6)t=k=1nf\|qk\|(c1n^kn^ktc2i), where c1 and c2 are scalar constants that depend on the dimensionality of the data [5, 30], nf is the number of quadrature filters and i is the identity matrix . The resulting tensor is phase invariant, as the magnitude of the quadrature filter response is invariant to the type of local neighbourhood (e.g., in 2d bright lines, dark lines, dark to bright edges, etc . ). This is in contrast to when the local structure tensor is estimated by using gradient operators, such as sobel filters . The number of filters that are required to estimate the tensor depends on the dimensionality of the data and is given by the number of independent components of the symmetric local structure tensor . The required number of filters is thus 3 for 2d, 6 for 3d and 10 for 4d . The given tensor formula, it is possible to spread 6 filters evenly in 3d, but it is not possible to spread 10 filters evenly in 4d . For this reason, 12 quadrature filters have to be used in 4d (i.e., a total of 24 filters in the spatial domain, 12 real valued and 12 complex valued). To apply 24 nonseparable filters to a 4d dataset requires a huge number of multiplications . In this paper a new type of filters, monomial filters, monomial filters also have one radial function r and one directional function d. the directional part of the monomial filters are products of positive integer powers of the components of the frequency variable u. the monomial filter matrices of order one, f1, and two, f2, are in the frequency domain defined as (7)f1,n = r(\|\|u\|\|)u^n, f2,mn = r(\|\|u\|\|)u^mu^n . The monomial filters are first described for 2d and then generalized to 4d . In 2d, the frequency variable is in this work defined as u = [u v]. The directional part of first - order monomial filters are x, y in the spatial domain and u, v in the frequency domain . Two - dimensional monomial filters of the first - order are given in figure 2 . The directional part of second - order monomial filters are xx, xy, yy in the spatial domain and uu, uv, vv in the frequency domain . The monomial filter response matrices q are either calculated by convolution in the spatial domain or by multiplication in the frequency domain . For a simple signal with phase (e.g., s(x) = acos (ux +)); the monomial filter response matrices of order one and two can be written as (8)q1=iasin ()[uv]t, q2=acos ()(uuuvuvvv). The first - order products are odd functions and are thereby related to the odd sine function, the second order products are even functions and are thereby related to the even cosine function (note the resemblance with quadrature filters that have one even real part and one odd imaginary part). By using the fact that u + v = 1, the outer products of the filter response matrices give (9)q1q1t = sin 2()\|a\|2(uuuvuvvv),q2q2t = cos 2()\|a\|2(uuuvuvvv). The local structure tensor t, it is clear that the estimated tensor, as previously, is phase invariant as the square of one odd part and the square of one even part are combined . For information about how to calculate a total of 14 nonseparable 4d monomial filters (4 odd of the first - order (x, y, z, t) and 10 even of the second - order (xx, xy, xz, xt, yy, yz, yt, zz, zt, tt)) with a spatial support of 7 7 7 7 time voxels are applied to the ct volumes . The filters have a lognormal radial function with centre frequency 3/5 and a bandwidth of 2.5 octaves . The filter kernels were optimized with respect to ideal frequency response, spatial locality, and expected signal - to - noise ratio [5, 33]. By using equation (10) for the 4d case, and replacing the frequency variables with the monomial filter responses, the 10 components of the structure tensor are calculated according to (11)t1=fr1fr1+fr5fr5+fr6fr6+fr7fr7 + fr8fr8,t2=fr1fr2+fr5fr6+fr6fr9+fr7fr10 + fr8fr11,t3=fr1fr3+fr5fr7+fr6fr10+fr7fr12 + fr8fr13,t4=fr1fr4+fr5fr8+fr6fr11+fr7fr13 + fr8fr14,t5=fr2fr2+fr6fr6+fr9fr9+fr10fr10 + fr11fr11,t6=fr2fr3+fr6fr7+fr9fr10+fr10fr12 + fr11fr13,t7=fr2fr4+fr6fr8+fr9fr11+fr10fr13 + fr11fr14,t8=fr3fr3+fr7fr7+fr10fr10+fr12fr12 + fr13fr13,t9=fr3fr4+fr7fr8+fr10fr11+fr12fr13 + fr13fr14,t10=fr4fr4+fr8fr8+fr11fr11+fr13fr13 + fr14fr14, where frk denotes the filter response for monomial filter k. the first term relates to q1q1, and the rest of the terms relate to q2q2, in total q1q1 + q2q2 . If monomial filters are used instead of quadrature filters, the required number of 4d filters is thus decreased from 24 to 14 . Another advantage is that the monomial filters require a smaller spatial support, which makes it easier to preserve details and contrast in the processing . A smaller spatial support also results in a lower number of filter coefficients, which decreases the processing time . When the local structure tensor t has been estimated, it is then mapped to a control tensor c, by mapping the magnitude (energy) and the isotropy of the tensor . The purpose of this mapping is to further improve the denoising . For 2d and 3d image denoising, this mapping can be done by first calculating the eigenvalues and eigenvectors of the structure tensor in each element of the data . The mapping is first described for 2d and then for 4d . In the 2d case, the magnitude 0 of the tensor is calculated as (12)0=12+22, where 1 and 2 are the two eigenvalues . The magnitude 0 is normalized to vary between 0 and 1 and is then mapped to with a so - called m - function according to (13)=(0 0 + +), where,, and are parameters that are used to control the mapping . The variable is directly proportional to the signal - to - noise (snr) ratio of the data and acts as a soft noise threshold, mainly controls the overshoot (that can be used for dynamic range compression or to amplify areas that have a magnitude slightly above the noise threshold), and mainly controls the slope / softness of the curve . The purpose of this mapping is to control the general usage of highpass information . The highpass information should only be used where there is a well - defined structure in the data . If the magnitude of the structure tensor is low, one can assume that the neighbourhood only contains noise . The isotropy 0 is in 2d calculated as (14)0=21 and is mapped to with a so called mu - function according to (15)=(0(1))(0(1))+((10)), where and are parameters that are used to control the mapping, mainly controls the transition of the curve and mainly controls the slope / softness . The purpose of this mapping is to control the usage of highpass information in the nondominant direction, that is, the direction that is given by the eigenvector corresponding to the smallest eigenvalue . This is done by making the tensor more isotropic if it is slightly isotropic, or making it even more anisotropic if it is anisotropic . The control tensor c is finally calculated as (16)c=e1e1t+e2e2 t, where e1 is the eigenvector corresponding to the largest eigenvalue 1 and e2 is the eigenvector corresponding to the smallest eigenvalue 2 . The mapping thus preserves the eigensystem, but changes the eigenvalues and thereby the shape of the tensor . For matrices of size 2 2 and 3 3, there are direct formulas for how to calculate the eigenvalues and eigenvectors, but for 4 4 matrices, there are no such formulas and this complicates the mapping . It would of course be possible to calculate the eigenvalues and eigenvectors by other approaches, such as the power iteration algorithm, but this would be extremely time consuming as the mapping to the control tensor has to be done in each time voxel . The mapping of the local structure tensor to the control tensor is in this work therefore performed in a way that does not explicitly need the calculation of eigenvalues and eigenvectors . The tensor magnitude is first calculated as (17)tmag=\|\|t8\|\|1/8, where \|\|\|\| denotes the frobenius norm . The exponent will determine how close to 1 the estimated tensor magnitude will be; a higher exponent will give better precision, but an exponent of 8 has proven to be sufficient in practice . To reduce the computational load, t is calculated as (18)t2=tt, t4=t2t2,t8=t4t4, where denotes matrix multiplication . 0 is then calculated as (19)0=tmagmax (tmag), where the max operator is for the entire data set, such that the maximum value of 0 will be 1, 0 is then mapped to by using the m - function . To map the isotropy, the structure tensor is first normalized as (20)t^=ttmag, such that the tensor only carries information about the anisotropy (shape). The fact that t^ and i - t^ have the same eigensystem is used, such that the control tensor can be calculated as (21)c=(i+(1)t^), where i is the identity matrix . The following formulas are an ad hoc modification of this basic idea, that do not explicitly need the calculation of the isotropy and that give good results for our ct data . The basic idea is that the ratio of the eigenvalues of the tensor change when the tensor is multiplied with itself a number of times, and thereby the shape of the tensor also changes . This approach does not give exactly the same results as the original isotropy mapping, but it circumvents the explicit calculation of eigenvalues and eigenvectors . A help variable t^f is first calculated as (22)t^f = t^2(i+2(it^)), and then the control tensor c is calculated as (23)c= (i(it^f)8(i+8t^f)). The resulting transfer function that maps each eigenvalue is given in figure 7 . Eigenvalues that are small become even smaller, and eigenvalues that are large become even larger . The result of this eigenvalue eleven nonseparable reconstruction filters, one lowpass filter h0 of the zeroth order and 10 highpass filters h2,mn of the second order, with a spatial support of 11 11 11 11 time voxels are applied to the ct volumes . The denoised 4d data i d is calculated as the sum of the lowpass - filtered data, ilp, and the highpass filtered data for each highpass - filter k, ihp(k), weighted with the components ck of the control tensor c, (24)id = ilp+k=110ckihp(k). The result is that the 4d data is lowpass filtered in all directions and then highpass information is put back where there is a well - defined structure . Highpass information is put back in the dominant direction of the local neighbourhood (given by the eigenvector related to the largest eigenvalue) if the tensor magnitude is high . Highpass information is put back in the nondominant directions (given by the eigenvectors related to the smaller eigenvalues) if the tensor magnitude is high and the anisotropy is low . All the processing steps of the denoising algorithm are given in table 2 . In our case the ct data does not contain any significant structural information in the frequencies over /2 in the spatial dimensions, the volumes are therefore lowpass filtered and then downsampled a factor 2 in x, y, z. when the local structure tensor has been estimated, it is lowpass filtered, with a separable lowpass filter of size 5 5 5 3, to improve the estimate in each time voxel and to make sure that the resulting reconstruction filter varies smoothly . Note that this smoothing does not decrease the resolution of the image data, but only the resolution of the tensor field . After the tensor mapping, the control tensor is interpolated to the original resolution of the ct data . While the presented algorithm is straightforward to implement, spatial filtering with 11 reconstruction filters of size 11 11 11 11 (14 641 filter coefficients) applied to a dataset of the resolution 512 512 445 20 requires about 375 000 billion multiplications . This is the reason why the gpu is needed in order to do the 4d denoising in a reasonable amount of time . One of the main drawbacks of the presented algorithm is that, using standard convolution, the number of valid elements in the z - direction (i.e., slices) decreases rapidly . If the algorithm is applied to a dataset of the resolution 512 512 34 20, two slices are first lost due to the convolution with the lowpass filter of size 3 3 3 . The monomial filters are of size 7 7 7 7, thereby only 10 of the filter response slices are valid . During the lowpass filtering of each structure tensor component, another four slices are lost and then another four are lost during lowpass filtering of the control tensor . The result is thus that only 2 valid slices are left after all the convolutions . The same problem could exist in the time dimension, but since the heart cycle is periodic it is natural to use circular convolution in the time direction, and thereby all the time points are valid . The loss of valid slices can be avoided by using normalized convolution, both for the lowpass filtering of the data before downsampling and the lowpass filtering of the tensor components . In normalized convolution, a certainty a certainty - weighted filter response cwr is calculated as (25)cwr=(cs)fcf, where c is the certainty, s is the signal, f is the filter, denotes pointwise multiplication, and denotes convolution . Note that this simple version of normalized convolution (normalized averaging) cannot be applied for the monomial filters and for the reconstruction filters, as these filters have both negative and positive coefficients . It is possible to apply the full normalized convolution approach for these filters, but it will significantly increase the computational load . In this section, the gpu implementation of the denoising algorithm will be described . The cuda (compute unified device architecture) programming language by nvidia, explained by kirk and hwu, has been used for the implementation . The open computing language (opencl) could be a better choice, as it makes it possible to run the same code on any hardware . The cuda programming language can easily generate 2d indices for each thread, for example, by using algorithm 1 . To generate 3d indices is harder, as each thread block can be three dimensional but the grid can only be two dimensional . To generate 4d indices is even more difficult . To navigate in the 4d data, the 3d indexing approach described above is used, and the kernel is then called once for each time point . Fast - fourier - transform (fft-) based filtering can be very efficient when large nonseparable filters of high dimension are to be applied to big datasets, but spatial filtering is generally faster if the filters are small or cartesian separable . The main advantage with fft - based filtering is that the processing time is the same regardless of the spatial size of the filter . The main disadvantage with fft - based filtering is however the memory requirements, as the filters need to be stored in the same resolution as the data, and also as a complex - valued number for each element . To see which kind of filtering that fits the gpu best, both spatial and fft - based filtering was therefore implemented . For filtering with the small separable lowpass filters (which are applied before the data is downsampled and to smooth the tensor components), one easy way to implement 2d and 3d filtering on the gpu is to take advantage of the cache of the texture memory and put the filter kernel in constant memory . The drawback with this approach is however that the implementation will be very limited by the memory bandwidth, and not by the computational performance . Another problem is that it is not possible to use 4d textures in the cuda programming language . One would have to store the 4d data as one big 1d texture or as several 2d or 3d textures . A better approach is to take advantage of the shared memory, which increased a factor 3 in size between the nvidia gtx 285 and the nvidia gtx 580 . The data is first read into the shared memory and then the filter responses are calculated in parallel . By using the shared memory, the threads can share the data in a very efficient way, which is beneficial as the filtering results for two neighbouring elements are calculated by mainly using the same data . As multidimensional filters can be separable or nonseparable (the monomial filters and the reconstruction filters are nonseparable, while the different lowpass filters are separable) two different spatial filtering functions were implemented . Our separable 4d convolver is implemented by first doing the filtering for all the rows, then for all the columns, then for all the rods and finally for all the time points . The data is first loaded into the shared memory and then the valid filter responses are calculated in parallel . For the four kernels, 16 kb of shared memory is used such that 3 thread blocks can run in parallel on each multiprocessor on the nvidia gtx 580 . The shared memory approach works rather well for nonseparable 2d filtering but not as well for nonseparable 3d and 4d filtering . The size of the shared memory on the nvidia gtx 580 is 48 kb for each multiprocessor, and it is thereby only possible to, for example, fit 11 11 11 9 float values into it . If the 4d filter is of size 9 9 9 9, only 3 3 3 1 = 27 valid filter responses can be generated for each multiprocessor . A better approach for nonseparable filtering in 4d is to instead use an optimized 2d filtering kernel, and then accumulate the filter responses by summing over the other dimensions by calling the 2d filtering function for each slice and each time point of the filter . Our nonseparable 2d convolver first reads 64 64 pixels into the shared memory, then calculates the valid filter responses for all the 14 monomial filters or all the 11 reconstruction filters at the same time, and finally writes the results to global memory . Two versions of the convolver were implemented, one that maximally supports 7 7 filters and one that maximally supports 11 11 filters . The first calculates 58 58 valid filter responses, and the second calculates 54 54 valid filter responses . As 64 64 float values only require 16 kb of memory, three thread blocks can run at the same time on each multiprocessor . This results in 58 58 3 = 10092 and 54 54 3 = 8748 valid filter responses per multiprocessor . For optimal performance, the 2d filtering loop was completely unrolled by generating the code with a matlab script . The 14 monomial filters are of size 7 7 7 7, this would require 135 kb of memory to be stored as floats, but the constant memory is only 64 kb . For this reason, 7 7 filter coefficients are stored at a time and are then updated for each time point and for each slice . It would be possible to store 7 7 7 filter coefficients at a time, but by only storing 7 7 coefficients, the size of the filters (2.75 kb) is small enough to always be in the cache of the constant memory (8 kb). The same approach is used for the 11 reconstruction filters of size 11 11 11 11 . While the cufft library by nvidia supports 1d, 2d, and 3d ffts, there is no direct support for 4d ffts . As the fft is cartesian separable, it is however possible to do a 4d fft by applying four consecutive 1d ffts . The cufft library supports launching a batch of 1d ffts, such that many 1d fft's can run in parallel . The batch of 1d ffts are applied along the first dimension in which the data is stored (e.g., along x if the data is stored as (x, y, z, t)). Between each 1d fft, it is thereby necessary to change the order of the data (e.g., from (x, y, z, t) to (y, z, t, x)). The drawback with this approach is that the time it takes to change order of the data can be longer than to actually perform the 1d fft . The most recent version of the cufft library supports launching a batch of 2d fft's . By applying two consecutive 2d fft's, it is sufficient to change the order of the data once, instead of three times . A filter is padded with zeros to the same resolution as the data and is then transformed to the frequency domain . To do the filtering, a complex - valued multiplication between the data and the filter is applied and then an inverse 4d fft is applied to the filter response . After the inverse transform, a fft shift is necessary; there is however no such functionality in the cufft library . When the tensor components and the denoised data are calculated, each of the four coordinates is shifted by using a help function, see algorithm 4 . As the monomial filters only have a real part or an imaginary part in the spatial domain, some additional time is saved by putting one monomial filter in the real part and another monomial filter in the imaginary part before the 4d fft is applied to the zero - padded filter . When the complex multiplication is performed in the frequency domain, two filters are thus applied at the same time . After the inverse 4d fft, the first filter response is extracted as the real part and second filter response is extracted as the imaginary part . The main problem of implementing the 4d denoising algorithm on the gpu is the limited size of the global memory (3 gb in our case). This is made even more difficult by the fact that the gpu driver can use as much as 100200 mb of the global memory . Storing all the ct data on the gpu at the same time is not possible, a single ct volume of the resolution 512 512 445 requires about 467 mb of memory if 32 bit floats are used . Storing the filter responses is even more problematic . To give an example, to store all the 11 reconstruction filter responses as floats for a dataset of the size 512 512 445 20 would require about 103 gb of memory . The denoising is therefore done for a number of slices (e.g., 16 or 32) at a time . For the spatial filtering, the algorithm is started with data of the resolution 512 512 51 20 and is downsampled to 256 256 26 20 . The control tensor is calculated for 256 256 20 20 time voxels, and the denoised data is calculated for 512 512 39 20 time voxels . To process all the 445 slices the algorithm is started with data of the resolution 512 512 31 20 and is downsampled to 256 256 16 20 . The control tensor is then calculated for 256 256 10 20 time voxels, and the denoised data is calculated for 512 512 18 20 time voxels . To process all the 445 slices requires 26 runs . To store the 10 components of the control tensor in the same resolution as the original ct data for one run with spatial filtering (512 512 39 20) would require about 12.2 gb of memory . As the control tensor needs to be interpolated a factor 2 in each spatial dimension, since it is estimated on downsampled data, another approach is used . Interpolating the tensor is a perfect task for the gpu, due to the hardware support for linear interpolation . The 10 tensor components, for one timepoint, are therefore stored in 10 textures and then the interpolation is done on the fly when the denoised data is calculated . By using this approach, only another 10 variables of the resolution 256 256 20 need to be stored at the same time . Table 2 states the in and out resolution of the data, the used equations, and the memory consumption at each step of the denoising algorithm, for spatial filtering and fft - based filtering . The out resolution refers to the resolution of the data that is valid after each processing step, as some data is regarded as non - valid after filtering operations . The reason why the memory consumption is larger for the fft - based filtering is that the spatial filtering can be done for one slice or one volume at a time, while the fft - based filtering has to be applied to a sufficiently large number of slices and time points at the same time . We were not able to use more than about 2 gb of memory for the fft - based filtering; one reason for this might be that the cufft functions internally use temporary variables that use some of the memory . Since the source code for the cufft library is unavailable, it is hard to further investigate this hypothesis . The 4d ct dataset that was used for testing our gpu implementation was collected with a siemens somatom definition flash dual - energy ct scanner at the center for medical image science and visualization (cmiv). The dataset contains almost 9000 dicom files and the resolution of the data is 512 512 445 20 time voxels . The spatial size of each voxel is 0.75 0.75 0.75 mm . During the image acquisition the tube current is modulated over the cardiac cycle with reduced radiation exposure during the systolic heart phase . Due to this, the amount of noise varies with time . A comparison between the processing times for our gpu implementation and for a cpu implementation was made . The used gpu was a nvidia gtx 580, equipped with 512 processor cores and 3 gb of memory (the nvidia gtx 580 is normally equipped with 1.5 gb of memory). The used cpu was an intel xeon 2.4 ghz with 4 processor cores and 12 mb of l3 cache, 12 gb of memory was used ., the openmp (open multiprocessing) library [38, 39] was used, such that all the 4 processor cores work in parallel . No other types of optimization for the cpu, such as sse2, were used . We are fully aware of the fact that it is possible to make a much better cpu implementation . The purpose of this comparison is rather to give an indication of the performance of the cpu and the gpu . If the cpu code would be vectorized, the cpu processing times can be divided by a factor 3 or 4 (except for the fft which already is very optimized). The processing times are given in tables 3, 4, 5, and 6 . The cpu processing times for the spatial filtering are estimates, since it takes several days to run the algorithm on the whole dataset . The processing times for a multi - gpu implementation would scale rather linearly with the number of gpus, since each gpu can work on different subsets of slices in parallel . As our computer contains three gpus, all the processing times for the gpu can thereby be divided by a factor 3 . To show the results of the 4d denoising, the original ct data was compared with the denoised data by applying volume rendering . Two volume renderers, one for the original data and one for the denoised data, run at the same time and were synced in terms of view angle and transfer function . Figure 8 shows volume renderings of the original and the denoised data for different time points and view angels . It is clear that a lot of noise is removed by the denoising, but since the denoising algorithm alters the histogram of the data, it is hard to make an objective comparison even if the same transfer function is applied . A movie where the original and the denoised data is explored with the two volume renderers was also made . For this video, the data was downsampled a factor 2 in the spatial dimensions, in order to decrease the memory usage . The video can be found at http://www.youtube.com/watch?v=wflbt2sv34m . By looking at the video, it is easy to see that the amount of noise in the original data varies with time . The result is that 4d image denoising becomes practically possible if the gpu is used and thereby the clinical value increases significantly . To make a completely fair comparison between the cpu and it has been debated if the gpu speedups that have been reported in the literature are plausible or if they are the result of comparisons with unoptimized cpu implementations . In our opinion, the theoretical and practical processing performance that can be achieved for different hardware is not the only interesting topic . In a research environment, the ratio between the achievable processing performance and the time it takes to do the implementation is also important . From this perspective, we think that our cpu - gpu comparison is rather fair, since about the same time was spent on doing the cpu and the gpu implementation . The cuda programming language was designed and developed for parallel calculations from the beginning, while different addons have been added to the c programming language to be able to do parallel calculations . While it is rather easy to make the cpu implementation multithreaded, for example, by using the openmp library, more advanced cpu optimization is often more difficult to include and often requires assembler programming . While spatial filtering can be significantly slower than fft - based filtering for nonseparable filters, there are some advantages (except for the lower memory usage). One is that a region of interest (roi) can be selected for the denoising, compared to doing the denoising on the whole dataset . Another advantage is that filter networks [41, 42] can be applied, such that the filter responses from many small filters are combined to the same filter response as from one large filter . Filter networks can reduce the number of multiplications as much as a factor 5 in 2d, 25 in 3d and 300 in 4d . To design and optimize a filter network another problem is that the memory usage increases significantly when filter networks are used, since many filter responses need to be stored in memory . Filter networks on the gpu is a promising area for future research . From our results, it is clear that fft - based filtering is faster than spatial filtering for large nonseparable filters . For data sizes that are not a power of two in each dimension, the fft based since medical doctors normally do not look at 3d or 4d data as volume renderings, but rather as 2d slices, the spatial filtering approach however has the advantage that the denoising can be done for a region of interest (e.g., a specific slice or volume). It is a waste of time to enhance the parts of the data that are not used by the medical doctor . The spatial filtering approach can also handle larger datasets than the fft - based approach, as it is sufficient to store the filter responses for one slice or one volume at a time . Recently, we acquired a ct data set with 100 time points, compared to 20 time points . It is not possible to use the fft - based approach for this data set . There are several reasons why the gpu speedup for the fft - based filtering is much smaller than the gpu speedup for the spatial filtering . First, the cufft library does not include any direct support for 4d fft's, and we had to implement our own 4d fft as two 2d fft's that are applied after each other . Between the 2d fft's the storage order of the data it can take a longer time to change the order of the data than to actually perform the fft . If nvidia includes direct support for 4d fft's in the cufft library, we are sure that their implementation would be much more efficient than ours . Second, the fft for the cpu is extremely optimized, as it is used in a lot of applications, and our convolver for the cpu is not fully optimized . The cuda programming language is only a few years old, and the gpu standard libraries are not as optimized as the cpu standard libraries . [44, 45] have created their own gpu fft library which has been proven to give better performance than the cufft library . They circumvent the problem of changing the order of the data and thereby achieve an implementation that is much more efficient . In 2008, their 3d fft was 5 - 6 times faster than the 3d fft in the cufft library . Third, due to the larger memory requirements of fft - based filtering it is not possible to achieve an as big speedup for the gpu implementation as for the cpu implementation . If a gpu with a higher amount of global memory would have been used, the fft - based implementation would have been more efficient . As previously discussed in the paper, 4d image processing in cuda is harder to implement than 2d and 3d image processing . There are, for example, no 4d textures, no 4d ffts, and there is no direct support for 4d (or 3d) indices . However, since fmri data also is 4d, we have previously gained some experience on how to do 4d image processing with cuda [1820]. The conclusions that we draw after implementing the 4d image denoising algorithm with the cuda programming language is thus that cuda is not perfectly suited for 4d image processing, but due to its flexibility, it was still possible to implement the algorithm rather easily . It might seem impossible to have medical image data with more than 4 dimensions, but some work has been done on how to collect 5d data . The five dimensions are the three spatial dimensions and two time dimensions, one for the breathing rhythm and one for the heart rhythm . One major advantage with 5d data is that the patient can breathe normally during the data acquisition, while the patient has to hold its breath during collection of 4d data . With 5d data, it is possible to, for example, fixate the heart and only see the lungs moving, or fixate the lungs to only see the heart beating . If the presented algorithm would be extended to 5d, it would be necessary to use a total of 20 monomial filters and 16 reconstruction filters . For a 5d dataset of the size 512 512 445 20 20, the required number of multiplications for spatial filtering with the reconstruction filters would increase from 375 000 billion for 4d to about 119 million billion (1.19 10) for 5d . The size of the reconstruction filter responses would increase from 103 gb for 4d to 2986 gb for 5d . This is still only one dataset for one patient, and we expect that both the spatial and the temporal resolution of all medical imaging modalities will increase even further in the future . Except for the 5 outer dimensions, it is also possible to collect data with more than one inner dimension . This is, for example, the case if the blood flow of the heart is to be studied . For flow data, a three - dimensional vector needs to be stored in each time voxel, instead of a single intensity value . To conclude, by using the gpu, true 4d image denoising becomes practically feasible . Our implementation can of course be applied to other modalities as well, such as ultrasound and mri, and not only to ct data . The short processing time also makes it practically possible to further improve the denoising algorithm and to tune the parameters that are used . The elapsed time between the development of practically feasible 2d and 3d image denoising techniques was about 10 years, from 3d to 4d the elapsed time was about 20 years . Due to the rapid development of gpus,
Igg4-related disease (igg4-rd) is a recently described systemic fibroinflammatory disease associated with elevated circulating levels of igg4 [14]. The pathologic lesion of igg4-rd is characterized by lymphoplasmacytic inflammation with increased numbers of igg4-positive plasma cells, fibrosis, and phlebitis . Although initial descriptions of this disorder focused on its pancreatic presentation (autoimmune pancreatitis), it has become apparent that igg4-rd is a systemic disease associated with a wide spectrum of clinical manifestations involving virtually any organ in the body . As initially observed in patients with autoimmune pancreatitis, the majority of patients with igg4-rd have an elevated serum igg4 level . Although serum igg4 level has been described to be the most sensitive and specific laboratory test for the diagnosis of igg4-rd, it is recognized that an elevated serum igg4 level can be encountered in other diseases such as pancreatic cancer, atopic diseases, and infections [7, 8]. Furthermore, serum igg4 level is elevated in up to 5% of the normal population [9, 10]. In this study, we sought to identify the spectrum of diseases associated with elevated serum igg4 levels in patients encountered in clinical practice and the frequency of igg4-rd in this population . Using a computer - assisted search, we identified all patients who had serum igg subclass levels determined on one or more occasions at mayo rochester, mn, usa, during the 2-year period from january 1, 2009 to december 31, 2010 and selected those with an elevated serum igg4 concentration (> 140 mg / dl) for analysis . The concentrations of igg subclass proteins in serum were measured in the mayo clinic clinical laboratory by automated nephelometry in which the concentrations of each protein were determined from standard curves . Human igg4 latex reagent (binding site group ltd, birmingham, uk) was used in quantifying the serum igg4 concentration . Medical records of those patients with elevated serum igg4 levels were reviewed to extract data regarding age, sex, clinical presentation, serum igg4 level, indication for serum igg subclass determination, imaging and biopsy results, and diagnoses . The main diagnosis that resulted from evaluation of the presenting clinical issue at the time of the serum igg subclass testing was identified . For this study, definite diagnosis of igg4-rd required the following criteria in addition to the known serum igg4 elevation: (1) clinical and/or radiologic evidence of lesions consistent with igg4-rd in one or more organs as previously described in the literature and (2) igg4 staining showing greater than 10 igg4 cells / high - power field and igg4/igg ratio greater than 40% in the presence of lymphoplasmacytic infiltration and fibrosis . Patients who fulfill the organ - specific criteria for igg4-related autoimmune pancreatitis, igg4-related mikulicz's disease, and igg4-related kidney disease were also designated as having definite igg4-rd . Those patients clinically diagnosed based on clinical and imaging features along with an elevated serum igg4 level but not fulfilling histopathologic criteria outlined above or in the absence of tissue biopsy and exhibiting improvement with corticosteroid therapy were designated as possible none of our patients met the criteria for probable igg4-rd outlined by umehara and colleagues . Non - igg4-rd diagnoses were determined based on the results of the diagnostic evaluation, the clinicians' diagnostic impression, and the subsequent clinical course . Approval was obtained from the mayo foundation institutional review board prior to beginning the study . Data are presented as mean sd and percentages for categorical variables unless stated otherwise . Means of continuous variables were compared between groups with a two - sample t - test . Serum igg4 levels between groups were compared using the wilcoxon rank - sum test . In all cases p - values we identified 3,300 consecutive patients who had their serum igg subclass testing performed on one or more occasions during the 2-year interval from january 1, 2009 to december 31, 2010; 158 patients (4.8%) had at least one high serum igg4 level (> 140 mg / dl). The demographic features and serum igg4 level of these 158 patients are outlined in table 1 . Indications for igg subclass testing were evaluation for possible igg4-rd in 104 patients (65.8%) and to assess for immunodeficiency (e.g., patients with recurrent or chronic infections) in 54 patients (34.2%). Twenty - nine patients (18.4%) met the criteria for definite or possible igg4-rd (table 2). The mean age of those with igg4-rd was older compared to those without igg4-rd (58.3 16.9 versus 49.9 20.8, p <0.05) but the gender distribution was not different (p = 0.29). The serum igg4 level was significantly higher in those with igg4-rd compared to those with non - igg4-rd diagnoses (p <0.001) (table 1). Furthermore, mean serum igg4 level was higher for those with definite igg4-rd (940 990) compared to possible igg4-rd (329 318) which, in turn, was higher compared to non - igg4-rd subgroup (226 127) (p <0.05) (figure 1). The mean serum igg4/igg ratio was significantly higher in patients with igg4-rd (definite and possible) compared to non - igg4-rd patients (0.263 0.239 versus 0.148 0.061, p <0.01), but there was substantial overlap in individual values between the two groups as shown in figure 2 . Among 29 patients with igg4-rd, 10 patients met the criteria for definite igg4-rd which included supportive histopathologic findings on tissue biopsy . Of 19 patients with possible igg4-rd, 12 had undergone biopsy procedures but the biopsy findings did not meet the criteria for definite or probable igg4-rd diagnosis . In the remaining 129 patients who did not have igg4-rd, the most common diagnoses were primary sclerosing cholangitis, bronchiectasis, non - igg4-related pancreatitis, vasculitis, chronic rhinosinusitis, and pancreatic or bile duct cancer . No specific diagnosis was achieved in 29 patients; most common presenting complaints in these patients included abdominal pain, fevers, and lymphadenopathy . Pancreas, bile ducts, and orbital structures were most commonly involved . However, these patients exhibited involvement in a number of other organs including the salivary glands, retroperitoneal region, lymph nodes, kidney, lung, pleura, sinuses, gastrointestinal tract, and testis . Patients with two or more organ involvements (n = 13) had a higher mean serum igg4 level (840 914) compared to those with single organ involvement (296 250) (p <0.01) (figure 3). All except one patient (surgical resection of a biliary lesion) with igg4-rd (definite or possible) were treated with prednisone and improved . Twelve of 28 patients initially treated with prednisone experienced subsequent relapses requiring reinstitution of prednisone alone or in combination with another immunosuppressive agent (including azathioprine, methotrexate, and rituximab). Median duration of followup was 23 months (range, 1 to 126 months). In this study we found a broad spectrum of diagnoses to be associated with elevated serum igg4 levels encountered in the clinical practice setting; less than one - fifth of these patients manifested evidence for igg4-rd . Igg4 subclass testing was ordered by clinicians for evaluation of possible igg4-rd or immunodeficiency, and thus it is not surprising that various types of non - igg4-related pancreatic and biliary tract disorders were included along with chronic infections, for example, bronchiectasis and sinusitis . At the present time, there is no published international consensus on the diagnostic criteria for igg4-rd . Most authors agree that definitive diagnosis of igg4-rd requires histologic confirmation that includes the presence of characteristic histopathologic features (lymphoplasmacytic infiltration, fibrosis, and obliterative phlebitis or arteritis) along with immunostaining that demonstrates increased numbers of igg4 cells . Various authors have used different cutoffs for igg4 staining criteria . For the purposes of this study, we used the diagnostic criteria recently published by umehara and colleagues for definite, probable, and possible, there are patients in whom the diagnosis of igg4-rd is likely and are empirically treated without biopsy confirmation . This may occur when patients decline invasive procedures or the initial biopsy specimen is nondiagnostic and additional biopsies are not pursued for based on patient preference, perceived risks, or lack of any other likely diagnosis . All patients included in this analysis exhibited elevated serum igg4 levels and the degree of this elevation was not used in determining the presence or absence of igg4-rd . Our patients with igg4-rd exhibited a significantly higher serum igg4 levels compared to those without igg4-rd although there was an overlap in their serum igg4 values . Some authors have suggested that a serum igg4 concentration that is more than twice the upper limit of normal (> 280 mg / dl) is highly specific for igg - rd . In this study 13 of 29 patients (45%) with igg4-rd and 18 of 129 patient (14%) without igg4-rd had a serum igg4 concentration that was> 280 mg / dl . The sensitivity of elevated serum igg4 levels in the diagnosis of igg4-rd has been reported to be in the range of 67% to 95% and specificity to be 90% to 97% [1, 2, 10, 1317]. Serum igg4 elevation is present in 5% of the normal population [9, 10] and has been observed in patients with other disorders . For example, serum igg4 levels have been reported to be elevated in 7% to 10% of patients with pancreatic cancer [5, 13]. Similarly, serum igg4 elevation has been seen in 5% to 9% of patients with other forms of pancreatitis and benign pancreatic tumors . These include skin diseases including atopic dermatitis and pemphigus vulgaris [18, 19], as well as parasitic diseases [7, 8]. Our study cohort included one patient with atopic dermatitis, but none had pemphigus or parasitic diseases . Van nieuwkoop and colleagues had previously described an association between a polyclonal increase in serum igg4 subclass with acquired respiratory diseases . Bronchiectasis and chronic rhinosinusitis were most respiratory disorders in our study cohort likely reflecting the fact that suspected immunodeficiency was one of the main indications for igg subclass testing in this population . Primary sclerosing cholangitis (psc) was the single most common diagnosis in the non - igg4-rd group . Elevated serum igg4 levels have been described in 9% to 12% of patients with psc [21, 22]. Since differentiation of autoimmune pancreatitis from pancreatic cancer and biliary tract disease is a common clinical indication for igg subclass testing, it seems reasonable to expect that patients with various types of biliary tract diseases including psc and cholangiocarcinoma will be encountered in those patients with elevated serum igg4 levels . Vascular involvement has been seen in igg4-rd mainly in the form of aortitis, periaortitis, and inflammatory abdominal aortic aneurysm [2, 23, 24]. Recently, elevated serum igg4 levels have been reported in patients with churg - strauss syndrome [25, 26] and hypocomplementemic urticarial vasculitis . Additionally, igg4 antiproteinase 3 autoantibodies have been demonstrated to stimulate neutrophils to undergo a proinflammatory response suggesting potential relevance in the pathogenesis of granulomatosis with polyangiitis (wegener's). In this regard, it is interesting to note that nine patients in our study cohort had systemic vasculitis including granulomatosis with polyangiitis and the churg - strauss syndrome . Prevalence of high serum igg4 level in patients with anca - associated vasculitis and the relevance of igg4 in the pathogenesis of these disorders need to be explored further . Although igg4-related hepatopathy and hepatic inflammatory pseudotumors have been described [2931], none of our patients with liver disease and elevated serum igg4 levels fulfilled the criteria for igg4-rd . They had other identifiable causes including hepatitis c, drugs, sarcoidosis, and primary biliary cirrhosis . As previously noted, serum igg4 level is elevated in 5% of the normal population [9, 10]. Similarly, 4.8% of 3,300 consecutive patients undergoing serum igg subclass testing at our institution over a 2-year period exhibited an elevated serum igg4 level . It seems reasonable to assume that elevated serum igg4 level will be found coincidentally in a small portion of various disease populations that are subjected to igg subclass testing, for example, patients with bronchiectasis . None of our 54 patients with high serum igg4 level who had undergone igg subclass testing for the indication of suspected immunodeficiency had evidence of igg4-rd . It appears unlikely that there is a causal relationship between the elevated serum igg4 level and many of the diseases listed in table 2 . It remains to be determined whether elevated serum igg4 level is a relevant finding in other disorders other than those recognized currently as igg4-rd . This study was a retrospective survey with analysis limited to the clinical data available in medical records and imaging studies . The diagnostic evaluation for these patients was performed by various clinicians at our institution according to their own clinical judgment and patient context . It is possible that some cases of igg4-rd may have been missed particularly in those patients without a specific diagnosis . In addition, the extent of organ involvement may have been underestimated in patients with igg4-rd due to lack of relevant imaging studies or biopsy specimens in the absence of standard methodical evaluation . We conclude that only a minority of patients with elevated serum igg4 levels encountered in clinical practice have igg4-rd . Furthermore, elevated serum igg4 levels can be seen in patients with many different diseases, most of which likely represent coincidental occurrence . Our findings reinforce the principle that an elevated serum igg4 level in isolation is of limited diagnostic utility.
Diosmin (3, 5, 7-trihydroxy-4-methoxyflavone 7-rutinoside = diosmetin 7-o - rutinoside) is a naturally occurring flavonoid glycoside found in various plant materials, mainly citrus fruits . Due to the high demand for the substance, it is obtained semi - synthetically from hesperidin (3, 5, 7-trihydroxy-4-methoxyflavanone 7-rutinoside = hesperitin 7-o - rutinoside). Diosmin (figure 1a) is a component of formulations used in the treatment of venous insufficiency, hemorrhoids, lymphedema, diabetes, melanoma, dermatitis, mastalgia, colitis, pre - menstrual syndrome and many other conditions (1, 2). Hesperidin (figure 1b) is also used with diosmin in various formulations and plant extracts . Hesperidin due to their low water solubility, both drugs are barely absorbed from the gastrointestinal tract; therefore, diosmin is more and more frequently micronised to obtain particles smaller than 2 - 20 m particles . The bioavailability of the formulations is thus much higher, as so their efficiency (3). Diosmin may be assayed spectrophotometrically with uv - vis detection (with 4-aminoantypirine using the berthelot reagent, with 3-methyl-2-benzothiazolinone hydrazone (mbth) or with 2, 4, 6-trimethylaniline) or densitometrically with uv detection . Traces of diosmin in pharmaceuticals are assayed voltamperometrically using a glass carbon electrode (4, 5, 6). Hesperidin, in turn, may be assayed spectrofluorimetrically, by measuring the intensity of the highly fluorescent complex between hesperidin and al (iii) ions in a micellar solvent (sodium dodecylsulfate in water) (7). Even though there are several distinct methods for the assay of diosmin and hesperidin in pharmaceutical formulations and body fluids, there is no accurate and rapid method for the co - assay of the substances . Diosmin and hesperidin may be assayed simultaneously by reversed - phase hplc with the following eluent: thf / water / acetic acid (21:77:2, v / v / v) at a wavelength of 280 nm (8) or methanol / water (60:40, v / v) at 345 nm (9). The most frequently used column packing for the assay of biological or pharmaceutical samples is silica modified by hydrophobic hydrocarbon chains of various lengths (usually ods) or by polar functional groups . When such packing types are used for the separation of certain analytes, in particular basic compounds, long retention times and peak tailing is observed . The effect results from adsorption interactions between free active silanols and the analyte being resolved, which significantly hinders analysis . In order to block active silanols, ionic liquids have been used as mobile phase additives in the separation of ephedrines (10), catecholamines (11), amines (12), basic drugs (13), phenoxy acid herbicides and phenols (14), vitamins (15) among other compounds . In this work, didecyldimethylammonium lactate (figure 2) is used as the mobile phase additive for diosmin and hesperidin resolution . Chemicals and reagents diosmin and hesperidin were obtained from lkt laboratories, saint paul, usa . The following solvents were used: dimethylsulfoxide (dmso), methanol and chloroform (high - performance liquid chromatography grade) were from poch, gliwice, poland . Juliusz pernak from the faculty of chemical technology, pozna university of technology, poland . Pharmaceutical formulations used in the study standards and samples stock standards (concentration: 1 mg / ml) were prepared separately by dissolving 0.0500 g of diosmin and hesperidin in dmso: methanol 1:1 . Working solutions were obtained by the successive dilution of the standard solution using the mobile phase used in the chromatographic analysis . Samples of the test products were prepared by dissolving 0.0500 g of the powdered product in dmso: methanol 1:1 . A hewlett packard liquid chromatograph (model hp 1050, waldbronn, germany) was used, composed of a quaternary pump, a variable - wavelength uv detector operating at 280 nm and a rheodyne model 7125 injection valve with a 20 l sample loop . The separation was performed on a lichrospher, rp-18 (5 m, 250 mm x 4.6 mm i d) stainless steel column (merck, darmstadt, germany). Before use, the mobile phase was vacuum - filtered through a 0.45 m cellulose filter and degassed with helium . The water was distilled and then purified by a milli - q water purification system (millipore, billerica, usa). Chromatography conditions the mobile phase consisted of methanol: water = 45:55%, v / v with 0.025% ionic liquid added . Analytical method validation parameters calibration curves were obtained based on the results of chromatographic analysis of diosmin and hesperidin standard solutions . The resulting solutions contained 2.5; 5; 10; 25; 50; 75 and 100 g of each compound per 1 ml of mobile phase . The regression equations were calculated in the form of y = ax + b, where y and x were peak area and sample concentrations, respectively . The repeatability of the chromatographic system was assessed under the chromatographic conditions previously selected by means of 10 replicate injections of a solution with 5 g / ml diosmin and 5 g / ml hesperidin and finding out the peak area by the proposed method . From this peak area% rsd day precision was determined by injecting three different concentrations (5, 50 and 100 g / ml) for three times in the same day . Inter - day precision was determined by injecting three different concentrations (5, 50 and 100 g / ml) for three days in a week . Limit of detection and quantification the limit of detection (lod) under the present chromatographic conditions is defined by the concentration of the analyte giving a signal to noise ratio of 3: 1 . The limit of quantification (loq) is the lowest analyte concentration which can be assayed with required precision and accuracy . Based on 6 parallel results obtained for each standard solution concentration, relative standard deviations were derived, and loq for rsd = 10% was derived from the relationship between rsd and test glycoside concentrations . The recovery value was determined by adding known quantities of the test compounds to the test formulations (standard addition method) (ich q2a, text of validation of analytical procedures, international conference on harmonization tripartite guidelines, adapted 27 oct . The recovery of the standards added (diosmin and hesperidin) was performed for three different concentration levels, corresponding to approx . Each sample was tested six times at each concentration level according to the above procedure . Optimization of chromatographic analysis reversed - phase chromatography was suggested as a suitable procedure for the co - assay of diosmin and hesperidin in complex matrices, such as pharmaceutical formulations . The optimization of the chromatographic process involved first the determination of the detection wavelength suitable for the co - assay of the test compounds and second, the selection of mobile phase parameters suitable for the total resolution of diosmin and hesperidin as rapidly as possible, with no interference from other ingredients of the test samples . The selection of appropriate conditions involved testing the effects on retention of type and concentration of organic solvent in the mobile phase, and the concentration of the ionic liquid added to the eluent on retention . The analysis of uv spectra for diosmin and hesperidin and initial hplc studies of the test matrices gave appropriate conditions for chromatographic resolution to facilitate the co - assay of both analytes . When an ionic liquid was added, analysis time was significantly reduced and peak symmetry was greatly improved . An example of the chromatograms obtained for a standard mixture of diosmin and hesperidin is shown in figure 3 . Chromatogram a was obtained without addition of ionic liquid and chromatogram b with 0.025% ionic liquid in mobile phase . The retention times of diosmin and hesperidin were 8.01 0.03 and 5.73 0.02 min, respectively . Chromatograms of diosmin d and hesperidin h for standard without addition of ionic liquid (a) and with 0.025% ionic liquid (b) in mobile phase we note very good peak symmetry and also the fact that the retention of the test compounds is repeatable with very good precision (low rsd values). The linearity of the method was tested for both flavonoid glycosides assayed in a concentration range of 2.5 to 100 g / ml by injecting standard solutions in the chromatographic conditions as above . The calibration curves for diosmin and hesperidin were derived by the least - squares method . Table 2 shows the linearity range tested and parameters of the fitted straight - lines . Calibration range and fitted parameters in the simultaneous determination of diosmin and hesperidin, the calibration curves were found to be linear for both compounds in the aforementioned concentrations and the correlations coefficients for the regression lines were 0.9983 and 0.9992 for diosmin and hesperidin, respectively . Repeatability expressed as relative standard deviation and determined for both analytes was 0.39 and 0.42% for diosmin and hesperidin, respectively . The precision of the method was demonstrated by inter day and intra day variation studies . In the intra day studies, six repeated injections of sample solutions were made and the mean area of analyte peaks and percentage rsd were calculated and presented in table 3 . In the inter day variation studies, six repeated injections of sample solutions were made for three consecutive days and mean area of analyte peaks and percentage rsd were calculated and presented in table 3 . Precision of the method from the data obtained, the developed hplc method was found to be precise . The limits of detection for diosmin and hesperidin were 2.5 and 1.2 g / ml, respectively, and the limits of quantification were 5.5 and 3.5 g / ml, respectively . The method was used for the assay of diosmin and hesperidin in four pharmaceutical products available in poland . Product compositions and manufacturers are listed in table 1 . The results of analyses calculated based on calibration curve equations are shown in table 2 . N.a. Data not available the results listed in table 4 show that the method can be used for the co - assay of diosmin and hesperidin in various pharmaceutical products . Rsd values for six independent determinations in the test pharmaceuticals were within a range of 1.10 to 3.21% and 0.57 to 3.37% of diosmin and hesperidin, respectively . Larger differences were noted between analyte quantities assayed and declared by the manufacturers . In order to account for the inconsistencies and to verify the correctness of our method, an experiment was performed to determine analytical accuracy . A solution of the test product was prepared with analyte concentration x within a range of concentrations used for the calibration curve and three series of identically prepared solutions to which diosmin and hesperidin with known concentrations a were added . The value of a for diosmin in pelethrocin and diosminex was 100, 200 and 300 mg per one tablet, respectively; for diohespan forte: 120, 240 and 360 mg per tablet; for belissa anti red: 15, 25 and 35 mg per tablet . The value of a for hesperidin in diosminex and diohespan forte was 2.5, 5.0 and 7.5 mg per tablet; for diohespan pelethrocin: 5.0, 7.5 and 10.0 mg per tablet; for belissa anti red: 10, 20 and 30 mg per tablet . The determinations were performed for all the test products and both analytes (six independent analyses for each series of solutions in the same conditions). Four series of results were obtained, x and x + a, listed in table 5 . The concentration values shown in table 4 correspond to diosmin and hesperidin concentrations with respect to one tablet . Method accuracy is determined by recovery values for quantities a of diosmin and hesperidin added to pharmaceutical product solutions . The recovery values are close to 100%, within a range of 96.9 to 103% and 96 to 102.9 for diosmin and hesperidin, respectively . Accuracy of diosmin and hesperidin assay in the test products (n = 6). The results shown in the tables confirm that our method for the co - assay of diosmin and hesperidin in pharmaceuticals has both high precision and accuracy . They confirm the correctness of our method and the procedure used; the discrepancies between the analytical results and the amounts declared by the manufacturers are consistent with the requirements specified by pharmacopoeia viii (diosmin should not contain more than 5% hesperidin, considered an impurity). The paper puts forward a method for the co - assay of diosmin and hesperidin in pharmaceutical products using hplc in which some ionic liquid is added to the mobile phase . When the ionic liquid was added, analysis time was reduced and resulting peak symmetry was improved . It is the first time an ionic liquid has been used for the resolution and assay of the compounds . The method has high precision from 1.10 to 3.21% and 0.57 to 3.37% for diosmin and hesperidin, respectively; accuracy from 96.9 to 103% and from 96 to 102.9 for diosmin and hesperidin, respectively and very good repeatability (0.39 and 0.42% for diosmin and hesperidin, respectively). The precision of the proposed method was carried in terms of the repeatability, inter - day and intra - day time periods . The low% rsd values of repeatability (0.39 and 0.42% for diosmin and hesperidin, respectively), intra - day (0,39% 1,12%) and inter - day (0.51% - 1.19%) variations reveal that the proposed method is precise . The limit of detection and quantification is 2.5 and 1.2 g / ml and 5.5 and 3.5 g / ml for diosmin and hesperidin, respectively . The results show that the method may be used for the control of diosmin and hesperidin contents in pharmaceutical products.
The anterior visual pathway (i.e., retina, optic nerves, chiasm, and optic tracts) represents a valuable model for understanding axonal and neuronal loss and clinical and functional correlates in persons with multiple sclerosis (ms). This pathway, particularly the optic nerve, is often affected by the ms pathological disease processes given its proximity with the vasculature around the ventricles of the brain . The integrity of the anterior visual pathway can be noninvasively imaged using optical coherence tomography (oct) of the retina . Oct provides metrics of retinal nerve fiber layer thickness (rnflt) and total macular volume (tmv). Rnflt reflects the integrity of nonmyelinated axonal tissue, whereas tmv reflects the integrity of nonmyelinated axonal tissue as well as all retinal layers including cellular segments . We further note that rnflt and tmv have been associated with lesion volume and brain atrophy metrics from magnetic resonance imaging in persons with ms [46]. This is important as imaging the anterior visual pathway may provide a correlate of the clinical and functional consequences of axonal and neuronal loss in ms . The integrity of the anterior visual pathway might be associated with walking function in persons with ms . Visual and ambulatory functions are integrally related in all persons, including those with ms, and represent two of the most valued functions that are compromised in the ms population [79]. Importantly, oct metrics have been associated with the expanded disability status scale (edss) scores in persons with ms (range: r = 0.30 through 0.70), and the edss is heavily weighted by ambulatory function in the middle range of scores based on 500-meter walk performance . Oct metrics have further been correlated with other measures of disability such as the multiple sclerosis functional composite (msfc) (r = 0.227), and the msfc has an ambulatory component based on the timed 25-foot walk (t25fw). Lastly, rnflt and tmv metrics from oct might reflect neurodegeneration on a more global level, thus supporting a possible association with walking impairment another global manifestation of ms . The motivation for considering a possible association between integrity of the anterior visual pathway and walking function is further rooted in the idea that oct metrics can serve as structural outcomes for trials examining neuroprotective and neuroreparative outcomes in ms . To that end, many rehabilitation trials, for example, clinical trials of exercise training, are targeted towards improving ambulatory function in ms . Such a beneficial effect might have a basis in structural adaptations involving axons and neurons [16, 17]. If true, then oct and its metrics might be considered for inclusion in clinical trials of rehabilitation for capturing the possible influence on central nervous system (cns) integrity in ms . This study examined the association between integrity of the anterior visual pathway and walking functions in persons with ms . We hypothesized that the metrics of rnflt and tmv would be associated with scores on well - characterized walking outcomes, namely, the six - minute walk (6mw) and the t25fw [14, 18], even after controlling for possible covariates (e.g., disease duration, edss, and age). We contacted and screened 113 possible participants residing in central illinois between the middle of september, 2010, through the last week of december, 2010 . The possible participants were not consecutive patients, but were referrals from the practices of three locally residing neurologists as well as our laboratory database . The final sample consisted of 58 patients with a definite diagnosis of ms who satisfied our inclusion criteria . The criteria for inclusion were the following: (a) capacity for independent ambulation or ambulation with an assistive device (e.g., cane, crutch, walking frame, or rollator walker); (b) relapse free during the previous 30-day period before testing; and (c) willingness to undergo testing . We excluded persons when conducting oct who had (a) ocular disease (prior history of optic neuritis, glaucoma, macular degeneration, etc .) Or (b) high myopia (minus 7.5 or higher) of both eyes based on a clinical examination from a neuro - ophthalmologist (john h. pula). Participants underwent time - domain oct scans (zeiss stratus oct 3, carl zeiss meditec, dublin, ca, usa) performed by a neuro - ophthalmologist (john h. pula). When necessary, 1% phenylephrine intraocular mydriatic drops were instilled prior to scanning . Throughout scanning, if a scan had poor image quality, defined as improper disc centering, poor reference image acquisition, or a signal strength score of less than 7, it was repeated until obtaining an adequate scan . Average rnflt was determined as the distance between the first reflection from the vitreoretinal interface and the anterior boundary of the second reflective layer, corresponding to bruch's membrane, and average tmv was recorded as an overall macular volume which did not contain the optic nerve head . The 6mw was performed in a rectangular corridor with hallways that exceed 50 m in length and that were clear of obstructions and foot traffic . We provided standardized instructions and emphasized walking as far and as fast as possible for 6 minutes . One researcher followed alongside of the participant for safety, while another researcher followed 35 feet behind the participant and recorded the distance travelled in feet using a measuring wheel (stanley mw50, new britain, ct, usa). The t25fw was performed along a clearly marked 25-foot long path on a carpeted corridor that was clear of obstructions and foot traffic . One researcher followed alongside the participant for safety, and another recorded the time in seconds by using a stopwatch . The t25fw was performed twice, and the average of the two trials was included for the analysis . Edss scoring includes tests of 8 different functional systems (fs), including visual, brainstem, pyramidal, cerebellar, sensory, bowel / bladder, cerebral, and ambulatory function . Each of these separate functional system scores received a step score, and, at the end, all step scores were combined with gait function (500-meter walk) into an overall edss score . Edss scores range from 0 (no disability) to 10 (death from ms). The procedure was approved by a university institutional review board (i.e., the united states equivalent of an ethics review committee), and all participants provided written informed consent . The measures were administered in a single session by the trained and experienced staff of an ms research center . The participants provided demographic information and underwent oct scanning and a neurological examination for generating the edss score . The data were analyzed using spss version 18 (ibm, chicago, il, usa). We provided descriptive statistics (e.g., mean, median, standard deviation (sd), and range of scores) for the demographic, clinical, oct, and ambulatory outcomes . We examined the associations between oct metrics (rnflt and tmv) with ambulatory outcomes (6mw and t25fw performance) using generalized estimating equation (gee) models . The gee models were adjusted for within - patient, inter - eye correlations, and controlled for age, disease duration, and edss scores when examining the linear associations between oct metrics and ambulatory outcomes . Participants were ambulatory women (n = 46) and men (n = 12) with a definite diagnosis of ms . The mean (sd) age was 52 (11) years, and the sample primarily had a relapsing - remitting clinical course (n = 44; 76%). The mean (sd) disease duration was 11 (10) years, and the sample had a median edss score of 4.5 (interquartile range = 2.0). The data for rnflt, tmv, t25fw, and 6mw are provided in table 1 . The mean (sd) values for rnflt and tmv of persons (without optic neuritis or any other ocular diseases) in the present study were similar to those in a previous research . The mean (sd) values for t25fw and 6mw were consistent with those in previous researches [19, 24]. The gee models accounting for inter - eye correlations and age, disease duration, and edss scores indicated that rnflt was not significantly associated with 6mw (p = 0.99) or t25fw (p = 0.57). By comparison, the gee models accounting for inter - eye correlations and age, disease duration, and edss scores indicated that tmv was significantly associated with 6mw (p = 0.023) and t25fw (p = 0.005). The coefficients indicated that unit differences in 6mw (100 feet) and t25fw (1 second) were associated with 0.040 and 0.048 unit differences in tmv (mm), respectively . Scatter plots of associations between rnflt and tmv with t25fw and 6mw are provided in figure 1 . These scatter plots confirm the presence of weak correlations between rnflt with t25fw and 6mw, but stronger correlations between tmv and the ambulatory outcomes . The present study demonstrated that integrity of anterior visual pathways based on oct metrics was associated with ambulatory outcomes in persons with ms . Those who had reduced tmv, in particular, walked a shorter distance during the 6mw and slower during the t25fw, independent of disease duration, edss, and age in the gee models . Collectively, this research indicates that oct metrics of anterior visual pathway integrity, particularly tmv, are associated with functional consequences, namely, reduced ambulatory performance, in ms . This might position oct and its metrics as an important outcome for inclusion within rehabilitation research of walking function; such metrics might provide indications of possible neuroprotective and neuroreparative consequences of rehabilitation based on integrity of the anterior visual pathway . Researchers have previously reported associations between oct metrics and edss as well as msfc scores in persons with ms [19, 25]. This is important as both the edss and msfc include ambulatory measures of the 500-meter walk and t25fw, respectively, when generating an overall score . Such observations, in part, motivated our interest in examining the associations between rnflt and tmv with ambulatory outcomes, and controlling for covariates, particularly edss scores . To that end, our results indicate an association between tmv and both 6mw and t25fw performance, independent of edss as well as age and ms duration . Accordingly, this is the first study, to our knowledge, that has reported an independent association between oct metrics of anterior visual pathway integrity and ambulatory performance in persons with ms . The mean values of rnflt, tmv, t25fw, and 6mw of persons with ms (without optic neuritis or any other ocular diseases) in the present study were consistent with those in previous researches [19, 23, 24]. For example, the mean (sd) rnflt in the present study was 90.9 (14.9) m, and this was consistent with the value of 95.6 (14.5) m from a previous research . The same consistency in mean values is seen with tmv . Regarding ambulatory function, the t25fw and 6mw values of 6.9 (3.5) seconds and 1,336 (441) feet, respectively, are consistent with the values of 6.4 (2.7) and 1,277 (255) in previous researches [19, 24]. Such observations are important for contextualizing our sample and results within the broader research on oct metrics and ambulatory function involving persons with ms . The integrity of the anterior visual pathway might be associated with ambulatory performance as vision is coupled with walking . Indeed, vision and gait are two of the most important bodily functions that are compromised, yet highly valued, across the early and late phases of ms . Visual dysfunction has further been identified as one of several possible impairments (e.g., weakness, sensory loss, and ataxia) that might influence walking in neurological diseases including ms . Overall, we are not surprised that integrity of the anterior visual pathway is a correlate of walking another global indicator of ms progression and this study provides the first data supporting the presumed association between visual and walking functions in ms . We believe that one possible implication of our study is that oct metrics might be included in rehabilitation trials for understanding adaptations within the cns that correspond with improvements in ambulatory function . Indeed, rehabilitation interventions that improve gait in ms might do so, in part, through neuroreparative and neuroprotective processes . If correct, then oct metrics might be considered for inclusion in clinical trials of rehabilitation approaches for improving ambulation in people with ms . Such an approach provides simple, affordable, and noninvasive metrics for documenting possible neuroreparative and neuroprotective outcomes . There are many strengths of the current study including the first examination of anterior visual pathway metrics and walking function controlling for edss and other covariates in ms . The most important limitation is that the study included time - domain oct, and this has poor resolution and reduced accuracy of outcomes . This would seemingly bias any associations with other outcomes towards the null and could be overcome in future research using spectral domain oct and better characterizing associations with ambulatory outcomes . We do not have data from spectral domain oct for addressing this limitation in the current study . The sample size might be considered small, but it is sufficient for a preliminary examination of oct metrics and ambulation . The sample mostly had relapsing - remitting ms, and this limits the generalizability of our data and results amongst those with progressive ms . The study included a cross - sectional design and did not provide data on changes in oct metrics and ambulatory outcomes over time . Lastly, the t25fw was performed on a carpeted surface, and this differs from the typical administration on a noncarpeted walkway . We are excited by the novel data indicating that oct metrics of anterior visual pathway integrity are associated with walking outcomes in ms.
Ad is a progressive and irreversible neurodegenerative disorder leading to cognitive, memory, and behavioral impairments . Cerebral elevation and accumulation of a are necessary steps in the pathogenesis of ad [13]. Sequential proteolytic cleavages of amyloid precursor protein (app) by membrane - bound -secretase and -secretase produce two major isoforms of a, a40, and a42 . Alternatively, app can be sequentially processed by -secretase, and -secretase, precluding a production (nonamyloidogenic pathway). Even though advanced age serves as a major risk factor, approximately 5% of ad cases are familial (fad), and some of them are attributable to autosomal dominant mutations in presenilin (ps) genes, ps1 and ps2 . Ps1 and ps2 function as catalytic subunits of -secretase, and fad mutations in pss affect app processing increasing the ratio of a42 to a40 [68]. Growing evidence indicates that dysregulation of lipid pathways have regulatory consequences for app processing and a generation . Especially, cholesterol has been suggested to participate in the etiology of ad by increasing the generation of a . Cholesterol can directly regulate the activities of -secretase or -secretase to alter amyloidogenesis [1113]. Alternatively, changes in cholesterol level may affect the lipid environment for app processing and a generation . App is located either within or outside of lipid rafts . Since bace1 (-secretase) is predominantly located in lipid rafts, app processing occurring within lipid rafts is amyloidogenic, whereas app processing occurring outside lipid rafts is considered nonamyloidogenic . When cholesterol is depleted, the association of bace1 with lipid rafts is decreased, producing less a [1517]. In contrast, increasing cholesterol induces the co - clustering of app and bace1, producing more a . From these results it could be hypothesized that high cholesterol levels may be responsible for initiating the pathogenesis of ad . However, it was recently demonstrated that lowering cholesterol levels results in increased -amyloid production in neurons . Abad - rodriguez et al . Reported that lowering the intracellular levels of cholesterol could increase the rate of amyloidogenic processing of app by placing the hydrolyzing enzyme (bace1) and app in close proximity within the same intracellular compartments . Therefore, more experiments will be needed to clarify the conflicting results about the role of cholesterol in pathogenesis of ad . Pip2 is known as one of phospholipid component of cell membrane, playing important regulatory roles in a variety of cell functions, such as rearrangement of the cytoskeleton and membrane trafficking . We have reported that fad - linked ps mutants down - regulate pip2 levels, and that pip2 levels are inversely correlated to the production of a42 . We also demonstrated that increased membrane cholesterol level decreases the level of pip2 via the activation of plc . Therefore, there exists a crosstalk between two plasma membrane - enriched lipids, cholesterol and pip2 . Considering the close relationship between pip2 levels and the production of a42, we suspected that increased membrane cholesterol levels affect the a42 production via down - regulating pip2 levels . In this study, we found that membrane cholesterol decreased pip2 levels and increased secreted a42 . Supplying pip2 by using a pip2-carrier system blocked the effect of cholesterol, which might indicate that the effect of cholesterol on a42 was by downregulation of pip2 levels . Enriching membrane with cholesterol increased the expression of some plc isoforms, such as plc1 and plc3 . Blocking the new protein synthesis prevented the effect of cholesterol on pip2 levels as well as on a42 production . These results suggest that increased membrane cholesterol levels, and fad - linked ps mutations may share the same molecular mechanism, that is, the downregulation of pip2, which may serve as the molecule linking cholesterol metabolism to the pathogenesis of ad . Hela cells stably transfected with app751 carrying the swedish mutation (appsw) were cultured at 37c, 5% co2, in dulbecco's modified eagle medium (dmem) supplemented with 10% heat - inactivated fetal bovine serum containing 100 units / ml penicillin, 100 g / ml streptomycin, 260 g / ml zeocin, and 400 g / ml g418 . Human neuroblastoma sh - sy5y cells were cultured in dmem containing 10% heat - inactivated fetal bovine serum, 100 units / ml penicillin, and 100 g / ml streptomycin . To enrich the cells with cholesterol, cells were exposed to dmem culture medium containing methyl--cyclodextrin (mcd, sigma, usa) saturated with cholesterol (water - soluble cholesterol). During the incubation, cells were maintained in a humidified co2 incubator at 37c . In some experiments, to avoid the use of mcd, cells were incubated with cholesterol which was solubilized by sonication . For this purpose, cholesterol in methanol / chloroform mixture (1: 1 v / v) was dried under nitrogen gas and sonicated for 2 min in phosphate - buffered saline before use . In some experiments, cells were pretreated with 10 m actinomycin - d (sigma) or 50 g / ml cyclohexamide (sigma) for 0.5 h before 75 m water - soluble cholesterol was added . Carrier - pip2 complex was incubated with app - transfected hela cells for 4 h in the absence or presence of 75 m water - soluble cholesterol . The antisense oligonucleotides (idt, usa) targeted at plc1 and plc3 were designed to be complementary to the 5 sequences and were phosphorothionated at all positions to minimize intracellular cleavage by enzymes and to enhance their stability (5-actccgggttgagccccggc-3 for plc1 and 5-tccaactgcagcgcgtggac-3 for plc3). Antisense oligonucleotide (5-gccccgtatgaccgcgccgg-3) having no target was used as a control in all of experiments . The app - transfected hela cells were plated at a density of 2 10 cells per 60 mm dish and incubated overnight and then treated with the 10 m antisense oligonucleotides for 4 h in dmem culture medium without serum . After treatment, the medium was replaced by a new medium containing 10 m antisense oligonucleotides with or without water - soluble cholesterol for 2 h. media were collected to measure levels of a, and cells were homogenized to confirm plc expression levels and pip2 levels . Filipin staining of cells (0.05%, dmso 1%) was performed for 1 h at room temperature after cholesterol enrichment to confirm the changes of free cholesterol levels at the plasma membrane . Fluorescence images were obtained using a lsm 710 confocal microscope (zeiss) using laser emitting at 351 nm . Images were quantified to obtain the mean fluorescence density values of plasma membrane from the edge of the cell to 500 nm inside using the imagej program . A elisa kits (usa) or wako -amyloid elisa kits (high - sensitive; japan). For sapp, sapp detection, app - transfected hela cells at 80% confluence in a 35 mm dish were cultured for 8 h with water - soluble cholesterol in dmem culture medium without serum . Control cells were treated similarly and incubated with serum - free dmem solution without any cholesterol . After exposure to cholesterol, supernatants were collected to measure levels of a, sapp, or sapp. To detect a from sh - sy5y cells, supernatants were desalted using pd-10 desalting column (ge healthcare, usa), dried, and reconstituted in water . The samples were then centrifuged at 1,000 g for 10 min at 4c to remove nuclei and debris . Supernatants were separated by centrifugation at 100,000 g for 1 h at 4c into membrane (pellet) and cytosol (supernatant) fractions . Whole cell lysates were prepared by homogenizing with lysis buffer (10 mm tris - hcl, 150 mm nacl, 1% triton x-100, 0.25% nonidet p-40, 2 mm edta, ph7.4) using a cell scraper . The lysed cells were centrifuged at 12,000 g for 10 min at 4c . The protein in the supernatant was determined by bradford assay (bio - rad, usa). Membranes were blocked with 5% nonfat milk powder in tris - buffered saline / tween 20 (tbst) for 1 h at room temperature, then incubated with rabbit polyclonal anti - plc1 (sc-9050), plc2 (sc-206), plc3 (sc-13958), plc4 (sc-20760), plc2 (sc-9015), mouse monoclonal anti - plc1 (sc-7290) antibodies (santa cruz biotechnology, usa), anti - app antibody (ln27, zymed), anti -actin (a5441, sigma), and rabbit anti -tubulin (t2200, sigma) for overnight at 4c . Dilutions were 1: 500 for plc isozymes and 1: 4000 for -tubulin, -actin, and app . After washing, membranes were incubated for 1 h at room temperature with horseradish peroxidase - conjugated goat anti - rabbit igg or goat anti - mouse igg antibodies (1: 2000 dilution; zymed, usa) and washed . Blots were quantified with the multi gauge software using a las-3000 system (fugifilm, japan). The amount of pip2 extracted from app - transfected hela cells were measured by using pip2 mass elisa kit (echelon biosciences inc . Pip2 was extracted from the control cells or cells treated with water - soluble cholesterol according to the supplier's instructions . Cellular pip2 quantities were estimated by comparing the values from the standard curve, which showed linear relationship at the range from 0.5 to 1000 pm concentrations . Statistical comparisons between the controls and treated experimental groups were performed using the student's t - test . Mcd, a water - soluble cyclic oligosaccharide, has hydrophobic cavity that is able to encapsulate insoluble compounds, thus enhances the solubility of cholesterol . Mcd saturated with cholesterol (water - soluble cholesterol) has been used to increase membrane cholesterol level, since it acts as a cholesterol donor [2426]. We incubated app - transfected hela cells with 15, 75, or 150 m water - soluble cholesterol for 8 h, and filipin staining was performed for 1 h at room temperature to monitor the membrane cholesterol level . Typical confocal image in figure 1(a) shows that the membrane cholesterol level increased by 75 m water - soluble cholesterol . The changes of cholesterol level were confirmed by quantifying the filipin fluorescent intensities from plasma membranes . By incubating cells with 15 and 75 m water - soluble cholesterol, the fluorescent intensities were increased by 58.5 5.8% and 83.3 15.9% we also tested the time - dependent accumulation of cholesterol in the membrane by incubating cells with 75 m water - soluble cholesterol . Cholesterol levels increased after 0.5 h, and it steadily increased further after 1.5 h or 5 h (supplementary figures 1(a) and 1(b), see supplementary material available online at http://dx.doi.org/10.1155/2013/407903). From these results we concluded that the direct administration of the water - soluble cholesterol leads to the increases in the membrane cholesterol levels . Recently, we have reported that augmentation of membrane cholesterol levels downregulates pip2 level . To validate this observation in the current system, app - transfected hela cells were incubated with 15, 75, or 150 m water - soluble cholesterol for 8 h, and the steady state levels of pip2 were measured using a pip2 elisa . Pip2 levels in 75 and 150 m cholesterol - treated cells were downregulated by 23.2 5.0% and 26.1 2.5% (n = 6), respectively (figure 1(c)). We also tested the time - dependent effect of increased membrane cholesterol on the levels of pip2 by incubating cells with 75 m water - soluble cholesterol . As shown in figure 1(d), the steady state levels of pip2 after 1.5 h and 5 h incubation time were downregulated by 20.3 5.1% and 26.3 5.3% (n = 6), respectively . Since we have reported that cellular pip2 levels are closely correlated with the a42 levels, we tested the effect of increased membrane cholesterol levels on secreted a. for this purpose, app - transfected hela cells were incubated for 8 h with 15, 75, or 150 m water - soluble cholesterol, and a levels were measured from the conditioned media by using an elisa kits specific for a40 or a42 . The secreted a40 levels were not changed by increased membrane cholesterol levels (open bars in figure 1(e)). However, a42 levels were increased by 28.1 8.4%, and 36.2 8.1% (n = 6) when cells were incubated with 75, and 150 m water - soluble cholesterol, respectively (closed bars in figure 1(e)). We also tested the effect of increased membrane cholesterol levels on the levels of secreted a from neuroblastoma sh - sy5y cells . The level of endogenous a42 increased significantly (closed bars in figure 1(f)), while the endogenous a40 level was not changed (open bars in figure 1(f)). Thus, these results suggest that the effect of cholesterol enrichment is specific to a42 levels, and is not cell - type specific . Alternatively, app can be cleaved sequentially by -secretase followed by -secretase precluding the production of a. thus, the effect of increased membrane cholesterol levels on a levels can occur in any of those processes . To begin to investigate the effects of membrane cholesterol on app processing increased membrane cholesterol levels led to a moderate increase in the full - length app levels (figure 2(a)). Then, we tested the effects of increased membrane cholesterol levels on the activities of -secretase and -secretase . For this purpose, we measured the levels of sapp and sapp from the conditioned media using specific elisa kits, since they are produced via the activities of -secretase and -secretase, respectively . In this experiment, we used sapp elisa kit for swedish mutant . As shown in figure 2(b), the levels of both sapp and sapp were also slightly increased by increased membrane cholesterol levels, which might be due to the increased level of their precursor, app . However, the amount of increased sapp level was not robust to explain the a42-selective changes associated with increased membrane cholesterol levels . Since membrane cholesterol levels affect a42 but not a40, it is conceivable that the effects of cholesterol may influence the specificity of -secretase - mediated cleavage of amyloidogenic app c - terminal fragments (e.g., c99). In order to elucidate the role of pip2 for the effect of increased membrane cholesterol levels on secreted a42, we used a pip2-carrier system for the intracellular delivery of pip2 . Because carrier compounds are charge - neutralization species after the carriers were added at a one - to - one molar ratio with pip2 at room temperature, the complex was diluted to the desired final concentration . Then, the carrier - pip2 complex was incubated with cells for 4 h before a levels were measured from the conditioned media . The presence of 2 m and 5 m carrier - pip2 complex decreased secreted a42 levels by 12.8 12.1% and 41.5 4.1% (n = 6), respectively (open bars in figure 2(c)). This result is consistent with our previous result showing the close correlation between pip2 levels and a42 production . In the absence of carrier - pip2 complex, incubating cells with 75 m water - soluble cholesterol for 4 h increased a42 levels by 17.7 2.9% (n = 6), which is consistent with the result in figure 1(e). However, the presence of either 2 m or 5 m carrier - pip2 complex completely prevented the effect of water - soluble cholesterol on a42 levels (closed bars in figure 2(c)). These results suggest that the relative levels of cholesterol and pip2 correlate closely with secreted a42 levels in a positive or negative manner, respectively . Unlike a42, the a40 levels were not affected by the presence of carrier - pip2 complex (open bars in figure 2(d)). Also, the a40 levels were not affected by increased membrane cholesterol levels in the presence of carrier - pip2 complex (closed bars in figure 2(d)), which was consistent with the specific effect of cholesterol on a42 level . Since we suspected that the effect of increased membrane cholesterol levels on the secreted a42 levels is due to the downregulation of pip2, we tested whether increased membrane cholesterol levels affect the activity of plc . We first examined the expression levels of plc isoforms by monitoring them using western blot analysis from cytosol and membrane fractions in app - transfected hela cells . The expression levels of plc1 were significantly increased by cholesterol only in membrane fractions as shown in figure 3(a). Densitometry analysis of the bands corresponding to plc1 clearly supports this conclusion (figure 3(b); n = 5). After 0.5 h incubation time with 75 m water - soluble cholesterol, expression levels of plc1 were increased by two folds in membrane fractions . The effect of cholesterol on plc1 expression lasted as long as 5 h. the expression level of plc3 was also increased in membrane fractions by cholesterol (figures 3(a) and 3(c)). However, the use of mcd may cause nonspecific effects in addition to enrichment of membrane cholesterol levels . To avoid the use of mcd, free cholesterol when cells were incubated with 75 m solubilized cholesterol for 1 h, the expression of plc1 in the membrane fraction was increased (supplementary figure 2). At 1 h incubation time, the expression of plc3 was not changed . These results indicated that the effect of cholesterol on plc1 expression was not due to the nonspecific effect of mcd . Cholesterol - induced increases in the levels of plc1 and plc3 were observed almost exclusively in the membrane fraction . Also, increased membrane cholesterol levels did not change the expression levels of plc2 as shown in figure 3(a) from a typical experiment . The expression levels of other plc isoforms (plc2, plc4, and plc1) were not changed either (supplementary figure 3(a)). We also observed specific increase of plc1 and plc3 expressions from sh - sy5y cells by enriching membrane cholesterol levels (supplementary figure 3(b)). To determine whether the effect of cholesterol on plc expression was due to increased transcription, cells were preincubated for 10 min with the transcription inhibitor, actinomycin - d (act - d, 10 m), or with the translation inhibitor, cyclohexamide (chx, 50 g / ml). Then, cells were incubated further for 0.5 h with 75 m water - soluble cholesterol . Representative western blots for plc1 and plc3 from membrane fractions are shown in figure 4(a). The effect of cholesterol on plc1 and plc3 expressions was completely prevented either by act - d or by chx, indicating that the effect of cholesterol on plc1 and plc3 expression was via the up - regulation of transcription . Then, we tested the effect of act - d on the steady state level of pip2 . As we expected, pip2 levels were downregulated by 18.9 0.3% (n = 6) when cells were treated with 75 m water - soluble cholesterol for 1 h in the absence of act - d (figure 4(b)). However, the effect of cholesterol on pip2 levels was completely prevented when cells were pretreated with act - d (n = 6). These results confirmed that increased membrane cholesterol levels increases the expression of plc1 and plc3, leading to the downregulation of pip2 levels . Next, we tested the effect of act - d on the level of secreted a42 . For this purpose, cells were incubated for 4 h with 75 m water - soluble cholesterol with or without act - d . As expected, cholesterol increased the levels of secreted a42 by 20.1 2.8% (n = 4) in the absence of act - d (figure 4(c)). However, the presence of act - d completely prevented the effect of increased membrane cholesterol levels on a42 (n = 4). These results suggest that the increase of plc transcription by cholesterol induced the downregulation of pip2 levels, which increased secreted a42 levels . We tested whether plc1 or plc3 is required for the observed effects of cholesterol on a42 . To avoid any further transfection, cells were pretreated with 10 m antisense oligonucleotides against plc isoforms for 4 h. then the media were replaced by fresh media containing the same antisense oligonucleotides with or without 75 m water - soluble cholesterol, followed by additional incubation for 2 h. antisense oligonucleotides having no specific target were used for controls in all of experiments . A typical western blot for plc1 is shown in figure 5(a), and the band densities are expressed relative to -tubulin density in figure 5(c) (n = 6). However, the presence of antisense oligonucleotides against plc1 (anti 1) blocked the effect of cholesterol . Similarly, an increase of plc3 expression by cholesterol was blocked by antisense oligonucleotides against plc3 (anti 3; figures 5(b) and 5(d)). These results demonstrated that these antisense oligonucleotides were very effective to block the effect of increased membrane cholesterol levels on the expression of plc1 and plc3 . Then, we tested the effect of these antisense oligonucleotides on the pip2 levels . In the presence of control antisense oligonucleotides (anti 1), cholesterol decreased pip2 levels by 12.1 2.8% (figure 6(a); n = 6). In the presence of antisense oligonucleotide against plc1 (+ anti 1), cholesterol decreased pip2 levels only by 2.5 1.0% (n = 6). In contrast, cholesterol decreased pip2 levels by 10 2.8% (n = 6) even in the presence of antisense oligonucleotide against plc3 (+ anti 3; figure 6(b)). Thus, inhibiting plc1 expression, but not inhibiting plc3 expression, prevented the effect of increased membrane cholesterol levels on pip2 levels . These results may suggest that the downregulation of pip2 levels by cholesterol enrichment is specifically via the increased expression of plc1 . Next, we tested the effect of increased membrane cholesterol levels on the levels of secreted a42 in the presence of these antisense oligonucleotides . As we expected, cholesterol increased the levels of secreted a42 by 25.7 5.5% (n = 8) in the presence of control antisense oligonucleotides (figure 6(c)). In the presence of antisense oligonucleotide against plc1, cholesterol increased the secreted a42 level only by 2.0 2.1% (n = 8). Thus, plc1 antisense oligonucleotide almost completely prevented the effect of cholesterol on the secreted a42 levels . In contrast, the presence of plc3 antisense oligonucleotides failed to prevent the effect of cholesterol . The a42 production was still increased by 16.7 3.4% (figure 6(d); n = 6). Together, these results strongly suggest that increased membrane cholesterol levels increased the levels of secreted a42 by down - regulating pip2 levels via specific enhancement of plc1 expression . We have previously reported that fad - linked ps mutants down - regulate pip2 levels, which are closely related to increased a42 level . In addition, up, or downregulation of pip2 levels by pharmacological means decreases or increases the production of a42, respectively . In this study, we showed that cholesterol enrichment increases the secreted a42 levels by down - regulating pip2 levels . Thus, there exists a close relationship between pip2 levels and a42 levels, consistent with our previous results . Recently, we demonstrated that enrichment of cholesterol decreases the levels of pip2 via the activation plc . Therefore, it was suggested that there exists a crosstalk between two plasma membrane - enriched lipids, cholesterol, and pip2 . In this paper, we confirmed that cholesterol decreases the level of pip2 via the activation plc . In addition, we showed that cholesterol specifically increases the expression levels of plc1 and plc3 . Consistent with this conclusion, inhibiting transcription prevented the effects of cholesterol not only on pip2 levels but also on the production of a42 . Also, the inhibition of plc1 expression, but not that of plc3, prevented the effects of cholesterol, indicating a close link between plc1 and regulation of pip2 levels . Although pip2 is a minor component in the plasma membrane, it plays important regulatory roles in a variety of cell functions, such as rearrangement of the cytoskeleton and membrane trafficking . The concept of spatially confined pip2 pools was proposed to explain the multiple roles of pip2 . Cholesterol- and sphingolipid - rich rafts may serve to confine pip2 within the plasma membrane, allowing pip2 hydrolysis to occur locally and restrict signaling mechanisms to the site of activation [30, 31]. In addition to this confined regulation of pip2, it is possible that the steady - state level of pip2 is dynamically determined by the concerted action of phosphoinositide kinases and phosphatases . In this study, we showed another way of regulating pip2 levels within specific microdomains: cholesterol content in a specific microdomain may regulate pip2 levels via plc activity . Interestingly, plc1 is shown to localize in detergent - resistant membrane microdomains prepared from the synaptic plasma membrane fraction of rat brain . For this reason, increase of plc1 expression by cholesterol enrichment may directly induce downregulation of pip2 in the confined microdomain . It was shown that cholesterol increases clathrin - dependent app endocytosis, and that it is likely the direct cause of the increased a42 secretion . Since pip2 is a key regulator for the rearrangement of the cytoskeleton and membrane trafficking, it is possible that the downregulation of pip2 may be the underlying mechanism for the increased clathrin - dependent app endocytosis by cholesterol enrichment . Alternatively, the downregulation of pip2 may directly activate -secretase since pip2 is shown to inhibit -secretase activity by suppressing its association with the substrate . It is also possible that pip2 induces changes in -secretase conformation to alter the generation of a42 . Recent reports show that the changes in ps1 conformation by various manipulations of ps1 itself, pen2, aph1, app, and pharmacological agents known as -secretase modulators can allosterically modify -secretase catalytic specificity, leading to increase of a42/a40 ratio [34, 35]. Further studies will be needed to clarify the role of pip2 in the production of a. the physiological relevance of our finding is not clear since a large increase of cholesterol in the membrane was required to induce meaningful downregulation of pip2 and up - regulation of a42 in hela cells (figures 1(c) and 1(e)). In sh - sy5y cells, however, a42 level was significantly increased even by 15 m water - soluble cholesterol (figure 1(f)). We also observed a significant increase of plc1 expression and downregulation of pip2 level by 15 m water - soluble cholesterol when we used hek cells (data not shown). Thus, it seems that the effective amount of cholesterol to induce changes in pip2 and a42 levels is cell type dependent . However, even the minimal increase of a42 by the mild cholesterol enrichment will have profound cytotoxic effects since the cytotoxicity of a42 is caused by its small molecular aggregates, such as dimer form of a42, and the produced a42 will accumulate . It has been demonstrated that cholesterol levels in the brains of ad patients are increased [36, 37]. The levels of cholesterol increase in the brain even during normal aging . In an ad brain, cholesterol homeostasis is impaired, and cholesterol retention likely enhances a production . These results may suggest that high cholesterol levels in the brain participate in the etiology of ad by increasing the generation of a. however, further work is needed to understand how cholesterol is implicated in ad pathogenesis . In this study, we showed that membrane cholesterol levels may share the same molecular mechanism with fad ps mutations, that is, downregulation of pip2, for the increased generation of a42 . Therefore, pip2 may serve as the molecule linking cholesterol metabolism to the pathogenesis of ad.
The range of such a situation can vary from unstable angina to the most acute condition, i.e., acute myocardial infarction and irreversible necrosis of the myocardium . About 1 million people are involved every year in acute or recurrent coronary syndrome in the usa . Based on who estimation, about 23.6 million people will have died of cardiovascular diseases by the end of 2030 . About 12 million people suffer from coronary artery diseases, of whom 600,000 die due to coronary arteri diseas . Despite the vast advancements concerning prevention, diagnosis, treatment, and rehabilitation of cardiac patients, these diseases account for a high mortality . One of the interventions used in improvement of cardiovascular diseases is cardiac rehabilitation programs, about which there is a bulk of studies, especially on the effect of such programs across the world . Cardiac rehabilitation is conducted through promotion and preservation of cardiovascular health through unique programs designed to improve patients physical, psychological, social, occupational, and emotional conditions . The goal of cardiac rehabilitation is speeding up the trend of secondary prevention and improvement of patients quality of life (qol). Qol not only refers to individuals personal health status but also to their physical and mental conditions as well as psychological factors such as social and functional interactions and their level of independency . Previous studies mostly measured the effect of rehabilitation programs on the physiological improvement and exercise tolerance, as well as modification of the risk factors . These studies revealed that cardiac rehabilitation activities have positive effects on mortality rate, physical health, socio - psychological function, levels of blood lipids, hypertension, dyspnea, weight loss, smoking, and level of stress . In recent years, some studies have been conducted on the effect of rehabilitation programs on patients qol . These studies are different concerning the type of intervention, length of intervention, study population, and subjects demographic characteristics and have shown controversial results . Shabani et al . Also reported the positive effect of cardiac rehabilitation on patients qol after coronary bypass or vascular reconstruction surgeries (p <0.05). Failde and soto, in a study conducted in spain, showed a significant reduction in the qol score 3 months after acute coronary syndrome incidence, in the domains of physical role, general health, and vitality . Reported that cardiac rehabilitation did not lead to an improvement in qol in the study group compared to the control group . Bettecourt et al ., in a study conducted in portugal, showed that there was no significant change in qol between rehabilitation and control groups . Cieslik et al ., in their study from turkey, reported no significant difference in qol between rehabilitation and control group (p> 0.05). With regard to the existing shortage in knowledge and related research and the reported controversial results on the effect of cardiac rehabilitation on qol, the researchers decided to design and conduct the present research . It is hoped that the obtained results can somehow modify the existing shortage of knowledge in this regard . This was a clinical trial conducted to investigate the effect of the independent variable of rehabilitation interventions on the dependent variable of qol . The present research was a two - group (study and control) two - stage (before - after) prospective study, with subjects random allocation conducted between oct 9 and feb 17, 2013 . The study population comprised 233 patients hospitalized in the ccus of selected hospitals affiliated to isfahan university of medical sciences (shohada lenjan hospital), with diagnosis of acute coronary syndrome . Inclusion criteria were: having no history of: joint disease, cardiac surgery, uncontrolled hypertension, complete heart block, uncontrolled arrhythmias and thrombophlebitis . In case of loss of interest at any stage to remain in the study or a change occurring in any of the inclusion criteria, finally, the study was conducted on 50 patients who had been hospitalized with diagnosis of acute coronary syndrome . Then, the subjects were randomly allocated to study and control groups by random numbers table . Data were collected through interviews, observation, and questioning and by use of patients medical files, demographic characteristics questionnaire, and qol questionnaire (sf-36). This questionnaire measures eight sub - scales including physical function index, physical role, emotional role, vitality, mental health, social function, pain, and general health status . Cronbach alpha of this questionnaire was calculated between 0.71 and 0.93 in eight sub - scales in the study of chan et al . Cronbach alpha of the persian version of this questionnaire was calculated between 0.70 and 0.85 in asghari - moghadam's study and its reliability was estimated between 0.77 and 0.9 in the study of montazeri et al . After obtaining a written consent from all the subjects, demographic characteristics and qol questionnaires physical rehabilitation interventions in phases 1 and 2 were administered to the subjects in the study group . Conventional rehabilitation (phase 1) was administered for the subjects in the control group . To administer rehabilitation in phase 1, the rehabilitation program was conducted for five straight days under the researcher's and a cardiologist's supervision in the hospital . This program was designed based on the amount of permitted energy consumption measured by met with regard to the number of hospitalization days . For instance, on the first day of hospitalization, the upmost permitted energy consumption was 1 met, and then the patient remained in complete bed rest for 12 h. on the second and third days, the amount of permitted energy consumption was at the most 2 mets . Then, the patient was permitted to have activities up to an optimum of 3 mets until the fifth day, if there was no chest pain, dyspnea, dizziness, and other signs . After the end of the first phase (at the time of patients discharge), the rehabilitation program was administered in phase 2 for four straight weeks . As shohada lenjan hospital lacks treadmill, barbell, stationary bicycle, and other professional rehabilitation devices, rehabilitation program in this phase was designed by simulation of rehabilitation activities according to the energy consumed based on met, with the cooperation of a cardiologist, a ccu nurse, and one of the academic members in the nursing school teaching cardiology subject . For instance, the patient was educated about what activities he / she was permitted to do in the first week and how to increase his / her activities in the absence of no abnormal signs in the following week . The subjects were followed up through phone calls in the second and third weeks to monitor the trend of rehabilitation and to supervise the appropriateness of administration of the prescribed activities during the second phase . The subjects were asked to refer to the hospital in the first and the fourth weeks . At any time of their referral, the patient was visited by a cardiologist and an ecg was taken, and after taking the cardiologist's permission, the patient was asked to walk a certain distance in the hospital hall for a certain period of time . Immediately after, the patient underwent cardiac monitoring for 10 min concerning arrhythmia, chest pain, dyspnea, and other related signs . Patients bp and pulse were measured before and after intervention . In case of patients being in stable condition, follow - up phone calls, the subjects were asked about their physician status and existing signs, and their questions in this regard were answered . Patients discussed their problems and were educated about appropriate activities based on their permitted and safe energy consumption . Therefore, in the present study, resuming the physical activities started from very light activities (1, 2, and 3 mets) and continued up to moderate level activities under the supervision of the related physician . If the patients did not feel any chest pain, fatigue, respiration distress, or abnormal changes in heart rhythm and rate while doing the activities, the severity of activities would be elevated to a higher level . The subjects in the control group underwent conventional and routine rehabilitation of phase 1 in the hospital and received no further interventions in the following 4 weeks after their discharge . One month after intervention, the questionnaire of qol was filled by the subjects in both groups . They were informed that participation was voluntary and that they could withdraw from the study at any time . It was emphasized that non of the informations would be identifiable and then informed consent was obtained . They were informed that participation was voluntary and that they could withdraw from the study at any time . It was emphasized that non of the informations would be identifiable and then informed consent was obtained . About 27 subjects were male and 23 were female, and 46 were married and 4 were either divorced or widowed . Statistical tests showed no significant difference in subjects age, gender, marital status, occupation, and level of education (p> 0.05). Regarding the cardiac risk factors (lack of exercise, hyperlipidemia, hypertension, diabetes, overweight, and smoking) independent t - test showed a significant difference in the scores of qol between the two groups before intervention in any of the domains . Paired t - test showed a significant difference in the domains of physical function, pain, and general health mean scores (p <0.05), but not in the other domains in the control group after intervention compared to before intervention . In the study group, mean scores of all qol domains increased after intervention compared to before intervention (p <0.05). On comparison of mean scores of qol in the two groups 1 month after intervention [table 1], it was found that these mean scores were significantly higher in its all domains (p <0.05) except general health and social function (p> 0.05) in the study group compared to control . Comparison of mean qol score changes in the two groups 1 month after intervention [table 2] showed that except in the domains of general health and social function (p <0.05), changes in mean scores were significant in the other domains (p <0.05). Comparison of qol domain scores before and after intervention in the two groups comparison of qol domains mean score changes 1 month after intervention in the two groups the findings of this study show that qol of acute coronary artery syndrome patients is significantly improved after cardiac rehabilitation (p <0.05). Yu et al . Showed that cardiac rehabilitation resulted in improvement of qol in the study group . Similar to the present study, in yu's study, comparison of general health domain in two groups showed no significant difference . In isfahan, iran, mostafavi et al . Showed that cardiac rehabilitation could improve patients qol (p <0.05). Although mostafavi's study was a retrospective, one - group study conducted on the medical files of 100 cardiac patients and was different from the present study, attarbashi - moghadam et al . And abbasi et al . These studies also reported an improvement of qol after rehabilitation . In the study of attarbashi - moghadam et al ., an improvement was observed in all domains of sf-36 questionnaire (p <0.005), although it was conducted on 44 patients and their qol was measured after a coronary bypass surgery . Meanwhile, the present study was conducted on 50 patients who had not undergone coronary bypass surgery as these cases were excluded from the study . Abbasi et al ., who investigated the effect of taking a walk on chronic heart failure patients, reported an improvement in patients qol (p <0.05). The questionnaire adopted in abbasi's study was minnesota, but their results are in line with those of the present study . The study of dugmore et al . Showed that rehabilitation activities could have positive effects on qol, well - being, and mental and psychological factors (p <0.05), although the qol was also significantly increased in the control group . The difference between their results and those of the present study may be due to the difference in the study populations . In dugmore's study, these were 122 male and 2 female subjects (subjects were mostly male). In addition, the length of monitoring was different . During 12 months, there might have been more adaptation with the disease condition in the control group, which might have resulted in improvement of qol . Grace et al . Found out that rehabilitation could lead to an increase in and improvement of qol and anxiety (p <0.05). They also reported that rehabilitation could also improve signs of depression (p <0.05). Although anxiety and depression were not investigated in the present study, a significant improvement was observed in the dimensions of exhilaration, vitality, and mental health . Briffia compared qol scores in the study and control groups after intervention and reported a significant difference just in physical function, which is consistent with the present study . Samartzis et al ., in a meta - analysis study on 1074 patients in the study group and 1106 patients in the control group, reported that cardiac rehabilitation improved patients qol through mental and psychological effects (p <0.05), which is in line with the present study . Koertage et al . Also reported positive effects of rehabilitation intervention on patients qol in the study group compared to control . In the literature review and meta - analysis studies, it was found that cardiac rehabilitation resulted in a reduction in mortality rate and a significant increase in o2 consumption and qol . Chan et al ., in a clinical trial conducted in hong kong, showed an improvement in subjects qol, regardless of them being in either control or study group, although the difference was not significant (p> 0.05). The reason for the difference in their results and those of the present study, especially in the control group, might have been the difference in the number of subjects and the length of monitoring (6 months) as well as a high number of subjects dropping out of the study . In portugal, bettencourt et al . Showed that cardiac rehabilitation had no significant effect on the domains of qol (p> 0.05), except on the dimensions of exhilaration and general health, possibly due to an equal distribution of the subjects into study and control groups (31 in study vs 95 in control group after subjects dropout). The results of most of the above - mentioned studies are in line with those of the present study . It seems that administration of cardiac rehabilitation activities could lead to improvement of qol in patients with acute coronary syndrome . As educating the nurses about cardiac rehabilitation and its administration in clinical setting does not impose high costs, their education in ccus can result in improvement of patients qol and their physical and mental health indexes . Patients education about the principles of rehabilitation and the gradual trend of resuming activities can improve their qol and prevent complications which result from their inadequate knowledge and disobedience of doing appropriate activities in their recovery period after discharge . As such a study was conducted for the first time in isfahan province in which cardiac patients have inadequate access to cardiac rehabilitation and as the study yielded positive results, cardiac rehabilitation is recommended to be administered in all provinces in iran to move toward promotion of public health . In this way the present study showed that cardiac rehabilitation could improve qol of the patients with acute coronary syndrome, although rehabilitation activities need patients education and supervision on their rehabilitation activities . One of the limitations to the present study was patients personal differences that may have affected their qol and were out of researchers control . Finally, the researchers suggest conducting such a study with a higher number of subjects and for a longer period of time.
Why are flies, worms, and humans subject to laws of age - specific adult mortality that are uncannily similar in shape? After suitable species - specific changes in scale, organisms with different environments, life histories, body plans, and lifespans turn out to resemble each other in the statistics of their demise . The hazard function is a summary measure of rates of death by age across a population, equal to the negative slope of the logarithm of the population survivorship function . Hazard functions for populations from many species show two common features, exponential increase with age over a stretch of ages and attenuated increase over later ages, generating the visual appearance of a plateau . The recognition of these commonalities goes back at least as far as pearl and miner (1935); the generalisation and quantitative elaboration have been signal achievements of the new biodemography, summed up by vaupel et al . (1998), carey (2003), wachter and finch (1997), carey and tuljapurkar (2003). Explanations for shared features of senescent mortality across species are sought in considerations from reliability engineering, from optimal life - history theory, and from evolutionary processes of antagonistic pleiotropy and mutation accumulation . Reliability engineering is a functional approach to senescence, picturing the organism as a machine with some component structure, attempting to derive the failure modes of the whole from some presumably simpler failure modes of the components . The aim is usually to draw inferences from qualitative classes of structures to general shapes of mortality curves . The enterprise is considered successful if the broad features common to many real - world mortality rates are reproduced in the model . Some examples are strehler and mildvan (1960), rosen (1978), gavrilov (1978), gavrilov and gavrilova (1991), weitz and fraser (2001), finkelstein and esaulova (2006). Functional models start from the structure of the organism, while evolutionary models pose prior questions: what kind of machine is the organism, and why is it put together the way it is? Many functional models lead to the same general pattern for mortality rates, after all, and each generic class of models can yield diverse shapes of age - specific mortality . Optimal life - history approaches try to narrow down the choices a priori, by explaining why a given structural framework, or a certain choice of parameters within the structural framework, might be evolutionarily preferred . Much work in this area (for example, rose 1985; schnebel and grossfield 1988; hedrick 1999; wachter 1999; williams and day 2003) builds on the concept of antagonistic pleiotropy, introduced into the theory of senescence by george williams (1957). Williams held that early reproduction and late survival would be negatively associated through direct genetic mechanisms . Recent research often abstracts from the genetic term pleiotropy, to contemplation of more general trade - offs and compromises that operate across time within the lifetime of an organism and across generations (cf . Toupance et al . 1998; hasty 2001; campisi 2003; nystrm 2003). The other side of the conventional evolutionary theory of aging, called mutation accumulation, views senescence not as an optimal trade - off between early- and late - life reproductive success, but rather as the age - specific effect of genetic load, a concept developed by peter medawar (1952). Ongoing random mutation spews mostly deleterious changes into the genome . Since the only genetic repair mechanism is the death of the organism carrying the defect, there is perpetually an overhang of deaths not yet realized, stretching from the time of the initial mutation until all descendants have died from the effect of the allele . Since an individual may not live long enough to experience the harm from a late - acting mutation, this provides another process through which natural selection reshapes demographic schedules . At equilibrium, the population observed at any given time will be found to be genetically heterogeneous, because new mutations with particular age effects are scattered independently across the individuals in a population and the mutations act together to alter each individual s internal susceptibilities to causes of death . All these approaches have something to contribute to an understanding of the central phenomenon at issue, that risks of impairment and death increase with age . This article treats mutation accumulation, but in a way that incorporates, as a start, one characteristic feature from reliability models, early - age concomitants of late - age debilitation . In future work we hope to tackle head - on the challenge of linking evolutionary models with mechanistic and physiological models . Trade - offs and impacts on age - specific mortality must be embodied in complex reliability structures . With the exception of pletcher and neuhauser (2000), the mathematical development on both sides decades of research have established, piece by piece, a mathematical framework for characterizing genetic load and the interplay between mutation, selection, and recombination . Developments through the end of the twentieth century are presented in an authoritative book by brger (2000). Early achievements addressed single - locus and several - locus systems with rich genetic structure, but did not attempt to superimpose demographic dimensions . During the 1990s, brian charlesworth (1994) succeeded in consolidating an age - specific demographic treatment based on a linear approximation . Charlesworth (2001) showed that both of the tell - tale common features of hazard functions across species, the exponential gompertzian rise and the eventual onset of plateaus, could be predicted by the linear approximate model from simple, minimalist assumptions . Three obstacles have hitherto blocked the path to a broader application of mutation - accumulation models: first, the limited versions of age - specific genetic harm under consideration, second, the assumption that genetic loci affecting different ranges of ages evolve independently, and, third, inattention to heterogeneity . The early work of w. d. hamilton (1966) posited mutations that apply a single bolus of mortality at one fixed age, what we call a point - mass model . B. charlesworth (2001) tried other stylized patterns: mortality increments within specified windows, in gaussian shapes around specified centers, or beyond specified ages of onset . He also tried coupling these age - specific patterns with an increment independent of age; our coupling of late - age with early age effects follows in this spirit precedent . A provision that late - acting effects carry with them some early manifestations is characteristic of reliability models for senescent mortality . A typical example is the model of gavrilov and gavrilova (2001), which posits an underlying structure of independent components and identifies death with the first component failure . Waiting time until death may have a mean or mode late in life, but its distribution will have a left - hand tail showing up in some early deaths . Building evolutionary structure directly into reliability models remains a project for the future . On the issue of independence, it is an essential feature of demographically - based models that the evolution at distinct sites fails to be independent even if sites act independently; that is, even if the mortality increment due to two alleles co - occurring is merely the sum of their individual effects . To put it simply, death comes to an individual only once, so that any mutation that increases mortality makes a second mutation that also increases mortality less costly, as measured in lost reproductive opportunity . Linearization, as previously employed, treats multiple mutations as though they were evolving independently and so misses the critical interaction effect in the cumulative demographic impact . On the issue of heterogeneity selection can only balance mutation when some members of the population carry more deleterious mutant alleles than others . The levels of the mean counts of mutant alleles at equilibrium are altered by the variability of counts about their means, the variability which drives the whole mutation - selection process . All three of these imperatives, flexible profiles for effects, interactions, and heterogeneity, call for a fully nonlinear model, such as the one applied here . The need for such a model seems to have been appreciated already in brian charlesworth s (2001) pathbreaking paper . In his section 4 he sought to incorporate nonlinear interactions through an iterative numerical procedure, making survivorship at each step in time depend on the previous mean accumulation of mutant alleles en route to an equilibrium . This procedure suppresses heterogeneity and leads to different answers from our fully nonlinear model, but in some circumstances it generates usable approximations . The point - mass setting, nonlinearity can produce outcomes qualitatively different from those predicted with the linearized approach, as shown in steinsaltz et al . The full model also makes it possible to prove conditions for the existence of equilibria and walls of death . In our application here, mutant alleles arise that each increase age - specific mortality rates according to the profile of a gamma probability density function . The model builds in the nonlinear demographic interactions among the accumulating mutant alleles, and takes explicit account of the heterogeneity in genetic endowment among individuals . Investigating a range of choices of parameter values, we show that the features of prime demographic interest, gompertzian stretches and late - age plateaus, can be produced within this setting . The gamma profiles adopted here are reminiscent of functional forms common in reliability models, but no precise analogy is intended . Our choice was guided by the idea that the essential organs of gavrilov and gavrilova (1991) might be replaced by a large number of useful organs, of similar internal redundancy, whose propensity to failure could be triggered or exacerbated by the presence of one or more mutant alleles . Ultimately we hope it will be feasible to situate reliability models explicitly within the context of mutation accumulation . The evolutionary unified failure theory of our aspiration would also need to incorporate elements of optimal life history, as well as accounting for the complex hierarchy of trade - offs, from the level of single genes and organelles up to ecosystems, and on timescales from the milliseconds of rna transcription to the millennia of evolutionary time . For our present, more modest, purposes the gamma family was chosen because it has the desired property that late - age increments in mortality are systematically tied to early - age increments in a fashion that varies smoothly with the mean age of effect . This specification takes us beyond the highly stylized setting of point - mass cases, while retaining enough familiarity for ready interpretation . We describe the model in the section headed the mutation - selection model, the formulas that go into demographic calculations in the section headed formulas, and the detailed specification of ingredients and parameters in the section headed specifications . We present the mortality outcomes predicted by the theory in the section headed predictions . Medawar s idea of mutation accumulation as a cause of senescence depends upon the action of large numbers of mutations, each with small deleterious effects on survival at specific ranges of age . Mutations which affect young ages are weeded out of the population quickly by natural selection, because members who carry them contribute fewer offspring to the next generation . Mutations affecting older individuals, with less reproductive potential remaining to lose, are weeded out less rapidly . While weeding progresses, mutant alleles accumulate until a balance is reached between the force of mutation and the force of selection . All things being equal, the less costly mutations those that produce their harm later will be more common at equilibrium . Our model for mutation accumulation is an infinite - population model in continuous time with large or infinite numbers of genetic loci, in the tradition of a famous paper by kimura and maruyama (1966). The version applied here incorporates what we call free recombination, in which recombination is assumed to operate on a more rapid time scale than mutation and selection . (2006) which shows that this version can be regarded as a limiting case of discrete - generation models in the limit of weak selection and mutation . A companion version in which recombination is assumed to be negligible is developed in steinsaltz et al . Our two treatments of recombination bracket a potential continuum of more complex treatments . For each version of the model, there are analytic solutions available to describe entire time trajectories for the population . In this paper, we are primarily concerned with equilibrium states . Equilibrium states are distributions of genotypes which are stable in time under the joint action of mutation and selection . In many situations, including those treated here, we can prove that there is a unique equilibrium state, and that this state represents the distribution to which the population converges over time . The accumulating mutations under study here are germ - line mutations maintained in the genome over long stretches of evolutionary time . Our framework may also have some application to somatic mutations accumulating within the cells of an individual individual organism during its lifecourse, but that is not our current focus . First is a set of profiles for the age - specific action of deleterious mutant alleles . Second is a specification of the rates at which mutant alleles of different kinds arise, the mutation part of mutation - selection balance . Third is a function determining selective cost, the selection part of mutation - selection balance . In this section our choices for the ingredients, which serve as illustrations in this section, are spelled out in detail in the section headed specifications . Examples of profiles are the four functions of age in fig . 1, a figure discussed further in the section headed specifications . A profile is added onto the age - specific hazard function for each mutant allele carried by an individual . 1gamma profiles for increments to the hazard function for four selected values of the mutation index m, namely 1.125, 2.250, 4.125, and 6.000 (from left to right as they rise from the axis). Background parameters are = 15 and = 1/20, and = 0.100 gamma profiles for increments to the hazard function for four selected values of the mutation index m, namely 1.125, 2.250, 4.125, and 6.000 (from left to right as they rise from the axis). Background parameters are = 15 and = 1/20, and = 0.100 in general, we posit a set \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\mathcal{m}$\end{document} of potential mutations fitted with a geometric structure to allow us to describe the process of picking a new set of random mutations which are passed on to the next generation . In our application, the profiles form a one - parameter family of curves, and we can identify \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\mathcal{m}$\end{document} with the interval of the real line containing the permitted values of the parameter . Picking a set of random mutations comes down to picking a random set of points from the real line, what probability theorists call a point process, in this case a poisson point process . No attempt is being made to identify alleles with genes on chromosomes or otherwise to model biological structures . Versatile age - specific structure is achieved in conjunction with a degree of stylization in the representation of the genome . Our second ingredient gives rates at which mutant alleles arise, rates expressed in general by a measure on \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\mathcal{m}$\end{document}. When we take \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\mathcal{m}$\end{document} to be a real interval, we can identify with a non - negative function, the density of the measure, and we often take constant over the interval for the sake of having a neutral choice . It is convenient to scale the time axis so that one unit of time corresponds to one generation in discrete settings . Our third ingredient is a selective cost function s. it evaluates (on a logarithmic scale) the loss in fitness produced by any batch of mutant alleles which an individual may carry . The alleles are dominant, back - mutation is not allowed, and costs are evaluated under the assumption that age - specific fertility rates fx are being rescaled to keep population size stationary and are otherwise exogenous . The general form of the model allows fertility as well as mortality to be shaped endogenously by the action of deleterious mutations with a background level of any chosen form, but those options are not pursued in this paper . In the present context, as discussed in wachter et al . (2008), following charlesworth (2000), page 930, there are good reasons for identifying the selective cost of a batch of mutations with the resulting lifetime loss of net reproduction, and we do so here . We now introduce formal notation and describe how the ingredients of our model fit together in terms of the formulas from which predictions are calculated . Genotype as shorthand to refer to the finite batch of mutant alleles carried by a member of the population . Alleles with the same profile of action are treated as copies of the same allele even though they are found at different sites in the genome . The survivorship function x(g) for a subpopulation of members with genotype g is the proportion of members of the subpopulation living beyond age x. when we take the logarithm of x(g) and multiply by 1, we obtain the cumulative hazard function, whose derivative, when it exists, is the hazard function itself, also called the force of mortality . The cumulative hazard function at age x is the area under the hazard function up to age x. we work with cumulative hazard functions in order to have simpler expressions for survivorships x, as well as to have formulas that apply without change to discrete - age and continuous - age cases . We write (m) (m, x) for the increment to the cumulative hazard function at age x produced by allele m from \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\mathcal{m}$\end{document}. The cumulative profile function (m, x)the area up to age x under a curve like the curves in fig . 1represents the shape of the age profile of mortality effects, normalised to have total effect 1: that is, with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\int_{0}^{\infty}\kappa(m, x)dx=1$\end{document}. The factor (m) then adjusts the overall size of the effect . To write the survivorship function, we start with an exogenous baseline cumulative hazard (x) and add to it a term (m)(m, x) for each m in the batch g to obtain the cumulative hazard . We multiply the cumulative hazard by 1 and apply the exponential function to obtain the survivorship . We write capital g for the random batch of mutant alleles carried by an individual selected at random from the population . The count of alleles in g with values of m in any given subset of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\mathcal{m}$\end{document} is thus a random variable . The mean of this random variable is just the population average number of mutant alleles in the interval (2, 3), and it equals the area within the interval (2,3) under a curve called the intensity of g. we now touch on some probability theory, the tool which enables us to go beyond linear approximations and treat interactions and heterogeneity . The intensity gives information about overall genetic load, but on its own it may not provide a complete description of the genetics of the population . The function only specifies for each region of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\mathcal{m}$\end{document} the population average number of mutant alleles in that region and a priori does not enable one to compute the proportion of the population that have more than some number of mutant alleles in a given region of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\mathcal{m}$\end{document} or to determine whether a randomly chosen individual who happens to have larger than expected numbers of mutant alleles in one region is more or less likely to have a larger than expected number in another region . In this model, there is a distribution of genotypes, which are batches of mutant alleles from \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\mathcal{m}$\end{document}; the intensity only describes the overall frequency of each mutation, with no information about its genetic partners . When selective costs are linear effectively, the non - epistatic case in which distinct loci evolve independently the genotype distribution is a poisson random measure, a mathematical construct whose properties are described, for instance, in kallenberg (1983). Intuitively, the genotype of an individual sampled at random from the population can be described by going through \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\mathcal{m}$\end{document} point by point, and taking mutations independently at random with probabilities governed by . In the setting of interest to us, however, when the selective cost is nonlinear, there will be a complex structure of interactions between mutations . It is surprising, then, that the simple poisson structure returns, regardless of the complexity of the epistasis, in our model with free recombination, as shown in evans et al . While distinct loci now do not evolve independently, the distribution at any given time does have a the structure of a poisson random measure and is completely described by the intensity alone . The population average or expectation value of any quantity of interest depends only on; we use the notation \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\mathbb{e}_{\rho}$\end{document}. Our goal, then, is to determine the intensity (m) of mutations at equilibrium . From it we can find the expected (aggregate) population survival curve \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\mathbb{e}_{\rho} \, [\ell_x(g)]$\end{document}, the proportion of the whole population living beyond age x. the selective cost s(g) for genotype g is calculated under our assumption of zero population growth and is given by the difference in its net reproduction ratio from the net reproduction ratio for the null genotype: 1\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\label{e: sofg} s(g): = \int \, f_x \, \ell_x(0) \, dx - \int \, f_x \, \ell_x(g) \, dx . $$\end{document}survivorship for genotype g is given by 2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\label{e: ellxofg} \ell_x(g) = \exp \left(- \lambda(x) - \sum\limits_{m \in g} \, \eta(m) \kappa(m, x) \right). (2006) along with properties of poisson point processes imply that aggregate survivorship is given by 3\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\label{e: elxg} \mathbb{e}_{\rho} \, [\ell_x(g)] = \ell_x(0) \exp \left (- \int (1- e^{-\eta \, \kappa(m, x)}) \, \rho(m) \, dm \right). $$\end{document} the slope of minus the logarithm of the left - hand side is the population hazard . The increment to the cumulative population hazard due to the accumulation of copies of allele m can be written 4\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\label{e: hmx} h(m, x) = (1- e^{-\eta \, \kappa(m, x)}) \, \rho(m). Takes the form \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\ell_x(0) \exp (- \int h(m, x) dm) $\end{document}. The equilibrium intensity has to satisfy 5\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\label{e: kapintegral} 0 = \nu(m) - \rho(m) \, \int \, (1 - e^{-\eta \kappa(m, x)}) \, f_x \mathbb{e}_{\rho} \, [\ell_x(g)] \, dx $$\end{document} the eq . We start out with 0 0, corresponding to the null genotype and, supposing we have already constructed the approximate solutions,...,, define by 6\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\rho^{n+1}(m): = \nu(m) \bigg, f_x \mathbb{e}_{\rho^n} \, [\ell_x(g)] \, dx \right]. $$\end{document}under appropriate conditions, it is possible to prove that this sequence does in fact converge to a solution of eq . 5 . For our numerical calculations, we approximate the continuous range of values of m by a grid with one thousand points and evaluate integrals over age by a grid with steps of 0.10 years . Calculations are implemented in the open - source r statistical system based on the computer system s developed at bell laboratories . We now turn to the detailed specification of the cases treated in this paper, going into the particular choices for the three ingredients of the model and accompanying parameters . The first ingredient, the profiles for mutational action, have been introduced in the section headed mutation accumulation . We set 7\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\label{e: gamma} \kappa(m, x) = \begin{cases} (1 / \gamma(m)) \, \int_{\alpha}^x \, \phi^{m} \, (y-\alpha)^{m-1} \, e^{-\phi (y-\alpha)} \, dy, & \quad x \ge \alpha, \\ 0, & \quad x <\alpha . \end{cases} $$\end{document}the profile function (m, x) is the cumulative distribution function for a shifted gamma probability distribution . The gamma shape parameter equals the index value m and varies from allele to allele . The shift for the origin is the age of maturity; alleles affect only adult mortality . Each effect is assigned an effect size (m) which adjusts the strength of the action . In addition to their association with reliability models, gamma distribution functions offer advantages of familiarity and flexibility . They offer a clear contrast to the point - mass profiles going back to w. d. hamilton already studied in wachter et al . (2008). In the point - mass setting, (m, x) is a unit step - function and m indexes the age at the step . In our present setting, higher values of m still correspond to later - acting alleles, but effects are spread across ages, with wider spread for later - acting alleles . Even late - acting alleles have some small effect at young adult ages, a salient difference from the point - mass case . The more the mutational effect is spread over older ages, the lower is the selective cost, and the more copies there will be of m on average when natural selection manages to balance recurrent mutation . The mean age of action for allele m is the mean of the shifted gamma distribution + m/, the mode is + (m 1)/, and the standard deviation in age of action is \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\sqrt{m}/\phi$\end{document}. Figure 1 shows the shapes of the age - specific increments to the hazard function for four typical alleles in our setting, with 0.100, = 15, and m equal to 1.125, 2.250, 4.125, and 6.000 . The value = 0.100 is a typical standard value . We discuss the impact of other values in the section headed predictions . Our second ingredient, the mutation rate (m), is taken to be constant over an interval [1,] and zero outside it . In the choice of a constant mutation rate we follow the practice of charlesworth (2001), seeking to keep our assumption about mutation as neutral as possible . The total rate per generation tot of the deleterious mutations treated in the model amounts to the length of the interval, 1, times the value in the interval . We consider cases with between 5 and 7 and tot between 0.120 and 0.170 per generation . The final ingredient of model specification is the selective cost of a batch of mutations . We assume that mutations affect an individual s fitness only through their effect on mortality rates, and that the cumulative mortality effects of multiple mutations are additive contributions to the hazard function . Our selective cost function is a difference in net reproduction ratios, quantities which depend on fertility as well as survival . We assume a fixed fertility schedule fx equal to 0 below an age of maturity and above a latest age at reproduction and equal to a non - zero constant between these ages . For the predictions of the section headed predictions, = 15 and = 50 . The inclusion of an upper age limit on fertility is important to the interpretation of our results . The values of the age - specific profile (m, x) for x above are irrelevant to the selective cost imposed by m and therefore to the equilibrium frequency of m, but they make significant contributions to the predicted post - reproductive hazard . It is the association between early - age and late - age hazards built into our family of profiles that drives the predicted outcomes . Biologically, we are assuming a correlation between young and old ages in phenotypic effects . Reasons for doing so, in relation to reliability models for aging, have been discussed in the section headed mutation accumulation . The correlation across ages prevents the occurrence of a wall of death at the end of reproduction, and shapes the old - age hazards . The selective cost function also depends on the choice of baseline survival schedule, the schedule for the null genotype . Following the lead of charlesworth (2001), we assume a constant baseline hazard above the age of maturity, corresponding to a cumulative baseline hazard (x) = (x) above and zero below . Since we are rescaling fertility to achieve stationarity, pre - reproductive mortality can be ignored . The baseline hazard can be taken to represent a minimum realizable rate, sometimes identified with the so - called the extrinsic mortality rate despite the problems inherent in this notion discussed by williams and day (2003). As with fertility, our choice of baseline hazard is intended to be as neutral as possible, in order to concentrate on structure arising from the dynamics of mutation and selection . We now examine predicted hazard functions at mutation - selection equilibrium when the age - specific action of mutant alleles takes the form of gamma profiles described in the section headed specifications . We begin with a case chosen to serve as a standard example, to which we shall compare other cases . 2predicted hazard for a standard example with = = 1/20, tot = 0.150, = 6, = 15, and = 50 predicted hazard for a standard example with = = 1/20, tot = 0.150, = 6, = 15, and = 50 for our standard example, we set the upper cutoff on shape parameters = 6, and the total mutation rate tot = 0.150, along with an effect size constant at 0.100 . A baseline mortality level = 1/20, and a rate parameter = 1/20 . The maximum increment at any one age associated with our gamma profiles is then a little more than three per thousand per year . For the sake of analogy with human life history, we set the age of initial reproduction (also the age of earliest action of the mortality profiles) to be = 15, and the age at end of reproduction to be = 50 . It rises slowly from the background level and then accelerates, giving the impression of a gompertz - makeham curve in the middle of the age range, and straightening out at older ages . About one in ten - thousand individuals survive beyond age 70 . In this illustration, the equilibrium density of mutations turns out to be closely approximated by an exponential function of the shape parameter, namely \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$\rho(m) \approx 0.170 \exp (1.377 \, m) $\end{document}. On average, individuals in the population carry about two mutant alleles with m <2.0, a bit over a dozen with 3.5 <m <4.0, and nearly three - hundred with 5.5 <m <6.0, for an average total of 526 . The poisson standard deviation of the total number of alleles across individuals is around 23 . The effect for a given m peaks at age 15 + (m 1)/. The effects for m <2.0 are peaking before age 35, an age to which most members of the population survive . The cost in net reproduction from an additional mutation affecting these ages is high, and selection keeps their equilibrium representation low . Effects for m> 5.5 only become substantial at ages at which most individuals have already died . Selective costs are low and copies persist long enough to be found at high numbers in the population despite the rarity of new mutations . Figure 3 shows the logarithm of the population hazard rate for three comparative cases . The dotted curve has tot = 0.170 and an upper shape parameter cutoff of = 5.5 . The dashed curve has tot = 0.120 and = 7 . These alternatives have been chosen from among cases for which the predicted equilibrium hazard is between 0.300 and 0.550 at the age to which one in ten thousand survive, respectively equal to 69.9, 71.9, and 66.7 years . 3logarithm of predicted hazard for three cases showing early upward bend, straight middle gompertzian stretch, and late downward bend . The cases all have = = 1/20 and 0.100 . The solid curve has tot = 0.150, and = 6.0; the dashed curve has tot = 0.170, and = 5.5; the dotted curve has tot = 0.120, and = 7.0 logarithm of predicted hazard for three cases showing early upward bend, straight middle gompertzian stretch, and late downward bend . The cases all have = = 1/20 and 0.100 . The solid curve has tot = 0.150, and = 6.0; the dashed curve has tot = 0.170, and = 5.5; the dotted curve has tot = 0.120, and = 7.0 the higher hazards at old ages in the dotted curve are due to the presence of later - acting alleles with m ranging up to 7 . These alleles have effects whose age - specific profiles increase throughout the range of ages to which population members survive . Although the mutation rate is lower for the dotted curve, the shapes of the age - specific effects lead to higher hazards toward the end of life . We see that mutation accumulation with the given profiles and parameters produces a long middle stretch of nearly loglinear hazards, corresponding to a gompertz form . At young ages the curves are convex on the logarithmic scale, bending upward, as effects of mutant alleles come into play . At older ages, accumulation of mutational effects concentrated at late ages is held in check by their small accompanying effects at young ages in this specification . The interactions among effects at different ages taken into account by the nonlinear model turn out to have a substantial impact on predictions, as expected from results in wachter et al . We compare predictions from the full nonlinear model to predictions from a linear approximate model of the kind on which earlier studies have relied . Figure 4 shows the population survivorship function for our standard example in a thick line, along with baseline survivorship in a dashed line, and survivorship from the linear approximate model between them in a dotted line . Only about a third of the reduction in life expectancy from 35.0 years to 28.8 years due to 4probability of survival by age for the standard example as predicted by the full nonlinear model (circles), by a linear approximate model (dots), and by the baseline model probability of survival by age for the standard example as predicted by the full nonlinear model (circles), by a linear approximate model (dots), and by the baseline model the sizes of effects, in contrast to their shapes, turn out to have only modest influence on the predictions . Alleles with smaller effects accumulate at equilibrium in greater numbers . Changes in the intensity roughly balance changes in effect size . Figure 5 shows the predicted hazard functions with parameters taken from our standard example but with different choices of . Fig . 5the influence of effect size is shown with predicted hazard functions from seven cases described in the text, some with indistinguishable outcomes, sharing all parameter values except effect size with the example of fig . 1 the influence of effect size is shown with predicted hazard functions from seven cases described in the text, some with indistinguishable outcomes, sharing all parameter values except effect size with the example of fig . 1 three uppermost curves, almost indistinguishable, have constant = 0.0001, = 0.001, and = 0.010 . The mean total number of mutant alleles runs to a bit over five hundred thousand in the first case, fifty - thousand in the second, and five thousand in the third . Three slightly lower curves, also hardly distinguishable, include our example with constant at 0.100 and two examples with changing, one rising linearly with the shape parameter from 0.020 to 0.200 and one falling linearly from 0.200 to 0.020 . Small effect sizes accompany larger mean numbers when they occur for alleles with late action . Only as becomes this large, outside the range of intended application of the model, do we see substantially different predicted hazard functions . The approximate invariance of predicted hazard functions with effect sizes is an expression of haldane s principle, enunciated by haldane (1937) and discussed in terms of our nonlinear models in wachter et al . 4, the contribution h(m, x) from allele m is nearly linear in (m) (m) for small, so scaling (m) up can be nearly compensated, allele by allele, by scaling (m). One of the most familiar general predictions of the evolutionary theory of senescence is a positive relationship between the extrinsic mortality rate associated with unavoidable risks in natural settings such as predation and accidents that are present even the young and healthy, and the rate of senescence measured by the slope of the logarithm of the hazard rate with respect to age . Our predictions hint at such a relationship, but only for substantial values of the baseline hazard . Figure 6 shows the logarithms of the predicted equilibrium hazard for our standard set of parameters as the level of the constant baseline hazard is raised from 0.020 to 0.050 and on to 0.080 . 6the influence of the level of extrinsic hazards on the pace of senescent mortality is shown by predicted log hazards from three cases sharing parameters with the standard example of fig . 1 except for, which ranges, from bottom to top, over values 0.020, 0.050, and 0.080 the influence of the level of extrinsic hazards on the pace of senescent mortality is shown by predicted log hazards from three cases sharing parameters with the standard example of fig . 1 except for, which ranges, from bottom to top, over values 0.020, 0.050, and 0.080 slopes computed over the middle range of ages from 30 to 50 to which a gompertz fit is roughly appropriate hover around 0.050 for the first two cases but rise to 0.074 as increases to 0.080 . In cases not shown here in which non - zero fertility extends to higher ages, there is a closer match between values of the slope and values of the parameter itself, paralleling a relationship found with linear approximate models in charlesworth (2001). The stretch of ages with exponentially increasing hazards, corresponding to linear increase in log hazards, visible in figs . 2 and 3 does not extend out to extreme ages . Attenuation of increase is already visible in the upper ages toward right of those figures . We focus on this attenuation in fig . 7, which shows the predicted hazard rate for the same standard example of fig . 2 but with a horizontal axis extending all the way out to an age of 120 years . Around 100 years, the hazard levels off, establishing a brief plateau phase, and by 120 years a declining hazard is apparent . Only one in ten billion survive to 100 years with our standard parameter choices, but the plateau and subsequent decline are to be expected with parameters leading to milder mortality regimes as well . Fig . 7a plateau in the predicted hazard function at extreme ages in the standard example of fig . 2 with a longer range of ages a plateau in the predicted hazard function at extreme ages in the standard example of fig . 2 with a longer range of ages the plateau at extreme ages is due to the property of the profiles for the age - specific action of mutant alleles to which we have already called attention, namely that even alleles whose action is spread over old ages all have some small effects at young ages . They prevent any wall of death; that is, any finite age at which the hazard rate goes to infinity and survivorship reaches zero . Walls of death occur in many elementary cases for profiles with hamilton - style, point - mass profiles, as shown in wachter et al . The proposal for generating plateaus by assuming some small effects at young ages for all mutant alleles was put forward by charlesworth (2001) and shown to be valid for the linear approximate model . We now see that these outcomes also hold in the full nonlinear model with the particular profiles we are studying . In summary, we have found that the process of mutation accumulation can readily produce predicted population hazard functions with the chief features highlighted by the cross - species comparisons of biodemographers . It can produce a stretch of ages with an exponential, gompertzian rise in hazards and it can produce a late - age hazard plateau . These outcomes arise from a set of assumptions about the age - specific action of mutant alleles that are suggested by examples from reliability theory and from the functional approach to the study of senescence . It remains, however, to develop comprehensive models in which the generic mutation accumulation machinery is driven by plausible genetic and physiological mechanisms, and in which age - specific tradeoffs are derived compellingly from reliability theory . It also remains to be determined whether the examples studied here are typical, or whether they represent peculiar outcomes of our specific choices of parameter values . More generally, the field is open for attempts to characterize the conditions under which the force of natural selection in the presence of recurring deleterious mutation will mold hazard functions into familiar forms.
Muscle activation varies according to the size and shape of the driver s hand grip1,2,3 and it affects the amount of discomfort and fatigue they experience while they are driving . Most of them use a spinner knob with their non - affected hand instead of a steering wheel . A spinner knob helps a driver control the steering wheel with only one hand . . However, there is a lack of research about analyzing the driving performance of drivers who have had a stroke . Therefore, the purpose of this study was to investigate the muscle activates of drivers with post - stoke hemiplegia while driving using a steering wheel and a spinner knob, and compare them with those of non - disabled drivers . While 10 participants were originally selected for this study, a total of nine drivers participated in this experiment: five drivers with post - stroke hemiplegia (experimental group) and four non - disabled drivers (control group). The stroke group did not have any neurological, musculoskeletal, cognitive, or perceptual problems except for the stroke . In terms of driving history, they had been driving for more than three years after they had a stroke . The control group consisted of four drivers of a similar age and gender as the experimental group . However, one driver in the control group elected to drop out of the study during the experimental process . This study was conducted with 3 males and 2 females in the experimental group and 3 males and 1 female in the control group . The average height of the experimental group was 163.5 cm, and that of the control group was 162.75 cm, and their average weights were 60.75 and 59.25 kg, respectively . The average driving experience of the experimental and control groups was 15 years and 6 months, and 14 years and 5 months, respectively . The subjects participated in this study after receiving an explanation of purpose of this study and had signed a consent form . The driver s seat angle was 1055 and the height of the seat was 20.8 inch6 . A researcher adjusted the distance between the driver and the steering wheel to maintain a 113 angle of the elbow when the participant held the steering wheel at a 30 angle to the right from the midline7 (fig . A spinner knob was set at an angle 30 clockwise from the center of the upper part of the steering wheel because the drivers in the experimental group had post stroke, left side hemiplegia . The speed limit was at least 10 km / h but not more than 20 km / h . The experimental task consisted of driving forward in a straight line for 5 m, turning right or left, and driving straight ahead for another 5 m. the participants in the experimental group and the control group were instructed to only use either the steering wheel or the spinner knob with only their right hand . Muscle activities were measured using surface emg (telemyo 2400 t g2, noraxon usa, inc ., the collected surface emg signals were converted into digital signals by a telemyo 2400 t g2, and the converted signals were processed using myoresearch master xp 1.07 software . Electrodes were placed over the anterior deltoid, biceps brachii and triceps brachii on the right - side according to the method suggested by mo et al8 . The emg data were converted to root - mean - square values that provided values close to the actual output values of the emg signals . For quantification, the emg signal volumes as a percentage of the reference voluntary contraction (% rvc) were measured . The reference voluntary contraction was measured in a sitting posture with the knees and hips flexed at 90. before beginning the experiment, emg was measured when the participant flexed the elbow and then flexed the shoulder, extending the elbow with a 2-kg dumbbell in the hand . Data were analyzed using spss 18.0 . To test the homogeneity of age, gender, driving history, car model, and driver licenses classification between the experimental group and the control group, the test was performed . To compare activation of the three muscles between the two groups, the mann - whitney u test was performed . To compare the muscle activation between the spinner knob and the steering wheel by group, the results of the comparison of the activation of the three muscles between the steering wheel and the spinner knob while the drivers in the experimental group turned the vehicle to the left were as follows . There was no significant difference in the activation of the three muscles between the spinner knob and the steering wheel when drivers in the experimental group turned to the left . When the drivers in the experimental group turned to the right, a significant difference in the activation of the muscles between the spinner knob and the steering was found only in the biceps brachii . The activities of the three muscles between the steering wheel and the spinner knob when the control group turned the vehicle to the left showed no significant differences . When the control group turned the vehicle to the right, there were no significant differences in activities of the three muscles between the spinner knob and the steering wheel . Activation of the biceps brachii while turning the vehicle to the right with the spinner knob was significantly lower in the experimental group than in the control group (table 1table 1.muscle activation during left and right turns made with a spinner knob and a steering wheel (n=10) (unit:% rms)strokecontrolspinner knobsteering wheelspinner knobsteering wheelturning to the leftanterior deltoid27.19.127.25.235.011.738.312.5biceps brachii17.81.715.94.122.910.337.54.7triceps brachii22.310.020.39.138.83.636.66.7turning to the rightanterior deltoid25.85.626.13.931.212.732.916.7biceps brachii15.13.222.13.8 38.79.9 * 36.28.2triceps brachii22.98.126.916.341.817.242.718.4*significant difference between stroke group and control group at 0.05 level . As the number of drivers with post - stroke hemiplegia grows, driving rehabilitation becomes more important . To help these drivers, occupational therapists need to analyze their driving performance . The aim of this study was to investigate and compare muscle activation differences between using a spinner knob and a steering wheel in drivers with post - stroke hemiplegia and non - disabled drivers . The experimental group was comprised of five drivers with post - stroke, left - side hemiplegia . Because the required devices, driving position, and other factors may vary depending on the affected side, this study included only drivers with left - side hemiplegia . When the drivers in the experimental group turned the vehicle to the right using a steering wheel or a spinner knob, the activation of the biceps brachii was significantly different . Mo et al.8 reported that the biceps brachii was activated when a vehicle was turned to the right, and the anterior deltoid and the triceps were also activated when engaging in that movement . However, in this study, activation of the triceps and the anterior deltoid was not observed . With regard to the control motion of a spinner knob or a steering wheel when turning a vehicle to the right therefore, only activation of the biceps brachii was found to be significantly different between using a steering wheel and a spinner knob . It is interesting to note that activation of the biceps brachii was not significantly different between using a steering wheel and a spinner knob in the control group, unlike drivers in the experimental group . This shows that a spinner knob requires less muscle activation than a steering wheel for drivers with stroke . The results of the comparison of the activation of the biceps brachii between the two groups show that, the biceps activation in the drivers in the experimental group was lower than that in the control group . This could be because four of the five drivers in the experimental group usually used a spinner knob, while none of the drivers in the control group used a spinner knob . Adaptation might influence activation of the related muscles9 . Because drivers with stroke have difficulty with bilateral hand use, a number of them use a spinner knob instead of a steering wheel . Occupational therapists must know how to analyze a stroke patient s driving performance and understand how an adaptive driving device can help them to drive safely . The results of this study indicate that a spinner knob requires less activation of the main muscles than a steering wheel, especially in drivers who have had a stroke . Therefore, in the future, research on the time it takes to adapt to using a spinner knob and other adaptive devices will be needed . Limitations of this study are that all the participants in the experimental group were drivers with left hemiplegia, and the number of participants was insufficient to generalize the results . Although there was a speed limit for the driving, the drivers could nt keep a consistent speed.
Copd is a large and growing public health problem; it is now the most costly respiratory disease in europe.1 copd is an irreversible and debilitating disease that progresses through different stages and has a huge impact on patients functional performance and quality of life.2 it is also characterized by episodes of acute exacerbation that are a particular problem in the control of the disease because of the negative impact on quality of life,3,4 prognosis, and costs.5,6 in addition to the severity of copd, there are other concomitant factors, such as comorbidities and unmet social / health needs due to poor self - management of the disease, which can affect rates of hospital admissions and use of health care resources for these patients.7 in this context, different nonpharmacological interventions involving self - management are being developed, based on chronic care models8 with the aim of improving prognosis and reducing the use of resources.9 self - management in copd patients was defined as active involvement in the management of their disease, based on sufficient coping behavior to achieve optimal compliance and be able to take action against symptoms of exacerbation.10 the term self - management plan can apply to any formal education program aimed at teaching the skills needed to follow specific medical regimens, guiding the change toward healthy attitudes and providing support to patients to help them manage their disease.11 to date, there are few data available regarding which methods are the most effective in improving self - management, especially in reducing the number of exacerbations . One potentially effective method might be to help patients recognize and anticipate the initial symptoms of an exacerbation by way of an action plan . There is only a relatively moderate amount of evidence on the efficacy of self - management programs in copd . Such programs have mainly been directed at other chronic conditions, such as heart failure, diabetes, and asthma, where, in fact, they have been successful in achieving significant improvements in many processes, although few studies have demonstrated significantly reduced costs.12,13 a cochrane systematic review including three controlled trials evaluated written action plans only as a single intervention component, with a relatively small number of patients and methodological limitations, showing no beneficial effects on clinical outcomes or resource use, although they did improve self - management strategies against exacerbations.14 in contrast, another systematic review on the effectiveness of the chronic care model in copd found that patients who received interventions with two or more components had lower health care resource use compared to controls.15 in the most recent review by zwerink et al,16 self - management interventions in patients with copd were associated with better quality of life, fewer hospital admissions and improved symptoms; however, the heterogeneity of the interventions applied and the outcome measures make it difficult to provide recommendations on which components were most effective . Data on the role of self - management plans for copd in improving the use of health care resources or patient health remain insufficient, and further research is needed in this area.17 this study evaluates the efficacy of a self - management intervention added to usual care in a randomized, controlled study . Our hypothesis is that a multiple component intervention called a copd self - management program (smp - copd), led by a multidisciplinary team, could significantly reduce the use of health care resources and, in particular, the number of exacerbations requiring hospital care . Patients were recruited between february 2012 and march 2013 from two hospitals (hospital morales meseguer for the health area vi and hospital arrixaca for the health area i) in the autonomous region of murcia (spain). Each hospital served mainly an urban area with a population of 250,000 people; they had accident and emergency (a&e) units and accepted general and specialized admissions . Patients were recruited from each hospital s database if they had been treated in the a&e or had been hospitalized for an exacerbation of copd at least once during the year prior to inclusion in the study . Patients were called to participate in the study if at least 3 months had elapsed since the episode of hospital care . Patients were included if they met the following criteria: 1) clinical stability (at least in the 3 months prior to randomization, with no change in medication or usual symptoms); 2) active smoker or prior history of smoking of at least 10 pack - years; 3) post - bronchodilator forced expiratory volume in 1 second / forced vital capacity ratio <70%; 4) normal cognitive status (assessed by the intersecting pentagons test18) to read and understand written texts, and receive training in inhalation techniques or self - care education sessions; 5) physical status that allows for regular walking or exercise; 6) no diagnoses of asthma, advanced heart failure, unstable ischemic heart disease, terminal disease, dementia, or uncontrolled psychiatric disorders; 7) ability to read texts; 8) no participation in any pulmonary rehabilitation program in the previous year . The study was approved by the institutional review boards of the hospitals comit tico de investigacin clnica del hospital general universitario jos mara morales meseguer and comit tico de investigacin clnica del hospital arrixaca . Controlled, randomized, parallel - group, single - blind study with follow - up of 1 year . After consenting to take part in the study, simple randomization was carried out separately at each site by means of a list of computer - generated random numbers, assigning the patients to two groups . The intervention group (ig) received the smp - copd program on an individualized basis . The comparison group, or control group (cg), received routine care and attended routine visits . Because double - blinding was not possible, an independent evaluator, who did not know the patients group assignments, was responsible for evaluating the outcome variables . Cg patients and ig patients continued to see their respective specialists and primary care physicians, and had access to health care or public health programs and home respiratory therapies . Each patient randomized to the ig received the smp - copd, which consisted of a group education session on the main characteristics of the disease, an individual training session on inhalation techniques according to the devices indicated for each patient, and an action plan with written material consisting of color - coded sheets with treatment instructions for the stable periods, including recommendations for physical exercise (green) and exacerbations (orange). In addition to the visits at the start and the end of the study (the same for both groups), the ig had two extra visits with a respiratory nurse (at 1 and 3 months into the study), primarily to check on the correct use of the treatment instructions sheets and inhalation techniques . Training in inhalation techniques was a systematic, protocol - based process for training all patients in the ig, individually teaching correct administration technique for each prescribed inhaler, with particular emphasis on both avoiding critical errors and adherence . The smp - copd was taught and supervised by professionals previously trained in the intervention s features: six nurses, two physiotherapists, and six medical specialists in respiratory medicine . The material in the group education session was designed specifically for the program and consisted of a powerpoint presentation with 20 slides on the main characteristics of the disease, symptoms of exacerbation, and inhaled medicines . At the end, there was a chance for questions and a physiotherapist demonstrated how to do a series of basic physical exercises . Each session was delivered by a previously trained nurse to a group of six to eight patients . The action plan consisted of a folder containing written material with four kinds of sheets . Sheets with instructions on treatment and physical exercises for stable periods (green), treatment sheets for exacerbations (orange), and a red sheet with instructions to follow in the case of their condition becoming serious or an emergency . The exacerbation sheets explained the symptoms of bronchial infection for which they should start antibiotics and that if the symptoms did not improve within 48 hours or they developed dyspnea, they should start a course of oral glucocorticoids for 6 days . There were also instruction sheets on inhalation techniques, which explained the correct use and main features of the type of inhaler indicated for each patient . To ensure proper understanding of the texts included in the sheets, their readability was assessed by the computer program microsoft word 2000 (19831999 microsoft corporation, redmon, wa, usa). All the study visits were conducted at one of the two participating hospitals (a or b). Sociodemographic variables were collected at the baseline visit and included: respiratory symptoms; medical history and physical examination; body mass index; smoking status and accumulated intake; spirometry parameters; dyspnea (modified medical research council dyspnea scale);19 quality of life20 (copd assessment test); severity of copd according to the global initiative for chronic obstructive lung disease;21 comorbidity index;22 respiratory medicines; and a social - risk questionnaire.23 the latter is a hetero - administered questionnaire validated for the detection of social risk that explores the five areas of family, financial and housing situations, social relationships, and social support networks (a score> 15 indicates social problems). Information was collected on the use of antibiotics and glucocorticoids, number of visits to a hospital, a&es, and admissions, including length of hospital stay for copd exacerbation during the follow - up period . The primary outcome was the combined number of hospital admissions and a&e visits for copd during the 12-month follow - up period . Secondary outcomes included individual components of the primary outcome: hospitalizations and a&e visits for copd exacerbations, lengths of stay, number of patients who received antibiotic or glucocorticoid treatment and all - cause mortality . Exacerbation of copd was defined as a sustained worsening of the patient s condition from a stable state, with acute onset and beyond normal day - to - day variations, which requires treatment or additional care.24 hospitalization due to copd was defined as any admission where a hospital bed was used, in any unit and of any duration, and for which the diagnosis was listed as copd aggravation or exacerbation . A visit to the hospital a&e for exacerbation of copd was defined as remaining in this area for over 8 hours and receiving treatment with bronchodilators, parenteral corticosteroids, and oxygen . Sample size: accepting an alpha risk of 0.05 and a beta risk of 0.2 in a two - tailed test, 89 subjects were needed in total to detect an absolute difference of 30% in the reduction of exacerbations with hospital care (visit to a&e or admission), with 70% of the patients expected to be treated in hospital for an exacerbation during the year of follow - up . The analysis of the results was performed using the intention - to - treat principle, where each patient was analyzed in the group to which they were initially randomized . Patients lost to follow - up (inability to follow up due to a change of address) were evaluated until the time of the last contact with them by the research staff . The results were expressed as mean standard deviation for quantitative variables and as percentages for qualitative variables, and 95% confidence intervals were calculated for the outcome variables . The comparison between qualitative variables was performed using pearson s chi - squared test or fisher s exact test . The comparison between quantitative and qualitative variables was performed using student s t - test or the mann whitney u - test, depending on whether the qualitative variable was distributed normally or not . The comparison between quantitative variables was performed with a test to compare the means for paired data . Mcnemar s test was used to compare related qualitative data in addition to the test for comparison of related means . The time - related outcome variables were compared using kaplan meier curves with the log - rank test . All comparisons were made using a two - tailed test . For the analysis, the statistical package spss version 15.0 (ibm spss ., chicago, il, usa) for windows was used . Patients were recruited between february 2012 and march 2013 from two hospitals (hospital morales meseguer for the health area vi and hospital arrixaca for the health area i) in the autonomous region of murcia (spain). Each hospital served mainly an urban area with a population of 250,000 people; they had accident and emergency (a&e) units and accepted general and specialized admissions . Patients were recruited from each hospital s database if they had been treated in the a&e or had been hospitalized for an exacerbation of copd at least once during the year prior to inclusion in the study . Patients were called to participate in the study if at least 3 months had elapsed since the episode of hospital care . Patients were included if they met the following criteria: 1) clinical stability (at least in the 3 months prior to randomization, with no change in medication or usual symptoms); 2) active smoker or prior history of smoking of at least 10 pack - years; 3) post - bronchodilator forced expiratory volume in 1 second / forced vital capacity ratio <70%; 4) normal cognitive status (assessed by the intersecting pentagons test18) to read and understand written texts, and receive training in inhalation techniques or self - care education sessions; 5) physical status that allows for regular walking or exercise; 6) no diagnoses of asthma, advanced heart failure, unstable ischemic heart disease, terminal disease, dementia, or uncontrolled psychiatric disorders; 7) ability to read texts; 8) no participation in any pulmonary rehabilitation program in the previous year . The study was approved by the institutional review boards of the hospitals comit tico de investigacin clnica del hospital general universitario jos mara morales meseguer and comit tico de investigacin clnica del hospital arrixaca . Controlled, randomized, parallel - group, single - blind study with follow - up of 1 year . After consenting to take part in the study, simple randomization was carried out separately at each site by means of a list of computer - generated random numbers, assigning the patients to two groups . The intervention group (ig) received the smp - copd program on an individualized basis . The comparison group, or control group (cg), received routine care and attended routine visits . Because double - blinding was not possible, an independent evaluator, who did not know the patients group assignments, was responsible for evaluating the outcome variables . Cg patients and ig patients continued to see their respective specialists and primary care physicians, and had access to health care or public health programs and home respiratory therapies . Each patient randomized to the ig received the smp - copd, which consisted of a group education session on the main characteristics of the disease, an individual training session on inhalation techniques according to the devices indicated for each patient, and an action plan with written material consisting of color - coded sheets with treatment instructions for the stable periods, including recommendations for physical exercise (green) and exacerbations (orange). In addition to the visits at the start and the end of the study (the same for both groups), the ig had two extra visits with a respiratory nurse (at 1 and 3 months into the study), primarily to check on the correct use of the treatment instructions sheets and inhalation techniques . Training in inhalation techniques was a systematic, protocol - based process for training all patients in the ig, individually teaching correct administration technique for each prescribed inhaler, with particular emphasis on both avoiding critical errors and adherence . The smp - copd was taught and supervised by professionals previously trained in the intervention s features: six nurses, two physiotherapists, and six medical specialists in respiratory medicine . The material in the group education session was designed specifically for the program and consisted of a powerpoint presentation with 20 slides on the main characteristics of the disease, symptoms of exacerbation, and inhaled medicines . At the end, there was a chance for questions and a physiotherapist demonstrated how to do a series of basic physical exercises . Each session was delivered by a previously trained nurse to a group of six to eight patients . The action plan consisted of a folder containing written material with four kinds of sheets . Sheets with instructions on treatment and physical exercises for stable periods (green), treatment sheets for exacerbations (orange), and a red sheet with instructions to follow in the case of their condition becoming serious or an emergency . The exacerbation sheets explained the symptoms of bronchial infection for which they should start antibiotics and that if the symptoms did not improve within 48 hours or they developed dyspnea, they should start a course of oral glucocorticoids for 6 days . There were also instruction sheets on inhalation techniques, which explained the correct use and main features of the type of inhaler indicated for each patient . To ensure proper understanding of the texts included in the sheets, their readability was assessed by the computer program microsoft word 2000 (19831999 microsoft corporation, redmon, wa, usa). All the study visits were conducted at one of the two participating hospitals (a or b). Sociodemographic variables were collected at the baseline visit and included: respiratory symptoms; medical history and physical examination; body mass index; smoking status and accumulated intake; spirometry parameters; dyspnea (modified medical research council dyspnea scale);19 quality of life20 (copd assessment test); severity of copd according to the global initiative for chronic obstructive lung disease;21 comorbidity index;22 respiratory medicines; and a social - risk questionnaire.23 the latter is a hetero - administered questionnaire validated for the detection of social risk that explores the five areas of family, financial and housing situations, social relationships, and social support networks (a score> 15 indicates social problems). Information was collected on the use of antibiotics and glucocorticoids, number of visits to a hospital, a&es, and admissions, including length of hospital stay for copd exacerbation during the follow - up period . The primary outcome was the combined number of hospital admissions and a&e visits for copd during the 12-month follow - up period . Secondary outcomes included individual components of the primary outcome: hospitalizations and a&e visits for copd exacerbations, lengths of stay, number of patients who received antibiotic or glucocorticoid treatment and all - cause mortality . Exacerbation of copd was defined as a sustained worsening of the patient s condition from a stable state, with acute onset and beyond normal day - to - day variations, which requires treatment or additional care.24 hospitalization due to copd was defined as any admission where a hospital bed was used, in any unit and of any duration, and for which the diagnosis was listed as copd aggravation or exacerbation . A visit to the hospital a&e for exacerbation of copd was defined as remaining in this area for over 8 hours and receiving treatment with bronchodilators, parenteral corticosteroids, and oxygen . All the study visits were conducted at one of the two participating hospitals (a or b). Sociodemographic variables were collected at the baseline visit and included: respiratory symptoms; medical history and physical examination; body mass index; smoking status and accumulated intake; spirometry parameters; dyspnea (modified medical research council dyspnea scale);19 quality of life20 (copd assessment test); severity of copd according to the global initiative for chronic obstructive lung disease;21 comorbidity index;22 respiratory medicines; and a social - risk questionnaire.23 the latter is a hetero - administered questionnaire validated for the detection of social risk that explores the five areas of family, financial and housing situations, social relationships, and social support networks (a score> 15 indicates social problems). Information was collected on the use of antibiotics and glucocorticoids, number of visits to a hospital, a&es, and admissions, including length of hospital stay for copd exacerbation during the follow - up period . The primary outcome was the combined number of hospital admissions and a&e visits for copd during the 12-month follow - up period . Secondary outcomes included individual components of the primary outcome: hospitalizations and a&e visits for copd exacerbations, lengths of stay, number of patients who received antibiotic or glucocorticoid treatment and all - cause mortality . Exacerbation of copd was defined as a sustained worsening of the patient s condition from a stable state, with acute onset and beyond normal day - to - day variations, which requires treatment or additional care.24 hospitalization due to copd was defined as any admission where a hospital bed was used, in any unit and of any duration, and for which the diagnosis was listed as copd aggravation or exacerbation . A visit to the hospital a&e for exacerbation of copd was defined as remaining in this area for over 8 hours and receiving treatment with bronchodilators, parenteral corticosteroids, and oxygen . Sample size: accepting an alpha risk of 0.05 and a beta risk of 0.2 in a two - tailed test, 89 subjects were needed in total to detect an absolute difference of 30% in the reduction of exacerbations with hospital care (visit to a&e or admission), with 70% of the patients expected to be treated in hospital for an exacerbation during the year of follow - up . A loss rate of 7% of patients was estimated . The analysis of the results was performed using the intention - to - treat principle, where each patient was analyzed in the group to which they were initially randomized . Patients lost to follow - up (inability to follow up due to a change of address) were evaluated until the time of the last contact with them by the research staff . The results were expressed as mean standard deviation for quantitative variables and as percentages for qualitative variables, and 95% confidence intervals were calculated for the outcome variables . The comparison between qualitative variables was performed using pearson s chi - squared test or fisher s exact test . The comparison between quantitative and qualitative variables was performed using student s t - test or the mann whitney u - test, depending on whether the qualitative variable was distributed normally or not . The comparison between quantitative variables was performed with a test to compare the means for paired data . Mcnemar s test was used to compare related qualitative data in addition to the test for comparison of related means . The time - related outcome variables were compared using kaplan meier curves with the log - rank test . For the analysis, the statistical package spss version 15.0 (ibm spss ., chicago, il, usa) for windows was used . The study patients were followed up from april 2013 to january 2014 . In all, 250 patients from two health regions were recruited . Number excluded: 149 (59.6%); 33 patients (22.1%) due to severe comorbidities (12 cancers, 13 advanced heart failure, six unstable coronary heart disease, and two renal failure); 37 patients (14.8%) who, in the investigator s opinion, were too physically disabled at the time of first visit to benefit from the exercise program in the written action plan and the group educational session; 23 patients (9.2%) in whom the diagnosis of copd was not confirmed; 16 patients (6.4%) due to cognitive impairment (pentagons test); 15 patients (6%) who refused to take part; and finally, 12 patients (4.8%) due to psychosocial problems . Of the 101 patients enrolled, five did not attend the randomization visit . In all, 96 patients (38.4%) were randomized, 45 of whom (46.8%) were included in the cg and 51 (53.1%) in the ig . In the end, 85 patients (88.5%) completed the study: 38 patients in the cg (18 [47.4%] in area vi and 20 [52.6%] in area i); and 47 patients in the ig (25 [53.2%] in area vi and 22 [46.8%] in area i; p=0.593). In the cg, seven patients were lost to follow - up (one protocol violation and six dropouts), and in the ig, four were lost (one protocol violation and three dropouts). The patients recruited for the study are shown in figure 1 . In all, 78 men (91.76%) were included and seven women (8.24%). Forty - two patients (43.7%) were recruited from hospital a and the other 54 (56.2%) from hospital b. there were no significant differences between the two groups at baseline in terms of age or sex, or on the social - risk scale . There were also no significant differences in other clinical characteristics, such as the charlson index, body mass index or dyspnea (modified medical research council) scale . Quality of life (copd assessment test) showed a mean score of 15.25.9 in the cg and 13.66.9 in the ig (p=0.286). In the cg, there were 14 cases (34.9%) of active smokers compared with 16 cases (38.1%) in the ig (p=0.546), and accumulated intake was comparable between the two groups (p=0.586). Functional parameters were also very similar in both groups, and almost all the patients had advanced copd, classified as very serious in 71% of cg patients and 61.7% of ig patients (p=0.151). Only four patients (10.5%) from the cg and three patients (6.3%) from the ig had mild or moderate copd (2007 global initiative for chronic obstructive lung disease classification . Www.goldcopd.org).21 table 1 shows the baseline characteristics of the two groups . The combined primary outcome variable, the rate of copd exacerbations with visit to a&e or hospitalization, fell from 1.37 to 0.89 (p=0.04) in the ig . The numbers of patients hospitalized, at 19 (40.4%) versus 20 (52.6%) (p=0.26), and those who went to a&e, at 9 (19.1%) versus 14 (36.8%) (p=0.06), due to exacerbation of copd were also lower in this group . The number of patients who needed antibiotics during the year of follow - up was higher in the ig; 27 (56.3%), compared to 18 (47.4%) in the cg, although this was not statistically significant (p=0.306). In contrast, glucocorticoid use was slightly higher in the cg (44.7%) than in the ig (37.5%) (p=0.41). The length of hospital stay was also shorter in patients who received the smp - copd . The main results are detailed in table 2 . On the cumulative survival curve (figure 2) showing the days from baseline to the first instance of hospital care for exacerbation of copd, the time to the first event was longer in the ig, although the difference was not significant (p=0.097). There were no differences in mortality, with two deaths (5.26%) recorded in the cg and none in the ig (p=0.338). The number needed to treat (nnt) was calculated using the formula ci = 1e, proposed by suissa,25 where ci is cumulative incidence, ir is incidence rate (person - year), and t is time (years). The number of patients with advanced copd to whom the smp - copd would have to be applied for 1 year to prevent the need for one instance of hospital care for exacerbation was 6.25 . The study patients were followed up from april 2013 to january 2014 . In all, 250 patients from two health regions were recruited . Number excluded: 149 (59.6%); 33 patients (22.1%) due to severe comorbidities (12 cancers, 13 advanced heart failure, six unstable coronary heart disease, and two renal failure); 37 patients (14.8%) who, in the investigator s opinion, were too physically disabled at the time of first visit to benefit from the exercise program in the written action plan and the group educational session; 23 patients (9.2%) in whom the diagnosis of copd was not confirmed; 16 patients (6.4%) due to cognitive impairment (pentagons test); 15 patients (6%) who refused to take part; and finally, 12 patients (4.8%) due to psychosocial problems . Of the 101 patients enrolled, five did not attend the randomization visit . In all, 96 patients (38.4%) were randomized, 45 of whom (46.8%) were included in the cg and 51 (53.1%) in the ig . In the end, 85 patients (88.5%) completed the study: 38 patients in the cg (18 [47.4%] in area vi and 20 [52.6%] in area i); and 47 patients in the ig (25 [53.2%] in area vi and 22 [46.8%] in area i; p=0.593). In the cg, seven patients were lost to follow - up (one protocol violation and six dropouts), and in the ig, four were lost (one protocol violation and three dropouts). In all, 78 men (91.76%) were included and seven women (8.24%). Forty - two patients (43.7%) were recruited from hospital a and the other 54 (56.2%) from hospital b. there were no significant differences between the two groups at baseline in terms of age or sex, or on the social - risk scale . There were also no significant differences in other clinical characteristics, such as the charlson index, body mass index or dyspnea (modified medical research council) scale . Quality of life (copd assessment test) showed a mean score of 15.25.9 in the cg and 13.66.9 in the ig (p=0.286). In the cg, there were 14 cases (34.9%) of active smokers compared with 16 cases (38.1%) in the ig (p=0.546), and accumulated intake was comparable between the two groups (p=0.586). Functional parameters were also very similar in both groups, and almost all the patients had advanced copd, classified as very serious in 71% of cg patients and 61.7% of ig patients (p=0.151). Only four patients (10.5%) from the cg and three patients (6.3%) from the ig had mild or moderate copd (2007 global initiative for chronic obstructive lung disease classification . The combined primary outcome variable, the rate of copd exacerbations with visit to a&e or hospitalization, fell from 1.37 to 0.89 (p=0.04) in the ig . The numbers of patients hospitalized, at 19 (40.4%) versus 20 (52.6%) (p=0.26), and those who went to a&e, at 9 (19.1%) versus 14 (36.8%) (p=0.06), due to exacerbation of copd were also lower in this group . The number of patients who needed antibiotics during the year of follow - up was higher in the ig; 27 (56.3%), compared to 18 (47.4%) in the cg, although this was not statistically significant (p=0.306). In contrast, glucocorticoid use was slightly higher in the cg (44.7%) than in the ig (37.5%) (p=0.41). The length of hospital stay was also shorter in patients who received the smp - copd . The main results are detailed in table 2 . On the cumulative survival curve (figure 2) showing the days from baseline to the first instance of hospital care for exacerbation of copd, the time to the first event was longer in the ig, although the difference was not significant (p=0.097). There were no differences in mortality, with two deaths (5.26%) recorded in the cg and none in the ig (p=0.338). The number needed to treat (nnt) was calculated using the formula ci = 1e, proposed by suissa,25 where ci is cumulative incidence, ir is incidence rate (person - year), and t is time (years). The number of patients with advanced copd to whom the smp - copd would have to be applied for 1 year to prevent the need for one instance of hospital care for exacerbation was 6.25 . Since the 1997 study by watson et al26 showing the favorable effects of self - management skills in copd acquired through a written action plan, many authors have conducted clinical trials to evaluate different variables, with conflicting results . Some confirm positive results following the implementation of such programs, although the type of interventions applied varies greatly.27,28 bourbeau et al29 found that a self - management program called living well with copd decreased the use of services and hospitalizations . However, this benefit has not been confirmed by other authors using similar interventions.27 it seems clear that interventions with a single component, such as written action plans or education sessions, do not produce relevant effects.30 the evidence suggests that the decisive factor in self - care programs is that they contain several types of components (multicomponent) and are geared toward improving patients skills.31 as in all the clinical trials published, in our study, it was not possible to blind the investigators to the self - management plan, because of its characteristics . By considering the existing evidence, the smp - copd was designed as a multicomponent plan aimed at improving patients skills in self - care and promoting treatment compliance . However, the high number of patients excluded, similar to that reported by other authors, indicates that these interventions may not be applied systematically to the entire copd population.32 the number of patients with severe disease (more than one - half had very severe copd) is higher than that reported in most studies.16 in line with the findings of other recent studies,33,34 the smp - copd led to significant decrease in our primary outcome variable of visits to a&e and hospitalization due to copd exacerbation . The number of days of hospitalization for respiratory cause was also lower and the time to the first instance of hospital care longer . In terms of the consumption of antibiotics and oral corticosteroids during exacerbations, overall, the ig patients did not use more drugs to treat exacerbations than the cg; antibiotic use by the ig was higher while glucocorticoid use was lower, but the differences were not significant . Some studies found a significant increase in consumption of drugs in the patients subject to self - management interventions,28,33,34 but this did not happen in our study . In other studies, the results for these same variables were neither significant nor consistent,35,36 but these differences can probably be explained by the heterogeneity in the design of the interventions . The guidelines for action in the case of worsening symptoms differ considerably from one study to another, and are not always clear in the methodology . In our study, the written action plan advised patients to first start on antibiotics and, in a second phase 48 hours later, and only if the symptoms of exacerbation persisted or did not improve, they were to start taking the glucocorticoids . These guidelines may have contributed to the greater use of antibiotics and less use of glucocorticoids in the ig . Patients in the ig were provided with prescriptions at the initial visit, but these were not included in the final analysis of drug consumption . Finally, there may be other confounding variables that influence the consumption of antibiotics and oral glucocorticoids but do not tend to be sufficiently controlled in the majority of studies, such as patient initiative and accessibility in terms of seeing primary care doctors.37 the nnt over 1 year with the self - management plan of 6.5 that we obtained was similar to the nnt of 8 reported in the most recent cochrane review in the group of patients at high risk of hospitalization.16 our study has a number of limitations . The sample size is insufficient to reach significance in other variables, such as time to first hospitalization for copd . Our patients were almost exclusively male (91%), which probably reflects the demographics of patients currently receiving care in our hospitals . This has also been found in other recent studies, such as rice et al34 where the percentage of males is even higher, with 97.6% in the ig and 98.4% in the cg . We restricted our study to patients with severe or very severe copd, and do not know whether the same intervention would be effective in patients with milder disease . We also cannot determine the benefits of individual components of the smp - copd without conducting trials to directly compare them . Whether or not a more intensive and supervised pulmonary rehabilitation component would add benefit to the management of patients with advanced copd is an important question that we are unable to answer . Another limitation of the study is the lack of information on the influence of smoking on the results . Data on smoking were obtained by co - oximetry at the initial visit, but unfortunately, none was obtained at the final visit, so they were not taken into account . The main reason for exclusion (24.8%) was severe physical deterioration . Within this category, we included patients who could not walk unaided or had lost autonomy for performing activities of daily living (washing and dressing). We know that this exclusion criterion may be controversial, as it is possible that some component of the self - management program could in fact be useful to them, but we considered the physical activity component to be an important part of our program, and that patients with that degree of locomotor disability would not be able to benefit from it, from the group educational session or the exercises contained in the action plan . Although it could be considered a restrictive approach and affect the external validity of the results, there are no reports in the literature of a copd population with such advanced disease as ours . It is therefore possible that physical deterioration is more prevalent in our population than in other series and this would, in part, explain the high number of patients excluded for this reason.16 one interesting aspect of the study was the fact that the methodological approach was that of a randomized study . Moreover, the interventions were designed rigorously and comprehensively to address the main components of self - management of the disease referred to in other studies,16 such as education about the disease; individual training sessions; provision of a written action plan for taking medication in stable periods and when symptoms of exacerbation develop; and for showing how to do physical exercises . Particular attention was also given to ensuring readability and understanding of the material contained in the action plan . Copd, higher than reported in other series, represents a copd subgroup with greater needs in terms of care . The fact that a minority of patients may benefit from this intervention should be considered hypothesis - generating from the point of view of what kind of components need to be applied in different disease phenotypes.32 self - management interventions require the availability of research resources and care tools specifically assigned to the copd population . Given the magnitude of the global burden of the disease, it seems particularly important to identify which self - management components have more relative effectiveness in terms of health outcomes, since this aspect is considered important forboth patients and health care professionals . Studies evaluating the use of health care resources require longer follow - up and need to be adequately powered to detect safety outcomes and improvements in mortality rates.30 because of the complexity of self - management interventions and the general challenges of research in this field, some authors point to the necessity of organizing an international consensus to identify the key components of interventions and establish the end points that should help focus future research.38 the sample size is insufficient to reach significance in other variables, such as time to first hospitalization for copd . Our patients were almost exclusively male (91%), which probably reflects the demographics of patients currently receiving care in our hospitals . This has also been found in other recent studies, such as rice et al34 where the percentage of males is even higher, with 97.6% in the ig and 98.4% in the cg . We restricted our study to patients with severe or very severe copd, and do not know whether the same intervention would be effective in patients with milder disease . We also cannot determine the benefits of individual components of the smp - copd without conducting trials to directly compare them . Whether or not a more intensive and supervised pulmonary rehabilitation component would add benefit to the management of patients with advanced copd is an important question that we are unable to answer . Another limitation of the study is the lack of information on the influence of smoking on the results . Data on smoking were obtained by co - oximetry at the initial visit, but unfortunately, none was obtained at the final visit, so they were not taken into account . The main reason for exclusion (24.8%) was severe physical deterioration . Within this category, we included patients who could not walk unaided or had lost autonomy for performing activities of daily living (washing and dressing). We know that this exclusion criterion may be controversial, as it is possible that some component of the self - management program could in fact be useful to them, but we considered the physical activity component to be an important part of our program, and that patients with that degree of locomotor disability would not be able to benefit from it, from the group educational session or the exercises contained in the action plan . Although it could be considered a restrictive approach and affect the external validity of the results, there are no reports in the literature of a copd population with such advanced disease as ours . It is therefore possible that physical deterioration is more prevalent in our population than in other series and this would, in part, explain the high number of patients excluded for this reason.16 one interesting aspect of the study was the fact that the methodological approach was that of a randomized study . Moreover, the interventions were designed rigorously and comprehensively to address the main components of self - management of the disease referred to in other studies,16 such as education about the disease; individual training sessions; provision of a written action plan for taking medication in stable periods and when symptoms of exacerbation develop; and for showing how to do physical exercises . Particular attention was also given to ensuring readability and understanding of the material contained in the action plan . Copd, higher than reported in other series, represents a copd subgroup with greater needs in terms of care . The fact that a minority of patients may benefit from this intervention should be considered hypothesis - generating from the point of view of what kind of components need to be applied in different disease phenotypes.32 self - management interventions require the availability of research resources and care tools specifically assigned to the copd population . Given the magnitude of the global burden of the disease, it seems particularly important to identify which self - management components have more relative effectiveness in terms of health outcomes, since this aspect is considered important forboth patients and health care professionals . Studies evaluating the use of health care resources require longer follow - up and need to be adequately powered to detect safety outcomes and improvements in mortality rates.30 because of the complexity of self - management interventions and the general challenges of research in this field, some authors point to the necessity of organizing an international consensus to identify the key components of interventions and establish the end points that should help focus future research.38 to sum up, after applying a self - management program in patients with advanced copd, there were fewer hospitalizations and a&e visits for exacerbation of the disease in the group that received the intervention . This type of program can be an effective care tool adapted to the needs of chronic patients . However, further research is required to identify which patients benefit most and which components have the best cost / benefit ratios, and to conduct prospective validations in realistic scenarios.
The rapidly increasing prevalence of diabetes mellitus worldwide is one of the most serious and challenging health problems in the 21st century . The number of people with diabetes grows faster than expected . In 2007, 246 million people (roughly 6%) were affected worldwide and it is estimated that this will increase to 380 million, or 7.3% by 2025 . Furthermore, it is estimated that there are even more people (308 million or 8.1%) with impaired glucose tolerance (igt). These people have a significant risk of developing type 2 diabetes mellitus (t2 dm). Diabetes is a metabolic disorder which is characterized by hyperglycemia and glucose intolerance due to insulin deficiency, impaired effectiveness of insulin action or, both . Type 1 diabetes mellitus (t1 dm) is caused by cellular - mediated autoimmune destruction of pancreatic islet beta - cells leading to loss of insulin production . Dm accounts for 90%95% of all diabetes and is more common in people older than 45 who are overweight . However, the prevalence of t2 dm is becoming higher in children and young adults because of the higher rate of obesity in this population . Central obesity and insulin resistance next to diabetes, high cholesterol and high blood pressure form the most important risk factors for cardiovascular disease (cvd). Diabetes is also the leading cause of blindness, renal failure, and lower limb amputations [1, 2]. Dysfunction of the endothelium is regarded as an important factor in the pathogenesis of vascular disease in diabetes mellitus [35]. The endothelium is the active inner monolayer of the blood vessels, forming an interface or barrier between circulating blood in the lumen and the rest of the vessel wall, and plays a critical role in vascular homeostasis . It actively regulates vascular tone and permeability, the balance between coagulation and fibrinolysis, the inflammatory activity and cell proliferation . The endothelium even affects the functions of other cell types, such as vascular smooth muscle cells (vsmc's), platelets, leukocytes, retinal pericytes, renal mesangial cells, and macrophages, amongst others through the production of several chemical mediators [38]. In health, an imbalance in repair and injury resulting in early microvascular changes, including apoptosis of microvascular cells, can be seen in both experimental diabetic animal models and humans with diabetes . Several studies indicate that microvascular cell apoptosis plays an important role in the development of early lesions [6, 8, 9]. We will review the role of endothelial dysfunction and especially inflammation - induced apoptosis of endothelial cells in obesity - related diabetes mellitus and its co - morbidities . To maintain vascular homeostasis, the endothelium produces components of the extracellular matrix such as collagen and a variety of regulatory chemical mediators, including nitric oxide (no), prostanoids (prostacycline), endothelin-1 (et-1), angiotensin ii (ang - ii), tissue - type plasminogen activator (t - pa), plasminogen activator inhibitor-1 (pai-1), von willebrand factor (vwf), adhesion molecules (vcam, lam, icam), and cytokines, among them tumor necrosis factor (tnf) (figure 1). The endothelium has a prominent role in maintaining blood fluidity and restoration of vessel wall integrity to avoid bleeding . It regulates fibrinolysis by producing t - pa and its inhibitor pai-1 and limits activation of the coagulation cascade by thrombomodulin / protein c, heparin sulphate / antithrombin and tissue factor / tissue factor inhibitor interactions . Through release of promoters and inhibitors of growth and differentiation of the vsmc, such as platelet - derived growth factor (pdgf) and ang - ii ang - ii exerts regulatory effects on several vsmc activities including contraction, growth, proliferation, and differentiation . By the production of adhesion molecules like leukocyte adhesion molecule (lam), intracellular adhesion molecule (icam), and vascular cell adhesion molecule (vcam), inflammatory cells are attracted and anchored, thereby playing a regulatory inflammatory role [12, 13]. Endothelial dysfunction is the change of these properties, either in the basal state or after stimulation, that is inappropriate with regard to the preservation of organ function . The kind of changes that occur, can depend on the type of injury and may depend on the intrinsic properties of the endothelium (venous versus arterial endothelium). Under physiological circumstances, there is a balanced release of endothelial - derived relaxing factors such as nitric oxide (no) and prostacyclin (pgi2), and contracting factors such as endothelin-1 (et-1), prostaglandins, and angiotensin ii (ang - ii). In endothelial dysfunction, this balance is altered, predisposing the onset and progression of atherosclerosis . Risk factors such as hypercholesterolemia, dyslipidemia, smoking, and diabetes initiate atherosclerosis through endothelial activation and therefore through endothelial dysfunction . Endothelial dysfunction is expressed in increased interactions with leukocytes, smooth muscle growth, vasoconstriction, impaired coagulation, vascular inflammation, thrombosis, and atherosclerosis . A very important mediator synthesized by endothelial cells is nitric oxide (no), because of its vasodilatory, antiplatelet, antiproliferative, permeability - decreasing, antiinflammatory, and antioxidant properties . No inhibits rolling and adhesion of leucocytes as well as cytokine - induced expression of vascular cell adhesion molecule-1 (vcam-1) and monocyte chemotactic protein-1 (mcp-1), probably through the inhibition of the transcription factor nuclear factor b (nf-b) [14, 18, 19]. No is produced through the conversion of the amino acid l - arginine to l - citrulline by the enzyme no - synthase (nos). There are several isoforms: nos1 isolated from the brain, nos2, or inos, produced by macrophages and nos3 or enos from endothelial cells . Enos produces no which diffuses to the vascular smooth muscle (vsm) where it activates the enzyme guanylate cyclase which in turn increases cyclic gmp and thereby induces relaxation of the vsm . In this way nos is regulated by bradykinin, which acts with b2 receptors on the endothelial cell surface membrane, increasing the production of no via nos activation . The local concentrations of bradykinin are regulated by the activity of angiotensin converting enzyme (ace), by breaking down bradykinin into inactive peptides [20, 21]. Endothelial dysfunction is associated with decreased no availability, either through loss of no production or through loss of no biological activity . Oxidative stress is caused by three factors: (1) an increase in oxidant generation, (2) a decrease in antioxidant protection, (3) a failure to repair oxidative damage . Cell damage is induced by reactive oxygen species (ros), which are either free radicals, reactive anions containing oxygen atoms, or molecules containing oxygen atoms that can either produce free radicals or are chemically activated by them . Normally these ros are scavenged by different intra- and extra cellular mechanisms, but in a situation of oxidative stress these mechanisms are insufficient to cope with the exaggerated generation of ros . No may react with some ros species to form peroxynitrite, in turn increasing the oxidative stress in the cell . Several cardiovascular risk factors like hyperglycemia, insulin resistance, dyslipidemia, inflammation, and also cigarette smoking may induce oxidative stress [5, 19]. Oxidative stress is an important factor which can induce cell apoptosis . In the next part apoptosis is the process in which a cell plays an active role in its own death . This is why it is also called cell suicide . Apoptosis differs from necrosis in the level of control of the process . Necrosis is an uncontrolled process of cell lysis leading to inflammation and destruction of tissue areas or even whole organs, which can cause serious health problems . Apoptosis, or programmed cell death, is a normal component of the development and health of multicellular organisms and continues throughout adult life . Apoptosis and proliferation are responsible for shaping tissues and organs in developing embryos . During adult life, apoptosis is a protection mechanism which eliminates old, useless, and damaged cells . In healthy organisms apoptosis and cell proliferation there is an imbalance whereby cells have undergone certain mutations that prevent them from undergoing apoptosis . In neurodegenerative diseases such as parkinson's disease apoptosis is thought to account for the excessive loss of neurons . There are extrinsic signals such as the binding of death inducing ligands to cell surface receptors also called death receptors . Some of these ligands are expressed on the surface of cytotoxic t lymphocytes, for example, when a cell is infected by a virus . Apoptosis can also be induced by intrinsic signals, that are produced following cellular stress . Cellular stress can be caused by oxidative stress through free radicals, deprivation of growth factor, or exposure to radiation or chemicals . The sensitivity of cells to these stimuli can vary depending on a number of factors, such as the expression of pro- and antiapoptotic proteins, the severity of the stimulus and the stage of the cell cycle . Very important death inducing ligands are the fas ligand, tnf and trail (tnf related apoptosis inducing ligand). When they bind their specific death receptor, apoptotic signals are transmitted in the cell and a caspase cascade is activated within seconds of ligand binding, inducing apoptosis in a very rapid way . The general signaling pathway that is activated through death receptor binding begins with the generation of ceramide, produced by acid sphingomyelinase . A conformational change in the intracellular domains of the death receptors reveals the presence of a death domain which allows the recruitment of various apoptotic proteins to the receptor . This is called the death inducing signaling complex (disc). As a final step, the disc recruits and activates procaspase 8 . The sensitivity of cells to apoptotic stimuli can depend on the balance of pro- and antiapoptotic bcl-2 proteins . Bcl-2 and bcl - xl are antiapoptotic, while bad, bax and bid are proapoptotic proteins [23, 24]. The proapoptotic bcl-2 proteins are often found in the cytosol acting as sensors of cellular damage or stress . In case of cell stress this interaction between pro- and antiapoptotic proteins leads to the formation of permeability transition pores (ptp) in the mitochondrial membranes . Recent evidence implies that there may also be a mitochondrial apoptotic pathway distinct from that activated by proaptotic bcl-2 family proteins, dependent on cyclophilin d . The mitochondria contains proapoptotic proteins such as apoptosis inducing factor (aif), smac / diablo, and cytochrome c, which are released through these pores, which in turn leads to the formation of the apoptosome and the activation of the caspase cascade [27, 28]. Once cytochrome c is released into the cytosol, it interacts with apoptotic peptidase activating factor-1 (apaf-1) and this leads to the recruitment of procaspase 9 into a multiprotein complex called the apoptosome . Nitric oxide has been demonstrated to inhibit apoptosis in a number of cell types including endothelial cells . The antiapoptotic effects can be mediated through mechanisms such as nitrosylation and inactivation of caspase 1, 3 and 8 . Other mechanisms include activating p53, upregulating heat shock protein 70, and upregulating antiapoptotic proteins bcl-2 and bcl - xl . Through activation of cgmp signaling, caspase activity is suppressed, cgmp - dependent protein kinases are activated and possibly the expression of antiapoptotic proteins increases . Apoptosis and especially apoptosis of endothelial cells may be highly significant in the development of diabetes and atherosclerosis . Dysfunction of endothelium in diabetes mellitus is characterized by changes in proliferation, barrier function, adhesion of other circulating cells, and sensitivity to apoptosis . Furthermore, it is suggested that diabetes mellitus modifies angiogenic and synthetic properties of endothelial cells [3036]. There is a lot of evidence that endothelial dysfunction is closely connected to the development of diabetic retinopathy, nephropathy, and atherosclerosis in both t1 dm and t2 dm [4, 37]. But, what are the specific mechanisms that cause this close association between diabetes and endothelial dysfunction? Large clinical trials in both t1 dm and t2 dm have shown that hyperglycemia plays a big part in the pathogenesis of microvascular complications and is a major causal factor in the development of endothelial dysfunction and endothelial cell apoptosis [5, 38, 39]. However, the exact mechanism of hyperglycemia - related tissue damage and clinical complications remains unclear . There is also a significant role for insulin and especially insulin resistance, as increasing evidence implies that the obesity - related progression of insulin resistance to t2 dm parallels the progression of endothelial dysfunction to atherosclerosis . Still this relationship has been difficult to prove because insulin resistance is often accompanied by a cluster of other risk factors as mentioned above . The role of endothelial dysfunction in t2 dm is very complicated, due to the many independent factors involved, including ageing, obesity, hyperlipidemia, hypertension, low grade inflammation, insulin resistance, and hyperglycemia . All of these factors are associated with the metabolic syndrome, which usually precedes t2 dm . The relationship of endothelial dysfunction and all of these factors is not completely understood despite extensive research . Even the question whether endothelial dysfunction is a consequence or the cause of all the changes occurring in the metabolic syndrome and diabetes cannot be answered easily . In the next few paragraphs we will discuss the relation between endothelial dysfunction and the individual factors mentioned above, starting with insulin resistance . Insulin resistance is defined as the decreased ability of insulin to promote glucose uptake in skeletal muscle and adipose tissue and the decreased hepatic output of glucose . This may be present years before the development of abnormal plasma glucose levels becomes evident [41, 42] (figure 2). Insulin resistance is associated with an increased free fatty acids (ffa) release from adipose tissue, which results in dyslipidemia, including vldl - hypertriglyceridemia, high plasma ffa, and low hdl - cholesterol concentrations . Ffa - mediated endothelial dysfunction is probably caused by reduced availability of l - arginine and/or no and oxidative stress . It has been proven that increased saturated and polyunsaturated ffa concentrations, except for oleic acid, directly induce cell cycle arrest and apoptosis in vascular endothelial cells . Insulin is a vasoactive hormone and enhances muscle blood flow and vasodilation via stimulation of no production . Insulin can also redirect blood flow in skeletal muscles so that more glucose can be uptaken by muscle cells . Insulin's vasodilator actions are impaired, probably for a large part because of low no action . Normally, stimulation of no production by insulin is mediated by signaling pathways involving activation of phosphoinositide-3 (pi-3) kinase leading to phosphorylation of enos . It is suggested that endothelial dysfunction and impaired capillary recruitment can cause insulin resistance because the microvascular endothelium cannot react properly to insulin and glucose disposal is decreased . This is called endothelial insulin resistance . How metabolic and endothelial insulin resistance originate and their exact relationship are not fully understood . Both tnf and nonesterified acids (nefas) can cause metabolic and endothelial insulin resistance . Inflammatory cytokines like tnf, can act as mediators of insulin resistance by impairing the tyrosine kinase activity of both the insulin receptor (ir) and insulin receptor substrate (irs-1), thus inhibiting insulin signaling . It is suggested that a bidirectional relationship exists between hyperinsulinemia and low - grade chronic inflammation, by which hyperinsulinemia can lead to vascular inflammation and vascular inflammation causes insulin resistance and finally compensatory hyperinsulinemia . At normal physiological concentrations insulin exerts prevailing antiinflammatory effects, while hyperinsulinemia increases levels of oxidative stress and inflammation . A recent study with human umbilical vein endothelial cells (huvecs) shows that insulin, at pathophysiological concentrations alone or in combination with low concentrations of tnf, has the ability to promote vcam-1 expression, through increasing the steady state levels of mrna via the activation of transcription factors, such as nf-b, which has been linked to vcam-1 transactivation before . This way, hyperinsulinemia leads to increased monocytoid cell adhesion to huvecs [5, 19, 45]. A very important effect of insulin resistance is the fact that the normal route for insulin to activate the pi-3 kinase and akt - dependent signaling pathways is impaired, whereas hyperinsulinemia overactivates mitogen activated protein kinases (mapk)-pathways, thereby creating an imbalance between pi-3 kinase and map - kinase - dependent functions of insulin . This probably leads to decreased no production and increased et-1 secretion, characteristic of endothelial dysfunction . Through activation of the map - kinase signaling pathways, hyperinsulinemia promotes secretion of et-1, activates cation pumps, and increases expression of vcam-1 and e - selectin . Et-1, a vasoconstrictor, can increase serine phosphorylation of irs-1, causing a decreased activity of pi-3 kinase in vascular smooth muscle cells . Moreover, et-1 may also impair insulin - stimulated translocation of glut-4 in adipocytes [47, 48]. Hypertension induces endothelial activation and probably also endothelial dysfunction and is a major determinant of microangiopathy and atherothrombosis in diabetes . Hypertension is associated with insulin resistance and this relation can partly be explained by decreased capillary density and impaired capillary recruitment seen in insulin resistant states . Another explanation is the fact that no availability is diminished and et-1 availability is increased in both insulin resistance and hypertension . The adipose tissue has become known to be a highly active endocrine organ, releasing hormones, cytokines, and enzymes with the tendency to impair insulin sensitivity . It is an important modulator of endothelial function via secretion of a variety of hormones, including adiponectin, resistin, leptin, pai-1, angiotensin, estradiol, and the cytokines tnf and interleukin-6 (il-6). Plasma adiponectin levels are reduced in people with obesity and also in people with diseases associated with obesity, like t2 dm and coronary artery disease . Adiponectin has antiinflammatory features and is inversely related to bmi, oxidized ldl, insulin resistance, and atherosclerosis . Low adiponectin levels are associated with an increased oxidative state in the arterial wall and systemic oxidative stress . In endothelial cells, adiponectin increases the production of nitric oxide and suppresses oxidative stress and the inflammatory signaling cascades via amp - activated protein kinases (ampk) and the cyclic amp - protein kinase a - linked pathway . Moreover, it reduces the attachment of monocytes to endothelial cells and inhibits the expression of adhesion molecules [5, 51]. The role of resistin in insulin resistance and diabetes is controversial since a number of studies have shown that resistin levels increase with increased central adiposity and other studies have demonstrated a significant decrease in resistin levels in increased adiposity . It has been linked to the increased occurrence of thrombosis in patients with these conditions . Angiotensin ii is also present in adipose tissue and has an important effect on endothelial function . When angiotensin ii binds the angiotensin ii type 1 receptor on endothelial cells, it stimulates the production of ros via nadph oxidase, increases expression of icam-1 and increases et-1 release from the endothelium [5254]. Angiotensin also activates jnk and mapk pathways in endothelial cells, which leads to increased serine phosphorylation of irs-1, impaired pi-3 kinase activity and finally endothelial dysfunction and probably apoptosis . This is one of the explanations why an ace inhibitor and angiotensin ii type 1 receptor blockers (arbs) protect against cardiovascular comorbidity in patients with diabetes and vice versa . Insulin receptor substrate 1 (irs-1) is a protein downstream of the insulin receptor, which is important for signaling to metabolic effects like glucose uptake in fat cells and no - production in endothelial cells . Irs-1 in endothelial cells and fat cells can be downregulated by stressors like hyperglycemia and dyslipidemia, causing insulin resistance and endothelial dysfunction . A low adipocyte irs-1 expression may thereby be a marker for insulin resistance [19, 56, 57]. Nowadays atherosclerosis is considered to be an inflammatory disease and the fact that atherosclerosis and resulting cardiovascular disease is more prevalent in patients with chronic inflammatory diseases like rheumatoid arthritis, systemic lupus erythematosus and ankylosing spondylitis than in the healthy population supports this statement . Inflammation is regarded as an important independent cardiovascular risk factor and is associated with endothelial dysfunction . Shows that patients with active ankylosing spondylitis, an inflammatory disease, also have impaired microvascular endothelium - dependent vasodilatation and capillary recruitment in skin, which improves after tnf-blocking therapy with etanercept . The existence of chronic inflammation in diabetes is mainly based on the increased plasma concentrations of c - reactive protein (crp), fibrinogen, interleukin-6 (il-6), interleukin-1 (il-1), and tnf [5961]. Inflammatory cytokines increase vascular permeability, change vasoregulatory responses, increase leukocyte adhesion to endothelium, and facilitate thrombus formation by inducing pro - coagulant activity, inhibiting anticoagulant pathways and impairing fibrinolysis via stimulation of pai-1 . Nf-b consists of a family of transcription factors, which regulate the inflammatory response of vascular cells, by transcription of various cytokines which causes an increased adhesion of monocytes, neutrophils, and macrophages, resulting in cell damage . On the other hand, nf-b is also a regulator of genes that control cell proliferation and cell survival and protects against apoptosis, amongst others by activating the antioxidant enzyme superoxide dismutase (sod). Nf-b is activated by tnf and il-1 next to hyperglycemia, ages, ang - ii, oxidized lipids, and insulin . Once activated, nf-b - regulated genes are vcam-1, e - selectin, icam-1, il-1, il-6, il-8, tissue factor, pai-1, and nos . The tnf - family of cytokines plays an important role in regulating the immune response, inflammation, and apoptosis . The first cytokine discovered is tnf, which is produced by neutrophils, macrophages, and adipocytes and can induce other powerful cytokines such as il-6, which in turn regulates the expression of c - reactive protein (crp). Crp increases the expression of endothelial icam-1, vcam-1, e - selectin, mcp-1 and increases the secretion of et1 . Moreover, crp decreases enos expression and elevates the expression of angiotensin receptor type 1 in the vessel wall [63, 64]. Tnf can induce insulin resistance and this is probably a part of the explanation why insulin resistance, endothelial dysfunction, and atherothrombosis are so closely related . Recent studies indicate that tnf is likely involved in the pathogenesis of diabetic nephropathy and retinopathy . A very recent study with t1 dm and t2 dm rats shows that tnf plays an important role in microvascular apoptosis in diabetes . When the diabetic rats were treated with pegsunercept, a tnf inhibitor, a significant reduction of the number of endothelial cells that expressed activated caspase-3 by 76% to 80% occurred . Tnf inhibition decreases intercellular adhesion molecule 1 (icam-1) levels and nf-b activity in diabetic retina . Another study in diabetic rats demonstrated that increased levels of tnf consequently enhanced foxo-1 mrna levels, nuclear translocation, and dna binding in retinas of t1 dm and t2 dm rats . It also showed that the transcription factor foxo-1, which regulates cell death; prevents cell cycle progression, modulates differentiation in various cell types, plays a critical role in diabetes - induced apoptosis and retinal microvascular cell loss . It is possible that tnf upregulation may contribute to increased apoptosis detected in other diabetes associated complications and tnf inhibition may be a potential therapeutic option in preventing this comorbidity . Tumor necrosis factor alpha - related apoptosis - inducing ligand (trail), also known as apo2l, is another member of the tnf family of cytokines and is a type ii membrane protein . When trail binds trail - r1 (dr4) and trail - r2 (dr5) apoptotic signals are transduced . Trail - r3 (dcr1), trail - r4 (dcr2), and osteoprogeterin (opg) lack an intracellular death domain and cannot induce apoptosis . Uniquely, trail can exert anticancer activity, while causing no or minimal organ toxicity and inflammation . Studies have shown that opg is remarkably increased in diabetic patients and even more so in patients with cardiovascular disease, like coronary artery disease or abdominal aortic aneurysm [68, 69]. In a study with szt - induced rats and a control group of healthy rats the opg / trail ratio was markedly increased in the diabetic animals with respect to the control animals . The next remarkable observation in this study was the ability of insulin to downregulate trail expression in rat aortas in vivo . Further investigation of the role of insulin in the trail expression in diabetes was done with vsmcs in vitro . High glucose levels did not show any significant effect on trail surface expression in both studies . These findings suggest that the downregulation of trail expression may play a role in diabetic vasculopathy . A possible explanation for these results is the upregulation of the transcription factor early growth response protein 1 (egr-1), which in turn downregulates trail expression in endothelial cells, by both hyperglycemia and insulin . A supportive finding for this hypothesis is the fact that vegf receptor 1 (flt1) and pai-1, both known egr-1 responsive genes, are also increased in the presence of glucose and insulin . Thus, egr-1 upregulation, which is frequently observed in atherosclerosis, is likely to be involved in insulin - mediated trail downregulation . Plasma levels of c - reactive protein (crp) are increased in both t1 dm and t2 dm . Crp plays a significant role in atherogenesis in endothelial cells, next to vascular smooth muscle cells and macrophages, and several studies have revealed that crp levels predict cardiovascular disease . Crp causes numerous proinflammatory and pro - atherogenic effects in endothelial cells, such as decreased no and prostacyclin, increased et-1, cell adhesion molecules, mcp-1, il-8, and pai-1 . Another important contribution to chronic inflammation in diabetes is caused by primed peripheral polymorphonuclear leukocytes (pmns). In a small study with t2 dm patients and a control group, it was shown that t2 dm patients are exposed to oxidative stress and chronic inflammation partially because of the primed state of their pmns, amongst others because these primed pmns release superoxide significantly faster than normal control pmns . Apoptosis in primed pmns was also higher in the diabetic patients, probably partly because of intracellular factors such as high cytosolic calcium concentrations . At the same time apoptosis of normal pmns of the control group was significantly higher in diabetic serum, suggesting leucoclastic activity of diabetic serum . This study also observed a decrease in plasma gluthathione (gsh), an intra- and extra cellular antioxidant, which neutralizes oxidants, including hydrogen peroxide and superoxide, by converting them to other oxidized forms . Dyslipidemia is characterized by low hdl - cholesterol levels and an excess of small, dense ldl and is associated with obesity, insulin resistance and diabetes in general . An increase in postprandial triacylglycerol - rich lipoproteins, like chilomicrons and -ldl particles, enhances oxidative stress and consequently causes endothelial dysfunction and increased apoptosis . The basis for the activation of these pathways is most likely the overproduction of ros in mitochondria induced by hyperglycemia (figure 3). In a lot of cells sorbitol is later metabolized to fructose by sorbitol dehydrogenase, the polyol pathway . At the same time it increases the oxidation of nadph to nadp+ and the reduction of nad+ to nadh, the co - factors, which in turn decreases no bioavailability . This causes a redox imbalance that resembles tissue hypoxia and is therefore called hyperglycemic pseudohypoxia . The increased sorbitol accumulation increases osmotic stress and decreases other osmolytes such as myo - inositol and taurine . A study in rat and human retinas produced evidence that the polyol pathway may have an important role in diabetic retinopathy . It also proved that the aldose reductase inhibitor (sorbinol) prevents vascular processes, culminating in the development of acellular capillaries [5, 75, 77]. However, the full impact of this pathway in the endothelial dysfunction is not completely understood yet . The hyperglycemia induced activation of the diacylglycerol (dag)-protein kinase c (pkc) pathway has multiple adverse effects on the vascular function . Hyperglycemia increases the levels of dag, which in turn activates pkc . In hyperglycemic circumstances dag is synthesized from the glycolytic intermediates dihydroxyacetone phosphate (dhap) and glycerylaldehyde-3-phosphate, by a de novo pathway . The pathogenic consequences of pkc activation include dysregulation of the vascular permeability through the induction of vascular endothelial growth factor (vegf) in smooth muscle cells, dysregulation of blood flow by decreasing endothelial nos activity and/or increasing et-1 synthesis, basement membrane thickening through transforming growth factor - beta (tgf-)-mediated increased production of type iv collagen and fibronectin, increased expression of pai-1 which causes impaired fibrinolysis and activation of superoxide producing enzymes like nadph as well as an increased expression of a dysfunctional, superoxide - producing, uncoupled endothelial nos, thus increasing oxidative stress . Recently, geraldes et al . Have identified a new signaling pathway by which hyperglycemia causes increased vascular cell pathology and apoptosis resulting in diabetic retinopathy in mouse retinas . They proved that hyperglycemia, especially in pericytes, activates pkc-, probably through an increase in transcription of the gene encoding pkc-. This as well as activation of p38 mapk leads to increased expression of scr homology-2 domain - containing phosphatase-1 (shp-1), which subsequently induces apoptosis via deactivation of platelet - derived growth factor (pdgf-). Non - enzymatic glycation products are a complex and heterogeneous group of compounds which accumulate in plasma and tissues in diabetes and renal failure . There is emerging evidence that these compounds play a role in the pathogenesis of chronic complications associated with diabetes and renal failure . Earlier research in both diabetic animals and humans revealed an association between the accumulation of age - modified proteins and the severity of microvascular complications . The second evidence stems from the fact that typical microvascular complications develop following injections of age - modified proteins in non - diabetic animals . The advanced glycation end - products (age) concept proposes that chemical modification and cross linking of tissue proteins, lipids, and dna affect their structure, function and turnover, contributing to a gradual decline in tissue function and to the pathogenesis of diabetic complications . Nonenzymatic glycation of proteins is a condensation reaction between the carbonyl group of free glucose and the n - terminus of reactive - protein amino groups, like lysine or arginine, yielding schiff - base intermediates that undergo amadori rearrangement to form stable proteinglucose adducts, for example glycated hemoglobin a1c (hba1c) and fructosamine (fructoselysine). Amadori - modified matrix proteins are increased in diabetes . Because amadori - adducts are relatively stable, only a small fraction undergoes rearrangements to irreversible ages . At first it was believed that ages are only formed on long - lived extra cellular molecules, because of the slow rate of reaction of glucose with proteins . However, other sugars like glucose-6-phosphate and glyceraldehyde-3-phophate can also create ages with intracellular and short - lived molecules and at a much faster rate than glucose . Ages can arise from the decomposition of amadori products, from fragmentation products of the polyol pathway, and as glycoxidative products which all react with protein amino groups . When oxidation is involved, the so - called glycoxidation products such as pentosidine and carboxymethyllysine are formed . It has recently been found that glucose can probably autoxidize to form reactive carbonyl compounds (glyoxal, methylglyoxal and 3-deoxyglucosone) which may react with protein to form glycoxidation products . In endothelial cells they can act as oxidants and cause generation of reactive oxygen species (ros). Ages can decrease arterial elasticy and age modified type i and iv collagen can prevent normal matrix formation and cross - linking . Interactions of mononuclear cells and macromolecules like ldl with the endothelial wall are stimulated by age - modified matrix, through increased expression of endothelial adhesion molecules . Ages can also impair the binding of heparan sulfate to the extra cellular matrix, which results in a loss of anionic sites and thus in an increase in endothelial permeability . Early diabetic micro angiopathy is characterized by vasodilation, increased blood flow, and increased capillary permeability . Age - modified proteins may lead to all these changes . When ages get into the blood circulation they are highly reactive but are often detoxified by various enzymes . When they are not eliminated by the kidneys, recirculating age peptides can generate new ages reacting with plasma or tissue components . At this stage glycation age - modified plasma proteins can bind to age receptors (rage = age - receptor, macrophage scavenger receptor a) on different cell types like endothelial cells, where it can adversely affect the expression of thrombomodulin, tissue factor, and vcam-1 genes . Rage - binding mediates signal transduction via a receptor - mediated induction of ros and activation of transcription factors nf-b and p21-ras, leading to apoptosis . The nonenzymatic glycation of ldl (gldl) and its role in the pathogenesis of atherosclerosis is a popular subject in studies of late . Due to hyperglycemia, ldl glycation is increased in diabetic patients, however nonenzymatic glycation of ldl happens naturally in all individuals . The modification of ldl by glycation leads to a decreased recognition of ldl by the ldl receptor (ldl - r) and in turn increases the relative circulation time of the lipoprotein, which may result in increased particle oxidation, the formation of ages, and the activation of alternative uptake mechanisms by non additionally, gldl prevents shear stress - mediated l - arginine uptake and nitric oxide formation and causes increased production of plasminogen - activator inhibitor 1 and prostaglandins, while inhibiting the expression of tissue plasminogen activator in endothelial cells [8587]. It has been proposed that these processes could contribute to the increased susceptibility of diabetic patients to atherosclerosis and coronary heart disease . So measurement of the products of nonenzymatic glycation has a two - fold meaning: on one hand, measurement of early glycation products can estimate the extent of exposure to glucose and the subjects of previous metabolic control; on the other hand, measurement of intermediate and late products of the glycation reaction is a precious instrument in verifying the relationship between glycation products and tissue modifications . The increased production of superoxide anion radicals by mitochondrial electron transport chain plays a key role in the activation of the above pathways . Hyperglycemia - induced superoxide overproduction inhibits gadph activity by 66%, which is a consequence of poly adp - ribosylation of gadph by poly adp - ribose polymerase (parp), which in turn is activated by dna strand - breaks synthesized by mitochondrial superoxide overproduction . This overproduction particularly happens in mitochondria that have been uncoupled by the flux of nadh from the hyperglycemia - enhanced glycolysis . Gadph inhibition causes accumulation of glycolysis intermediates . In aortic endothelial cells, the hyperglycemia induced increased mitochondrial superoxide production and prevented enos activity and expression . In addition to mitochondrial uncoupling there are other mechanisms that can contribute to superoxide production in diabetes, namely, uncoupling of enos, increased peroxidation and glycoxidation, activation of nadph oxidases, decreased clearance of superoxide, and impaired antioxidant status . Oxidative stress is probably a key event in endothelial dysfunction since inhibition of hyperglycemia, induced, ros production prevents activation of the aldose reductase, hexosamine pathways, pkc activation, and age formation [77, 89]. Ros at low concentrations can function as signaling molecules and participate as signaling intermediates in the regulation of fundamental cell activities, such as cell growth and cell adaptation responses . At higher concentrations they can cause oxidative stress, cellular injury, and apoptosis [7, 90]. Ros can effect many signaling pathways, including g - proteins, protein kinases, ion channels and transcription factors . Finally ros can modify endothelial function by a variety of mechanisms, like peroxidation of membrane lipids, activation of nf-b, and decreasing the availability of no . A recently published study showed that transient exposure of cultured human aortic endothelial to hyperglycemia induces persistent epigenetic changes in the promoter of the nf-b p65 subunit . In the proximal promoter region of p65, increased monomethylation of histone 3 lysine 4 by the histone methyltransferase set 7 caused a continuing increase in p65 gene expression, leading to a sustained increase in the expression of the nf-b - responsive proatherogenic genes mcp-1 and vcam-1 . The cause of these changes was found in the increased generation of methylglyoxal and hyperglycemic - induced ros formation by the mitochondrial electron transport chain . This means that transient hyperglycemia can cause persistent atherogenic effects during normoglycemia by inducing long lasting chromatin remodeling and vascular epigenetic changes . These results provide a molecular basis for better understanding of the variation in risk for diabetic complications, which cannot be explained by hba1c . Cellular senescence or cellular ageing is the phenomenon where normal diploid differentiated cells lose the ability to divide . This phenomenon is also known as replicative senescence or the hayflick phenomenon . In response to dna damage (including shortened telomeres) cells either age or go into apoptosis if the damage cannot be repaired . There is strong evidence as mentioned above, that oxidative stress is increased in diabetic patients . Other studies have revealed that endothelial cells in atherosclerotic lesions show features of cellular senescence, like senescence associated -galactosidase (sa--gal) staining and telomere shortening . More importantly, senescence enhances vascular inflammation and thrombosis in vessels, promoting the development of cardiovascular events . There is also evidence that senescence is more accelerated in patients with diabetes compared to healthy individuals . One study demonstrated that high glucose induced premature cellular senescence in huvecs through the activation of the apoptosis signal - regulating kinase 1 (ask1). Activation of ask-1 also upregulated pai-1 expression in the huvecs and this plus senescence was also observed in aortas of stz - diabetic wild type mice, whereas this was not seen in stz - diabetic ask-1 knock - out mice . Pai-1 is known to play an important role in the pathogenesis of atherosclerosis and thrombosis . The number of (in vitro) studies delivering evidence that hyperglycemia can induce endothelial cell apoptosis [30, 90, 94] has increased extensively over the last few years . These studies have focused mainly on human or animal endothelial cells of kidney, retina, myocardium, and human umbilical vein endothelial cells (huvecs). Thanks to these studies, the mechanisms by which hyperglycemia initiates apoptosis are better understood . These mechanisms include oxidative stress, increased intracellular ca, mitochondrial dysfunction otherwise known as the mitochondria apoptosis pathway, changes in intracellular fatty acid metabolism, activation of mitogen activated protein kinases (mapk) signaling pathways, and impaired phosphorylation activation of the protein kinase akt [24, 31] (figure 4). One specific study with huvecs demonstrated that elevated glucose induces apoptosis and downregulates vegf in huvecs by inhibiting p42/44 map kinase activation . High glucose also significantly increased bax protein but did not affect bcl-2, thereby elevating the bax / bcl-2 ratio which activates cleavage of procaspase 3 into active caspase-3, in turn triggering apoptosis in huvecs . When vegf was added to the huvecs exposed to high glucose, apoptosis was prevented through inhibition of elevated ros generation, calcium overload and activation of the mitochondria apoptosis pathway . Vegf significantly decreased bax expression without affecting the bcl-2 level and attenuated the increase in caspase 3 activity . Vegf in huvecs could also decrease h2o2 production at 48 hours high glucose stimulation, suggesting that it inhibits the ros / nf-b / jnk / caspase-3 pathway . One earlier study with human aortic endothelial cells and bovine aortic endothelial cells exposed to high d - glucose also showed a significant increase in the bax / bcl-2 ratio followed by an increase in caspase-3 activity and cell death . They proved that bax inserts the mitochondrial membranes, triggering a transformation of mitochondrial function after high d - glucose treatment of the human aortic endothelial cells . This study also demonstrated that high d - glucose leads to phosphorylation of p38 mitogen - activated protein kinase (p38 mapk) mediated by mek - kinase1 (mekk1) downstream of bax - caspase proteases and thereby causes apoptosis of aortic endothelial cells . Another study with huvecs also investigated the role of the three mapk pathways: the extra cellular signal - regulated kinases (erk), the c - jun nh 2-terminal kinase /stress - activated protein kinases (jnk / sapk), and p38 mapk . This study showed no significant role for the other two mapk pathways . Later in 2005 this research group found that hyperglycemia induces ros generation through a pi3k - dependent pathway . They observed that hyperglycemia causes a pi3k / akt - dependent upregulation of cyclooxygenase 2 (cox-2) expression and thereby an increase of prostaglandin e2 (pge2) production and subsequently a caspase-3 activation and facilitation of apoptosis in huvecs . These findings were supported by the fact that ly294002 or wortmann (both pi3k / akt inhibitors) prevented the cox-2 mediated pge2 production and subsequently the caspase-3 activity, and apoptosis . Inhibition of cox-2 with a selective cox-2 inhibitor ns398 also inhibited pge2 production, caspase-3 activity and apoptosis in huvecs treated with high glucose levels . Moreover they found that hyperglycemia could trigger nf-b activation and that dominant - negative ikb could prevent cox-2 expression and apoptosis, implying that nf-b activation can lead to cox-2 mediated pge2 production and apoptosis in huvecs exposed to hyperglycemia . There are several studies with huvecs that prove that high glucose - induced apoptosis is associated with an increase in ca current, resulting from ca entry mediated by store - operated channels . Ca accumulation in mitochondria is one of the primary causes for mitochondrial permeability transition, through the opening of the pt - pore and this is an important key factor in the apoptotic pathway . The involvement of the intracellular fatty acid metabolism is suggested by a study in which huvecs were treated with high glucose concentrations for 24 hours and showed inhibition of fatty acid oxidation, increases in fatty acid esterification and the concentration of malonyl - coa before apoptosis was induced . This finding suggests a causal relation of alterations in intracellular fatty acid and apoptosis in hyperglycemia . All these metabolic alterations are associated with an increase in caspase-3 activity and an impaired ability of insulin at a physiological concentration to activate akt . Finally an antiapoptotic role for ampk is suggested in this study because incubation of the huvecs with 5-aminoimidazole-4-carboxamide - riboside (aicar), an ampk activator, prevented all of the above changes . Likewise, a similar decrease in caspase-3 activity was observed when ampk activity was increased by infecting huvec with constitutively active ampk using an adenoviral vector . Recently, a study with human pancreatic islet microvascular endothelial cells (mecs) proved that sustained hyperglycemia progressively affects cellular survival and proliferation and increases apoptosis of cultured mecs . After 24 to 48 hours, apoptosis was detected in high glucose both by dna fragmentation and activation of the caspase family . In this study they found that the islet mecs, under conditions of sustained hyperglycemia, showed a progressively reduced phosphorylation of akt, suggesting an interference with the pathways involved in akt activation . It is known that phosphorylated nephrin associates with pi3k and activates the multifunctional akt - dependent pathways . This suggests that hyperglycemia - induced apoptosis of islet endothelium likely involves the nephrin - mediated signaling cascade, wherein phosphorylation of the tyrosine sites within the intracytoplasmic c terminal domain of nephrin activates mitogen - activated protein kinase p38 and jnk and thereby the transcription factor activating protein-1 (ap-1)/c - jun, which modulates apoptosis . The study with islet mecs also detected an increased production of the proinflammatory cytokine il-1, which can induce fas expression enabling fas - mediated apoptosis . The relation between diabetic micro- and macroangiopathy and endothelial dysfunction is complex and is still a subject of extensive research . Especially in type 2 diabetes a lot of factors are involved including hyperglycemia, hyperinsulinemia, insulin resistance, dyslipidemia, hypertension, and obesity, which all influence each other and probably intensify each others actions . More insights into the exact mechanisms underlying endothelial dysfunction may lead to important treatment strategies which can significantly reduce the morbidity and mortality rate caused by endothelial dysfunction especially in diabetes patients . Although apoptosis is a natural phenomenon in all multicellular organisms, an increased and accelerated rate of apoptosis of endothelial cells is probably a crucial factor in diabetic co - morbidity . There are many pathways involved in activating endothelial cell apoptosis and all of these pathways can be activated in multiple ways . A common mechanism causing endothelial several studies show contradictory results regarding a possible role for antioxidants in the treatment to prevent micro- and macroangiopathy . However a treatment aimed at reducing oxidative stress in endothelial cells may be an answer to this major problem, especially since diabetes will soon become an even bigger health problem involving more than 5% of the world population.
The neuronal migration disorders, x - linked lissencephaly syndrome (xlis) and subcortical band heterotopia (sbh), also called " double cortex ", have been linked to missense, nonsense, aberrant splicing, deletion, and insertion mutations in doublecortin (dcx) in families and sporadic cases (1 - 8). Most dcx mutations identified to date are located in two evolutionarily conserved domains (7, 9). The prevalence of dcx mutations is 38 - 85% in sporadic sbh patients and 100% in all multiplex families with xlis / sbh (7, 10). Because sbh is typically seen in women with heterozygous dcx mutations and is a less severe phenotype than lissencephaly, which is a typical phenotype of men with hemizygous mutations, sbh is considered a mosaic phenotype that involves lyonization (5). These findings suggest that the conserved domains in dcx play a major role in neuronal migration during neural system development . The precise explanation for the function of the dcx protein in neuronal migration, however, remains unknown . We performed mutation analysis of dcx in two korean patients with sbh and report a novel mutation of dcx that is responsible for sbh . Two unrelated korean patients with epilepsy and sbh (patients 1 and 2) and their clinically unaffected family members were studied after obtaining informed consent . The sbh patients had mild to moderate developmental delays, drug - resistant generalized seizures, and diffuse thin (fig . Dna was extracted from peripheral blood using a standard protocol, and all coding regions of dcx (exons 1 to 7; exon numbering according to refseq aj003112.1, gen - bank) were amplified by pcr . Pcr assays were performed by using 1.25 u of amplitaq polymerase gold (applied biosystem, foster city, ca, u.s.a . ), 100 ng genomic dna, 2.0 mm mgcl2, and a final concentration of 10 m for each set of primers (table 2). Amplification conditions were as follows: an initial denaturing cycle at 95 for 7 min followed by 35 cycles of denaturation at 94 for 30 sec, annealing at 62 for 30 sec, and extension at 72 for 1 min . A final extension step of 72 for 7 min was used . The pcr products were electrophoresed in a 1.2% agarose gel, and the amplified genomic dna fragments were extracted and purified using the manufacturer's recommended protocol (qiaquick gel extraction kit; qiagen, germany). Direct sequencing of both strands was performed with bigdye terminator chemistry (pe biosystems, foster city, ca, u.s.a . ). As the mutation found in patient 1 destroys an mnl1 restriction site, restriction fragment length polymorphism (rflp) analysis was performed using mnl1 enzyme (new england biolabs, beverly, ma, u.s.a .) For the pcr products from both the family members of patient 1 and 60 unaffected, unrelated korean women . Two unrelated korean patients with epilepsy and sbh (patients 1 and 2) and their clinically unaffected family members were studied after obtaining informed consent . The sbh patients had mild to moderate developmental delays, drug - resistant generalized seizures, and diffuse thin (fig . Dna was extracted from peripheral blood using a standard protocol, and all coding regions of dcx (exons 1 to 7; exon numbering according to refseq aj003112.1, gen - bank) were amplified by pcr . Pcr assays were performed by using 1.25 u of amplitaq polymerase gold (applied biosystem, foster city, ca, u.s.a . ), 100 ng genomic dna, 2.0 mm mgcl2, and a final concentration of 10 m for each set of primers (table 2). Amplification conditions were as follows: an initial denaturing cycle at 95 for 7 min followed by 35 cycles of denaturation at 94 for 30 sec, annealing at 62 for 30 sec, and extension at 72 for 1 min . A final extension step of 72 for the pcr products were electrophoresed in a 1.2% agarose gel, and the amplified genomic dna fragments were extracted and purified using the manufacturer's recommended protocol (qiaquick gel extraction kit; qiagen, germany). Direct sequencing of both strands was performed with bigdye terminator chemistry (pe biosystems, foster city, ca, u.s.a . ). As the mutation found in patient 1 destroys an mnl1 restriction site, restriction fragment length polymorphism (rflp) analysis was performed using mnl1 enzyme (new england biolabs, beverly, ma, u.s.a .) For the pcr products from both the family members of patient 1 and 60 unaffected, unrelated korean women . Sequence analysis of the dcx coding region for patient 1 revealed a c.386c> t change in exon 3 (fig . The sequence variation results in a serine to leucine amino acid change at position 129 (s129l), which has not been found in other family members of patient 1, including maternal grandmother, parents, or siblings . Rflp analysis was conducted to confirm the sequencing result and to exclude the possibility that the genetic variation is a polymorphism . The heterozygous mutant band pattern was observed in patient 1, whereas it was not found in other family members of patient 1 or in a control population of 60 healthy, unrelated korean women (fig . No genetic variation in dcx coding region was found for patient 2 and her mother . In the developing cortex, cortical neurons must migrate long distances to reach the site of their final differentiation . The protein encoded by the dcx gene is a cytoplasmic protein that appears to direct neuronal migration by regulating the organization and stability of microtubules (10). Mutation clusters in the dcx gene were previously identified in exons 2 and 3, and 3 and 4, overlapping significantly with two proposed evolutionarily conserved domains of the dcx protein (7, 9). The first conserved domain binds tubulin and enhances microtubule polymerization, and the second binds tubulin less well and does not enhance microtubule assembly . Both domains seem necessary for optimal dcx protein function, and differences in the location and type of mutations in these regions may produce variable functional disturbances . The mutation identified in the present study is located in the first preserved domain of the dcx protein, which not been reported previously . In the setting of a family with a single girl affected with sbh, it is hard to determine the risk for sbh or xlis in future children . Because of x - linkage, a male with a dcx mutation should be clinically affected while a female with the mutation may be unaffected due to germline or somatic mosaicism or skewed x inactivation (5, 11 - 13). In the present study, the genetic analysis of the dcx mutation c.386c> t was negative in the maternal grandmother, mother, and two siblings of patient 1 who are free from sbh or xlis . Although patient 2 in the present study had similar clinical and radiological features to those of patient 1, she did not carry any genetic variation in the coding region of dcx . This suggests that the non - coding region of dcx, including the promoter region and introns, or other genes implicated in neuronal migration disorders may be responsible for this patient's condition . We identified a previously unreported missense mutation in the first preserved domain of the dcx protein . Second, it is absent from a large panel of 120 control x - chromosomes . Third, it occurs in a highly conserved position - the first domain of the dcx protein where the majority of the known dcx mutations cluster . The discovery of the new mutation reported here supports existing evidence that mutation in the first conserved domain of the dcx protein is important in the pathogenesis of sbh.
Transmission electron microscopy (tem) is one of the main tools to investigate the morphology of materials, from subnanometer to micrometer length scales . However, the interaction of electrons with materials, and especially beam sensitive structures such as polymers, biological materials, or zeolites, causes different types of radiation damage, e.g., atomic displacement, electrostatic charging, sputtering, radiolysis, and knock - on damage . These mechanisms operate at different length scales: at subnanometer length scales, knock - on damage and atomic displacement can result in distorted crystal lattices, while morphology changes due to heating, electrostatic charging and sputtering are visible at nanometer and micrometer length scales . Hence, for beam sensitive materials, beam damage constitutes a physical limit and determines the resolution that can be achieved in imaging, diffraction, and electron tomography . Therefore, it is important to analyze and understand beam damage to facilitate the study of these (beam sensitive) materials with a minimum amount of artifacts . The degradation of crystal lattices, at subnanometer length scales, can be studied by electron diffraction . It is well established that electron beam damage causes the fading of diffraction spots and rings in protein crystallography, and it has been shown that it is affected by the temperature of the material, the electron flux through the material, and the accumulated amount of electrons transmitting the material . Furthermore, this effect is easily quantified by following the intensity of diffraction rings or spots as a function of accumulated dose . The effects of beam damage at nanometer and micrometer length scales are dependent on and specific to the utilized imaging mode but mainly relate to materials loss by sputtering, and shrinkage . The degradation of materials causes artifacts in the form of changes in contrast or local intensity, while mass loss will result in an increase in overall intensity . In the literature, this phenomenon is mostly studied by using the difference image of two images with different accumulated electron doses . These techniques, however, is not commonly used to follow beam damage over a multitude of images, which is essential for e.g. Electron tomography . The analysis of organic photovoltaics (opvs) is a good example where beam sensitivity plays an important part . Since the morphology of this photoactive layer, i.e., the distribution of molecules, including their nanoscale phase separation, has a large impact on the overall efficiency of opvs, tem has become a standard characterization tool . Common opv materials are e.g. Poly(3-hexylthiophene) (p3ht) and phenyl - c61-butyric acid methyl ester (pcbm). These materials show diffraction rings instead of diffraction spots, resulting from the random orientation of crystallites or the presence of amorphous phases . Therefore, radial averaging of diffraction patterns is a suitable way to record and analyze the intensity change of p3ht and pcbm diffraction signals upon electron beam damage . In the analysis of opvs, tem samples are usually created by floating the photoactive layer on water from a water - soluble polymer film . The resulting water that is present in the sample might play a role in beam damage mechanisms as well, for example, by creating oxygen radicals . An oxygen- and water - free sample preparation method is therefore introduced to specifically study the influence of sample preparation . In this paper, beam damage is studied in both diffraction and bright field imaging, investigating the effect of electron dose rate, temperature, and sample preparation . More specifically, (1) beam damage of the crystallinity is quantified via radially averaged diffraction patterns, (2) mass loss is assessed via changes in intensity and standard deviation of the mean intensity, and (3) shrinkage is determined by monitoring the movement of markers, while a method is introduced to determine shrinkage or expansion via the use of the normalized cross - correlation . Poly(3-hexylthiophene) (p3ht, plexcore os2100) was supplied by plextronics, while pcbm ((6,6)-phenyl - c61-butyric acid methyl ester, purity 99%) was purchased from solenne b.v . And the solvent o - dichlorobenzene (odcb) from sigma - aldrich . Pedot: pss (clevios p vpal 4083) was purchased from heraues gmbh . Tem grids (quantifoil, r2\2 200-mesh cu) was purchased form quantifoil micro tools gmbh . P3ht and pcbm were dissolved in odcb, resulting in a solution with 1 wt% p3ht and 1 wt% pcbm . The solution was stirred at 70 c for 24 h to ensure complete dissolving of the materials . A custom - made specimen holder was used to facilitate direct spin - coating on tem grids . Four recesses were laser ablated (20 m deep with a diameter of 3 mm) on 5, 10, 15, and 20 mm from the center of a 50 50 mm glass plate . A custom spring was designed to lock the tem grids in the recesses, with minimal altering of flow behavior of the solution during spin - coating . Glass plates were cleaned by (1) ultrasonication in acetone for 30 min, (2) rubbing with soap and rinsing with demineralized water, (3) ultrasonication in isopropanol for 30 min, and (4) uv - ozone for 30 min . First, pedot: pss was spin - coated on 25 25 mm glass plates, at 3000 rpm for 60 s, resulting in a 50 nm thick water - soluble layer . The p3ht: pcbm solution was subsequently spin - coated at 2000 rpm for 120 s, which results in a 25 nm thick layer . The opv thin films prepared on the pedot: pss spin - coated glass plates were submerged in water to dissolve the pedot: pss layer, resulting in an opv thin film floating on a water surface . This film was picked up on a glow - discharged tem grid, dried at room temperature, and subsequently annealed in air at 120 c for 10 min . After floating of the opv layer on water and pickup by the tem grid, there is a small chance that some pedot: pss will remain on the sample surface . However, due to the small surface - to - volume ratio, the diffraction and imaging experiments are dominated by the bulk information, including oxygen and water which can penetrate into the bulk . Furthermore, this adds a significant advantage to the new direct spin - coating method, since there certainly will be no pedot: pss present during this method . In one of the conventional prepared samples, silver particles (10 nm diameter, dissolved in toluene with oleylamine as capping agent) were added to the p3ht: pcbm solution before spin - coating . To exclude water and oxygen being present in the opv thin film, another approach was followed . Here, the opv thin film was directly spin - coated on a tem grid inside a glovebox (m - braun labmaster glove box system) at 500 rpm for 10 min . The directly spin - coated samples were annealed at 120 c for 10 min inside a glovebox . Note that the direct spin - coating method is not used as an alternative to opv device fabrication but as an alternative tem sample preparation method . It is not possible to achieve water- and oxygen - free tem analysis by preparing a sample using the common floating method, hence the need for a direct spin - coating method . For clarity, the sample code used in the results section the samples were loaded into the tu / e cryo titan (fei company; now thermo fisher scientific), which was operated at 300 kv and is equipped with a field emission gun . Diffraction patterns were acquired at dose rates of 0.1, 1, 10 e/(s), with exposure times of 5, 0.5, and 0.05 s (keeping the total dose per pattern constant), at a camera length of 1.15 m. for the bright field imaging experiments, the dose rates were set at 1, 10, and 100 e/(s), with exposure times of 5, 0.5, and 0.05 s (again, keeping the total dose per image constant), respectively, at a magnification of 18k . For high accumulated dose experiments, the screen was flipped down to realize constant irradiation and flipped up to acquire intermediate images . Diffraction patterns were radially averaged using an in - house matlab script (details in the supporting information, section 1) and fitted by a nonlinear least - squares method using the lsqnonlin command in matlab (details in results section and figure 1b). The signal intensities of p3ht (at 0.256) and pcbm (at 0.217) were normalized to their initial intensity and followed as a function of accumulated electron dose . (a) fading of diffraction rings of a p3ht: pcbm bulk heterojunction at room temperature, before and after exposure to 50 e / . (b) radial average of diffraction pattern as shown in (a) decomposed in its different components by least - squares fitting . Prior to further analysis, bright field images were aligned via cross - correlation to avoid processing artifacts from sample drift . The aligned data stack was cropped to the overlap area present in all images for analysis . From the resulting aligned stack of images, the average intensity, standard deviation, and the normalized cross - correlation coefficient with the first image of the series were measured as a function of accumulated electron dose . Finally, to assess local deformations, the aligned stack was cut into 16 subareas of equal size . These subarea stacks were individually aligned via cross - correlation . From this alignment, the shift of each individual subarea with respect to the first image in the x and y direction was used to calculate the displacement of each subarea . Furthermore, the relative displacement from the center of the total image was calculated by measuring the distance between the center of the subarea and the center of the total image . More details can be found in the results section and supporting information section 2 . Figure 1a shows a diffraction pattern obtained from a typical p3ht: pcbm bulk heterojunction prepared via the conventional sample preparation (cf / rt/10), before and after exposure to 50 e / at room temperature . It is evident that the sharp outer ring, depicting the (020) lattice spacing in p3ht, is decreasing in intensity faster than the broad inner ring, characterizing pcbm nanocrystals, which is in agreement with previous studies . Since the pcbm is inherently broader than the p3ht ring, indicating a large part of pcbm being amorphous, a slower decrease in diffraction intensity for pcbm the radially averaged diffraction pattern of the p3ht: pcbm bulk heterojunction is presented . To quantitatively analyze the decrease in intensity as a function of accumulated electron dose, it is necessary to separate neighboring peaks . Therefore, to independently address the intensity of the pcbm peak at 0.217 and the p3ht peak at 0.256, the spectrum was fitted to a model function by least - squares fitting . This function contains (in the same order as in eq 1) a gaussian describing the pcbm peak, a gaussian describing the p3ht peak, a gaussian located between 0.285 and 0.323 describing (inseparable) secondary components of both p3ht and pcbm, a power law to estimate the inelastic background, and a constant representing residual dark current in the diffraction patterns . The variables a1 and a2 describe the intensity of the pcbm and p3ht signal, respectively.1 to study the effect of electron dose rate on the decrease of intensity of p3ht and pcbm diffraction rings, dose series were acquired at various dose rates, i.e., 0.1, 1, and 10 e/(s). The dose per image was kept constant at 0.5 e / by adjusting the exposure time accordingly . Because of differences in absolute peak height for pcbm and p3ht, and changes in pcbm / p3ht ratio in different regions of the sample, all peaks were normalized to the peak intensity of pcbm (a10) and p3ht (a20) of the initial diffraction pattern (a1,20). Figure 2 shows the decrease in relative intensity (a1,2i / a1,20) for p3ht (a) and pcbm (b) acquired at three different dose rates (cf / rt / x), in the presence of water and oxygen . The results confirm that the relative intensity of the p3ht diffraction ring decreases faster than the relative intensity of the pcbm diffraction ring, as seen in figure 1a . In all three cases the critical dose for p3ht, which we define by the accumulated electron dose at which the relative intensity decreases to 1/e (37%), is about 1619 e / (table 2), as calculated from an exponential decay fit . Most notably, changing the electron dose rate from 0.1 to 10 e/(s) has no significant effect on the fading of the diffraction rings, i.e., the critical dose . Fading of relative diffraction intensity (a / a) as a function of accumulated dose for p3ht (a) and pcbm (b) at different dose rates . For visibility reasons the critical dose of pcbm was obtained by an exponential decay extrapolation of the curves in figure 2b (details are presented in the supporting information, section 3). Therefore, the critical dose for pcbm is less well - defined than the critical dose for p3ht . Table 2 shows that this critical dose is between 265 and 412 e / . Despite the large error due to extrapolations, it is clearly shown that the critical dose of pcbm is more than 10 times larger than the critical dose of p3ht . Therefore, in this system, the stability of p3ht is the limiting factor . The results show that, using a dose of 0.1 e / per image, at least 160 diffraction patterns can be acquired, opening up the possibility of using high electron dose techniques such as electron diffraction tomography for organic crystals . To complement the presented results, studies on beam damage effects for each component individually and nanocomposites of different ratios should be considered ., dose rates between 0.1 and 10 e/(s) do not have an effect on the rate of relative intensity loss of both p3ht and pcbm . This is in agreement with previous studies on the decrease of features in the eels spectrum of other organic materials, but in contrast with observations of karuppasamy et al . In single - particle cryo - electron microscopy . Note that karuppasamy et al . Used an electron microscope operating at 120 kv, significantly decreasing the inelastic mean free path . At 300 kv, as in our experiments, changing the sample thickness or increasing the electron dose rate to values above 10 e/(s) might therefore cause an electron dose rate effect on sample damage . Nonetheless, this falls beyond the scope of this paper and is therefore not investigated . As the decrease in diffraction intensity is related to the breaking of chemical bonds, beam damage will change the homo and lumo levels of opv materials, which can be measured with electron energy loss spectroscopy (eels). Based on the low - loss eels results presented in ref (17), a critical dose can be estimated which is similar to our diffraction results . This clearly illustrates the close relationship between diffraction intensities and energy levels in opv materials with low - dose diffraction being somewhat simpler to carry out and more broadly available for critical dose measurements . The above results were obtained with a standard opv bulk heterojunction sample imaged at room temperature . Additional dose series were acquired in cryogenic conditions, as this has been shown to reduce beam damage in beam sensitive materials . We furthermore studied the effect of sample preparation using either the conventional sample preparation (cf) or by direct spin - coating (ds) on a tem grid in a water and oxygen - free environment (glovebox), facilitated by a custom - made tem specimen holder as described in the methods section . Figure 3 shows the influence of temperature and sample preparation on the decrease of relative intensity of the p3ht (a) and the pcbm (b) signal, with a dose per image of 0.5 e / acquired with a dose rate of 10 e/(s). It is clearly visible that the sample cf / cryo/10 shows a slower fading of the diffraction signal for both the pcbm and the p3ht signal . The critical dose increases from 16 e / at room temperature to 108 e / at 80 k (table 3). Hence, cryo - preservation is shown again to be essential in the analysis of beam sensitive materials . More importantly, having a quantitative criterion or threshold for determining exposure limits as presented here will provide a framework to maximize useful information before beam damage influences the validity of acquired data . Fading of relative diffraction intensity (a / a) for p3ht (a) and pcbm (b) as a function of accumulated dose, showing the effect of sample preparation at room temperature (rt) and at 80 k (cryo). Sample preparation effects, i.e., the presence or absence of oxygen and water, are only minor at room temperature . However, at cryogenic conditions, the rate of relative intensity loss for p3ht (figure 3a, purple triangles) is significantly decreased by a water- and oxygen - free sample preparation method (ds / cryo/10), increasing the critical dose from 108 to 188 e / (table 3). A detailed analysis of the significance of these results is presented in the supporting information, section 4 . The observed difference in critical dose for p3ht and pcbm with or without oxygen and water being present at low temperatures these molecules will form radicals upon electron beam irradiation, thus decreasing the crystalline order at lower accumulated electron doses . Because of the fact that the samples have been in high vacuum for at least 30 min before imaging, it is expected that water and oxygen will diffuse out of the material at rt conditions and thus only be present in small amounts in the imaged thin film . This explains why sample preparation seems to have little influence when measuring at room temperature . For cryogenic conditions the exclusion of water and oxygen significantly improves the stability of p3ht exposed to an electron beam . Furthermore, for the room temperature experiments, annealing in air might cause minor damage . This also could explain the observed critical dose, causing the critical dose for the cf / rt/10 to be lower than the critical dose for ds / rt/10 . The values in table 3 also suggest that a water- and oxygen - free method increases the critical dose of pcbm in cryogenic conditions . In this case, a linear extrapolation was used, since the adjusted r value for the exponential decay fits were too low (0.85 (exponential fit) compared to 0.95 (linear fit)). Since the pcbm intensity decay will probably not behave in a linear fashion, one could suggest that the difference between the pcbm critical doses at cryogenic conditions is therefore not significant . Since pcbm is more hydrophobic than p3ht, and it is known that p3ht forms a charge - transfer complex with oxygen, it can indeed be expected that the presence of water and oxygen has less influence on the decay of the pcbm diffraction ring . In imaging mode, beam damage can present itself as among others (1) shrinkage or expansion of an organic thin film and (2) sample thinning due to mass loss . Both effects were measured by acquiring dose series of samples prepared by cf and ds methods . These series of images were acquired at different dose rates and at either room temperature or cryogenic conditions . To study sample deformations, the normalized cross - correlation coefficient (nccc) of an image with respect to the first image within the series was calculated . Sample thinning due to mass loss was assessed by the relative decrease in average pixel intensity and the accompanying standard deviation . Figure 4a shows the decrease of the nccc with respect to the original image as a function of accumulated dose at dose rates of 1, 10, and 100 e/(s) (cf / rt / x). Local differences in thickness, small deviations in intensity due to microscope instabilities, and high contrast contaminations cause the nccc to start at different values for each series . Therefore, the trend of the curves is studies instead of the absolute nccc values . It is clearly visible that the nccc drops quickly before a more stable regime is reached after approximately 150180 e /, as calculated from the intersect of two linear fits: one fitted to the first 50 e / and one fitted between 750 and 1000 e / (for details, see supporting information, section 5). Identical fit regimes were chosen in every experiment to ensure a fair comparison between data sets, at room temperature and at cryogenic conditions . (a) cross - correlation coefficient with respect to the first image of three dose series (1, 10, and 100 e/(s)) as a function of accumulated dose . For visibility reasons, (b) cross - correlation coefficient with the first image of a dose series acquired at 10 e / at room temperature, starting at 750 e / . An exponential fit is added to calculate the critical dose . For cf / rt/100, the quick initial drop is less evident (a slope of (6.73 0.04) 10 as compared to (1.32 0.04) 10 and (1.67 0.05) 10 for cf / rt/1 and cf / rt/10, respectively), which is likely caused by pre - exposure with a higher dose before starting the dose series . An exposure of 0.25 s to check that there was no carbon film in the entire field of view caused a pre - exposure of 0.25 and 2.5 e / for the cf / rt/1 and cf / rt/10 samples, respectively, which is small compared to the dose limit . However, for the cf / rt/100, this pre - exposure will be 25 e / . This will cause significant damage, and therefore the nccc for the last image will be higher, as the first image is already more damaged . After the cutoff dose is reached, a more stable second regime starts, as indicated by a much smaller slope (1 10). Since the cutoff dose and the slope of the second regime are fairly similar between dose rates of 1 to 100 e/(s), it is concluded that the dose rate effect is negligible, similar to the diffraction data . Although the slope of the second damage regime is much smaller, beam damage still occurs . In a similar way as introduced for the diffraction data, a quantitative criterion can be set to calculate the critical dose, at which the initial nccc decreases with a factor of e. to estimate this critical dose for imaging, a dose series is acquired at 10 e/(s), reaching an accumulated dose of 30 000 e / . An exponential decay curve is fitted to the nccc decay of this data set, starting at 750 e / to exclude effects from the first damage regime . Calculating the critical dose imaging results in a value of (2.6 0.2) 10 e / (for details, since the nccc is a measure of similarity, it will entail among others sample deformation and contrast loss or intermixing . To show that the initial drop in cross - correlation is caused by sample deformations, the stack of aligned images acquired at 10 e/(s) was cut into 16 equal subimages (figure 5a), and these different subimages were aligned by the normalized cross - correlation to the next image of the dose series . This approach facilitates the measurement of displacement of each subimage, i.e., expansion or contraction of the thin film as a function of accumulated dose . It is clearly visible that the displacement of the subimages at the corners and edges of the stack is larger than the displacement of the subimages at the center of the stack (figure 5b), indicating movement to or from the center of the stack . Calculating the relative increase in distance between the center of each subimage and the center of the total image results in a shrinkage of 0.70 0.14% at an accumulated dose of 1000 e / . (a) schematic representation of cutting a stack of images in 16 parts (left to middle) and using the cross - correlation to find the shift in 1 part of the image compared to the original image (right), as a representation of shrinkage or expansion . (b) the average shift of the 4 corner subareas, the 8 edge subareas, and the 4 center subareas, acquired at 10 e/(s). (c) relative decrease in standard deviation of the mean intensity as a function of accumulated dose for samples prepared by the common floating approach at room temperature, at three different dose rates . For visibility reasons, the validity and accuracy of the method above has been proven against an approach that uses the movement of high contrast silver makers to determine shift, as presented in the supporting information, section 2 . As a different measure to assess beam damage, we quantify the change in average intensity that can be interpreted as a change in mass thickness due to either mass loss or deformations of the thin film . To minimize the effects of microscope instabilities, two low - magnification overview images were taken, before and after the dose series . From these two images the intensity ratio of the nonilluminated and illuminated region before and after the series was calculated (table 4). Because of the occurrence of lateral shrinkage, one would expect the sample to become thicker (assuming volume preservation), since material is moving toward the center . However, table 4 shows an insignificant change in intensity, which can be explained by the fact that the relative shrinkage is small . Furthermore, thinning caused by mass loss will increase the average intensity, compensating for the thickening effect of shrinkage . Before and after images are shown in the supporting information, section 7 . Last, figure 5c shows the relative decrease in standard deviation of the mean intensity of the aligned images . Shrinkage would result in a higher standard deviation, since more data will move into the field of view . It is, however, clear that the standard deviation decreases, again pointing out that mass loss plays a significant role in beam damage processes . The fast drop in relative standard deviation at the onset of beam damage might be caused by quick mass loss of material at the surface of the sample removing sample roughness . After this initial drop, a more stable regime is reached where mass loss plays a less significant role since the sample has reached a more equilibrated state . From this, we can again conclude that in imaging mode a dose rate between 1 and 100 e/(s) has a negligible effect on beam damage . Furthermore, we can conclude that the minimal pre - exposure of polymer thin films or microtome sections, often applied prior to acquiring electron tomography data sets, can now be quantified to limit artifacts in imaging . During this regime, mass loss and shrinkage occur, while after this regime, a second regime will set in, significantly reducing the rate of electron beam damage at higher doses . Furthermore, a critical dose in imaging is calculated, which results in a critical dose of more than 2500 e / . Even when the cutoff dose of the first damage regime is subtracted, this leaves more than enough dose available for acquiring tomography data sets (e.g., tilting from 70 to + 70 in 1 steps, with 10 e / per image results in a total dose of 1410 e /). Figure 6a shows the decline of the nccc with respect to the first image as a function of accumulated dose, for series acquired at room temperature and cryogenic conditions, and prepared by either the cf or ds methods . Comparing the room temperature measurements, one can see that the decrease in nccc in the first regime does not significantly change for ds method, as the calculated slope is (1.57 0.12) 10 compared to (1.67 0.05) 10 for the cf method . However, the cutoff between the two damage regimes decreases from 156 9 to 96 12 e / . This indicates that the rate of damage is similar for both the cf and the ds method but that the total damage in the first regime will be smaller for the ds method (the relative decay of nccc at the cutoff dose decreases from 24% for the cf method to 15% for the ds method), indicating a more stable system . (a) decrease in nccc with respect to the first image for dose series acquired at 10 e/(s), at room temperature and at cryogenic conditions, from samples prepared by either the conventional floating method or the oxygen- and water - free method . (b) cross - correlation with the first image of a dose series acquired at 10 e / at room temperature of a sample prepared by the oxygen- and water - free method . (c) mean - squared displacement of the 12 edge subareas and the 4 center subareas calculated by alignment via cross - correlation . The images were acquired at 10 e / at room temperature from a sample prepared by either the common floating approach or the oxygen- and water - free method . (d) see part c, but for images acquired at cryogenic conditions . For visibility reasons, every fifth data point is shown . The second, more stable regime shows a relatively constant nccc for the ds method, since the slope of this regime decreases from (12.7 0.3) 10 for the cf method to (6.9 0.2) 10 without water and oxygen present . The critical dose therefore increases from (2.6 0.2) 10 e / for the cf method to (5.0 0.4) 10 e / for the ds method (for details, see supporting information, sections 5 and 6). This again indicates that the ds method will create a more stable system during electron beam radiation . Figure 6c shows the displacement of parts of the images acquired at room temperature by using the alignment method as described before . It is clearly visible that by using the ds method, the shrinkage of the thin film decreases significantly, from 0.70 0.18% to 0.40 0.14% . The sample prepared by the ds method is more stable, since it is clear that the absolute decrease in relative standard deviation is smaller for this method . Just as the nccc behavior, a second more stable regime sets in . The smaller slope in this regime for the ds method (figure 6b, black squares vs red circles) indicates a larger stability . All in all, this leads to the conclusion that at room temperature an oxygen- and water - free sample preparation method significantly enhances sample stability . After subtracting the initial pre - exposure regime, an electron dose of 5 10 e / is still available before reaching the critical dose imaging, which is about twice as much as for a sample prepared by the cf method . Furthermore, since the relative decay of nccc at the cutoff dose is smaller for the ds method, the acquired data set in the second regime will show the most resemblance to the sample in the initial, nondamaged state . Figure 6a also shows the evolution of the nccc as a function of accumulated dose in cryogenic conditions . Clearly, the two damage regimes remain visible . The slope of the first regime decreases from (1.57 0.12) 10 for ds / rt/10 to (9.25 0.76) 10 for ds / cryo/10 and to a minimum of (9.53 0.57) 10 for cf / cryo/10 . This indicates that cryo - preservation could be useful when acquiring small data sets with a low total dose, since the nccc changes more slowly, and one can therefore acquire images as close as possible to the original, nondamaged state . The cutoff between the two damage regimes increases from 156 9 e / for cf / rt/10 to 219 19 and 271 28 e / for cf / cryo/10 and ds / cryo/10, respectively . With a relative decay of 19% at the cutoff dose for cf / cryo/10, in comparison to 37% for ds / cryo/10, it can be concluded that for small data sets with relatively low doses a conventional floating method at cryogenic conditions is best applicable, since the nccc decreases slowly over a larger amount of dose . In cryogenic conditions, the second regime behaves differently in comparison to room temperature conditions, since the nccc is increasing again . Therefore, it was impossible to determine a useful critical dose imaging, based on an nccc decay by a factor of e. the increase in nccc can be explained by an inversion of the displacement, i.e., initial shrinkage followed by expansion or vice versa . This is confirmed by the displacement of subareas, shown in figure 6d . At an accumulated dose of 400 e /, the relative shrinkage is calculated to be 0.39 0.16% and 0.79 0.42% for cf / cryo/10 and ds / cryo/10, respectively . This means that the sample will expand first, before it starts to shrink, in contrast to room temperature conditions where shrinkage occurs directly from the onset of beam damage . Whether a stable regime will appear after shrinkage in cryogenic conditions, just as in room temperature conditions, is not investigated . Because of the initial expansion, one would expect an initial decrease in standard deviation, since the information on less material will be spread over the same amount of pixels . This effect is seen in figure 6b . However, for the cf / cryo/10 sample, this effect is minimal . Local mass loss, which is expected since oxygen and water can be removed easily, will increase the standard deviation, explaining the smaller decrease in relative standard deviation . In cryogenic conditions, in contrast with room temperature conditions, the sample seems to be destabilized by an oxygen- and water - free sample preparation method . Furthermore, even without oxygen and water being present, the stability at high doses is less in cryogenic conditions than at room temperature . Further details on intensity changes of areas before and after exposure can be found in supporting information section 7 . By combining diffraction and imaging data, we have investigated electron beam damage effects in p3ht - pcbm thin films over multiple length scales . Analysis of the fading of electron diffraction rings allows us to quantify the beam sensitivity of each component of the nanocomposite individually . This shows that pcbm is 15 times more stable based on the criterion of the critical dose diffraction, i.e., the accumulated electron dose at which the intensity of the corresponding diffraction ring decreases with a factor of e. for electron dose rates between 0.1 and 10 e/(s), no dose rate effects are observed . Cryo - preservation increases beam stability of both materials with the critical dose for p3ht significantly increasing from 16 to 108 e /, while the critical dose for pcbm increases from 392 to 596 e / . Most importantly, it is shown that excluding water and oxygen during sample preparation further improves the beam stability of these materials, reaching a critical dose of 188 e / for p3ht, which corresponds to a 10-fold increase over conventional sample preparation and room temperature diffraction . Beam damage in imaging mode was analyzed in terms of mass loss and thin film deformations . The normalized cross - correlation coefficient (nccc) between images contains contributions from among others contrast loss and intermixing of phases . Here, the nccc is used to assess sample deformation, as the nccc is followed as a function of accumulated dose . First, it is shown that the dose rate effect is negligible for dose rates between 1 and 100 e/(s). Second, experiments at room temperature show a quick initial decrease in nccc before a stable second regime is reached . In this first regime, shrinkage and mass loss occurs, as shown via the movement of subimages and changes in relative standard deviation . The second, more stable regime is used to calculate a critical dose imaging, defined by the dose at which the initial nccc is decreased with a factor of e. an oxygen- and water - free sample preparation method increases this critical dose imaging from 2600 to 5000 e / . Furthermore, since the relative decay of the nccc at the cutoff dose is lowest in oxygen and water free conditions, data acquired in this second regime will be closest to the sample in a nondamaged state . In cryogenic conditions, the first regime shows a slower decrease of the nccc, indicating that the first regime is more stable than in room temperature conditions . In the second regime, however, the nccc increases again . This is due to the fact that after an initial expansion of the thin film the sample starts to shrink . In summary, we suggest to use an oxygen- and water - free sample preparation method combined with cryogenic conditions in diffraction mode, while for imaging an oxygen- and water - free sample preparation method is best suited combined with data acquisition at room temperature (high total doses, large data sets) and a conventional floating method is best suited for cryogenic condition (small total doses, small data sets). We believe that the quantitative analysis of tem beam damage as illustrated here will become a standard tool to optimize tem imaging conditions and sample preparation protocols in the future.
First - line procedural investigations for non - specific epigastric pain include endoscopy and endosonography, combined with biopsy of suspicious areas in the upper gastrointestinal tract . Despite high sensitivity and specifity, obtaining a definite histopathologic differentiation is sometimes unsuccessful, particularly if biopsy is not representative, e.g. Because of tumor bleeding into the affected tissue . In such a case, the single remaining diagnostic option is a curative laparoscopic endoscopically - assisted wedge - resection of the stomach . We present an interdisciplinary case report in which a tumor of the stomach, suspicious for gist, was removed surgically and was found to be a benign glomus tumor by definitive histology . A 44-year old patient presented with relapsing epigastric pain of variable intensity and tarry stool, beginning three days earlier . There were no clinical or laboratory findings to suggest an acute posthemorrhagic anemia (hemoglobin 14.7 g / dl, hematocrit 43.2%, pt 118%, aptt 27 s). Upper endoscopy and endosonography were performed, revealing a spherical, submucosal, solid tumor approximately 50 mm in diameter located in the pyloric antrum (fig . Overlying ulceration was considered the likely source of bleeding, and fine needle biopsy of the lesion (19 g) was performed, followed by preventive clip - application . An endosonographic picture with a poorly reflective, non - homogeneous pattern and echo - free areas was compatible with a 35 30 mm gist (fig . Investigations thus far were inadequate to exclude a malignant process, and surgery (endoscopically - assisted laparoscopic wedge - resection of the stomach) was indicated . Histological section of the surgical specimen revealed a rare benign glomus tumor (positive reaction on markers specific for glomus tumor (vimentin / actin), fig . Glomus tumor is a (quite) rare neoplasm and despite local invasion of vessels is mostly benign . It is often found in the skin (particularly in the dermis / subcutaneous tissues of the limbs), but can also be found in the gastrointestinal tract (usually intramurally in the mucosa / submucosa and serosa) as well as other solid organs . The present assumption that the first glomus tumor of the stomach was identified in 1948 and described with another two cases in 1951 smol'iannikov wrote that the first glomus tumor of the stomach was clearly described in a 64-year old man in 1928 by talijeva . Furthermore, the first malignant glomus tumor of the stomach was diagnosed in 1939 by kirschbaum et al . In a 40-year old man . There are case reports of malignant glomus tumors of the stomach in girls aged 1214 years, published by yannopoulos et al . . In addition, there have been a number of reports pertaining to glomus tumors of solid abdominal / retroperitoneal organs and of the colon [6, 7, 8]. There is no gender bias in the incidence of glomus tumors but their peak incidence occurs between the fourth and sixth decade of life . Immunohistochemically, most glomus tumors show a positive expression of vimentin / actin without expressing chromogranin a, neuronspecific enolase (nse), carcinoembryonic antigen (cea) or epithelial membrane antigen (ema). The rarity of glomus tumor, its variable organ involvement, its non - specific symptoms at presentation and the often equivocal results of standard first line investigations all contribute to diagnostic difficulty . Endoscopic and endosonographic images in glomus tumors of the stomach show a solid, submucosal tumor with or without ulceration and do not differentiate it from other important diagnoses, e.g. Gist, neuro - endocrine neoplasia (carcinoid), angiomyoma or lymphoma . Hence, presurgical diagnostic confirmation is often impossible . Only immunohistochemical analysis of representative biopsies (gist: positive reaction with cd-117 antibodies and missing expression of glomus tumor typical actin / vimentin) can confirm the diagnosis, and hence aid the clinician in determining appropriate therapy and prognosis [9, 10, 11]. Compounding pre - surgical diagnostic difficulties, there have also been reports of malignant transformation of glomus tumor . Therefore, surgery or en bloc endoscopic enucleation is in most cases the remaining diagnostic and therapeutic option [12, 13]. A definite immunohistochemical confirmation of the diagnosis is essential, because the prognosis of a potentially malignant lesion is otherwise unpredictable . In the case presented, a patient with recurring epigastric pain and melaena, there was an endoscopic and endosonographic finding of a submucosal tumor of the gastric antrum . Given the non - diagnostic biopsy result and the ongoing risk of gastrointestinal bleeding, endoscopically assisted laparoscopic wedge - resection of the stomach was performed as a combined diagnostic and therapeutic procedure . Histological and immunohistochemical analysis of the resected tissue showed an entirely removed (ro) glomus tumor . Unlike gist, if complete removal of a benign glomus tumor (ro) is verified histologically, there is no indication for further specific therapy [14, 15]. Sonographic (for solid organs) and endoscopic follow - up for early detection of recurrence or metastasis constitutes the most reasonable postoperative follow - up.
Prompt multidisciplinary management of hand infections is essential to prevent stiffness, loss of function and long - term disability . Although hand infections are relatively common in young patients particularly males engaged in sports, recurrent hand infections are not . Panton - valentine leukocidin (pvl) is a toxin produced by some strains of s. aureus and usually implicated in skin and soft tissue infections . It can also cause invasive infections, in particular haemorrhagic pneumonia . To date, there is only one previous case describing recurrent hand infections associated with panton - valentine leukocidin - positive staphylococcus aureus (pvl - sa). This case highlights the need for multidisciplinary management of these patients as the diagnosis of pvl - sa may have been missed with potentially devastating consequences . A 25-year - old right - hand dominant male warehouse operative presented to the emergency department (ed) with a three - day history of redness, pain and swelling in the right palm . He removed a wooden splinter from the palmar aspect of the metacarpophalangeal joint of his right middle finger 24 h before the symptom onset . In his past medical history, he had childhood - onset eczema and recalled always having cracked hands he was not currently managing his eczema with medical therapy but described several flare - ups of his eczema since childhood . There was no recent history of skin and soft tissue infection in his household contacts . He was on noregular medications, had no allergies and smoked 10 cigarettes a day . On examination, his temperature was 37.5c and he had tenderness, swelling and overlying erythema localised to his palm and extending to the proximal interphalangeal joint of his middle finger . Admission blood tests showed a serum white cell count of 10.71 10/l (reference range 3.510.5 10/l) and c - reactive protein of 14.1 mg / specimens cultured methicillin susceptible s. aureus (mssa) for which he completed a 14-day course of intravenous flucloxacillin 1 g qds . Following surgical drainage the wound was left open and dressed daily with bethidine wicks . Twelve weeks later he represented to his general practitioner (gp) with a four - day history of increasing pain, redness and swelling over his dorsal left wrist . He was referred to the ed 48 h later when he failed to respond to oral flucloxacillin . On examination he had a 3 3 cm fluctuant swelling over his dorsal wrist with surrounding cellulitis (figure 2a). G / l and c - reactive protein of 57.8 mg / l . Figure 2. (a) patient s second presentation with abscess dorsal left wrist and surrounding erythema . (a) patient s second presentation with abscess dorsal left wrist and surrounding erythema . Further investigations were requested in the context of a recurrent infection in a healthy, young patient . These included an oral glucose tolerance test, a mantoux test, serum hiv antibodies, immunoglobulin levels, complement levels, antibodies to atypical infections and a transesophageal echocardiogram . Similar to his previous admission, he required incision and drainage of the abscess followed by daily bethidine wick dressings (figure 2b). On this occasion pus was sent to the national methicillin resistant s. aureus (mrsa) reference laboratory and was reported positive for pvl - sa . The patient completed a 10-day course of intravenous flucloxacillin 1 g qds with five days of clindamycin 600 mg qds for anti - toxin cover . A decolonization regimen of five days 4% chlorhexidine wash and intranasal mupirocin was prescribed once his wounds had fully healed . He was advised not to share bedding or towels and the same decolonisation regimen was advised for his household contacts . He was discharged following completion of antibiotic therapy and followed - up in the outpatient clinic (figure 3). Pvl is a cytotoxin produced by 5% of staphylococci (both mssa and mrsa). It primarily occurs sporadically in young healthy patients and as a consequence can be missed . Although, predominately detected in isolates causing skin and soft tissue infections, pvl - sa strains can become invasive leading to necrotising fasciitis, purpura fulminans and necrotising pneumonia with a high mortality rate; up to 75% in necrotising pneumonia despite medical treatment . Pvl - sa infections are thought to be more highly transmissible than pvl - negative infections . Healthcare workers, contact sports players and closed communities are considered as high - risk groups for transmission . Pvl - sa infections have been described since the 1930s, but within the last decade their incidence in the community is rapidly increasing . Although surveillance and improved case recognition may have contributed, this rapid increase is of growing international concern . In the united states, mrsa isolates increased from 24% to 54% and mssa isolates overall increased from 9% to 12% . In ireland, an increased prevalence of pvl - positive mrsa (from 0.2% to 8.8%) and a decrease in pvl - positive mssa (from 20% to 2.5%) from 2004 to 2011 have been reported . This contrasts to uk where pvl - positive mrsa infections are rare (0.8% of all isolates) but pvl - positive mssa are more common (9.0% of all specimens). Mssa isolates are not routinely referred to the reference laboratory for analysis in ireland, which may partially explain these discrepancies . To date, one case of recurrent pvl - sa hand abscesses has been reported in the literature . In 2013, described a pvl - sa right palmar abscess in a healthy 24-year - old who developed a second abscess six weeks later in the contralateral palm . Although pvl - positive mrsa was identified, the age, immune status and clinical course were comparable to this case . Similar to wearn et al ., we consider our patient's second presentation to be a recurrence of pvl - sa . Following the initial infection he likely remained colonised with pvl - sa and his eczematous skin then facilitated further entry of pvl - sa with development of the second abscess . In addition to appropriate surgical and antibiotic management of this infection, management of his underlying skin condition, patient education regarding appropriate hygiene and decolonisation of the patient and his contacts facilitated prevention of a further recurrence . Clinical suspicion of pvl production is key and if suspected, advice should be sought from the clinical microbiological service regarding patient management and referral of positive specimens to a reference laboratory for pvl testing . The recommended management of pvl - sa infection includes treatment of infection (abscess drainage and antibiotic therapy), decolonisation of the index patient, increased individual (covering wounds and hand hygiene) and environmental hygiene and appropriate management of close contacts . The main strategy to prevent reinfection with or transmission of pvl - sa is stringent hygiene combined with decolonisation treatment . In this case guidance from the centers for disease control and prevention it refers to the five cs of risk factors for pvl - related infection: contaminated items, close contact, crowding, cleanliness, and cuts and compromised skin integrity . Particular emphasis was placed on the management of our patient s eczema to optimise skin integrity . Health protection agency (hpa) guidelines recommend that all patients and their close contacts undergo decolonisation (chlorhexidine wash 4% with intranasal mupirocin), although internationally there is no consensus on this approach . This relates to individual countries testing policies for the pvl toxin . In the uk where cases are considered rare and severe, testing is recommended as it is an aggressive approach to treatment including decolonisation . Despite these published guidelines, risk factors and recommendations to prevent recurrence recurrent primary hand abscesses in the young, healthy patient are uncommon and should raise suspicion of an underlying condition, atypical infection or pvl - sa . Prompt multidisciplinary management of these infections is needed to prevent stiffness, loss of function and long - term disability . The authors report no conflicts of interest . The authors alone are responsible for the content and writing of this article.
Enterococci are pervasive and predominant inhabitants of the gastrointestinal tract of humans and animals, found in soil, water and food (1). For many years, they were considered as normal flora and unharmful to man (2). However, over the last years, enterococci have emerged as major nosocomial pathogens, representing an increasingly important problem for public health (3). Indeed, these bacteria have a great ability to acquire resistance to some antimicrobial agents such as glycopeptides, in particular vancomycin, which are important for human therapy (4). Although enterococci are normally of relatively low virulence, they can transfer their antimicrobial resistance genes and virulence factors to other intestinal microflora and/or virulent bacteria, resulting in an increased pathogenicity (5). The climbing incidence of antibiotic resistant enterococcus spp . Is the result of increased use of antibiotics in human health care system and animal growth promoters (6). The presence of large numbers of enterococci, in particular multidrug resistant ones, occurs commonly in vegetables, dairy and animal products (7). This dilemma, in part, is because of extensive usage of antimicrobial agents in modern farm industry (8). Due to limited therapeutic options for treating vancomycin - resistant enterococci (vre), they are considered as a major cause of concern . Vre has been isolated from hospital sources, food animals, environment, and waste water . Previously, we investigated more about vre from clinical samples, surface water and sewage treatment plants in iran . The results of our prior studies showed isolation of a high number of vre isolates from water and sewage (9, 10). In view of the lack of information about vre isolates in food samples, we studied the occurrence of vre in meat, chicken and cheese which were sold in local markets . Thirty food samples, each 10 from chicken, meat and cheese were collected from tehran local markets from april to september 2010 . The samples were sealed in a plastic bag, labeled immediately and transported to a microbiology laboratory in a cold cycle . Ten grams of each sample were suspended in 90 ml of saline and then heavily vortexed . The mixture was filtered using 0.45 filter membrane (millipore, sparks, md, usa). The filters were then transferred to m - enterococcus agar (becton dickinson and co., sparks, md, usa) supplemented with 4 g / ml vancomycin and incubated for 48 hours at 37c . Colonies suspected to be enterococcus were subjected to identification tests using the following characteristics: growth and hydrolysis of bile - esculin agar, growth in the presence of 6.5% nacl, absence of catalase, presence of pyrrolidonyle arylamidase, 0.04% tellurite reduction, arabinose utilization, arginine dehydrolase activity, methyl - a - d - glucopyranoside acidification, and motility and pigmentation . Species identifications were confirmed by pcr using specific primers (11). Minimum inhibitory concentration (mic) for vancomycin, teicoplanin and gentamicin were determined using the e test (biodisk ab, solna, sweden). Susceptibility tests to ampicillin (10 g), ciprofloxacin (5 g), gentamicin (120 g), erythromycin (15 g), tetracycline (30 g), chloramphenicol (30 g), linezolid (30 g), and quinupristin / dalfopristin (q - d) (15 g), (mast diagnostics ltd ., bootle, merseyside, uk) were performed by disc diffusion method according to the clinical laboratory standard institute (clsi) guidelines (12). All the vre isolates were investigated for vancomycin resistance genes . For dna extraction, one isolated colony from each plate the mixture was centrifuged and 10 l of the supernatant was used as the dna template in the pcr mix . Identification of van genotypes (vana, vanb) for each isolate of vre was performed by a separate pcr with specific primers as follows: vana, 5-catgaatagaataaaagttgcaata-3, 5-cccctttaacgctaatacgatcaa-3; vanb, 5-gtgacaaaccggaggcgagga-3, 5-ccgccatcctcctgcaaaaaa-3. The pcr assay was performed in a total volume of 25 l containing 10 mm tris - hcl (ph = 8.3), 1.5 mm mgcl2, 0.2 mm each dntps, 0.5 u taqdna polymerase (ht biotechnology, cambridge, uk), and each primer (40 pmol). The pcr cycle was as follows; initial denaturation at 94c for five minutes, 30 cycles of denaturation at 94c for one minute, annealing at 54c for one minute and extension at 72c for one minute, with a final extension at 72c for 10 minutes (13). The pcr was performed with an eppendorf mastercycler (eppendorf, hauppauge, ny). All the isolated vre were typed using a high - resolution biochemical fingerprinting method, phene plate (phplate) system, which is specifically developed for typing of enterococci strains (the phplate ab, stockholm, sweden). Briefly, a loopful of a fresh bacterial culture was inoculated in phplate growth media containing 0.2% (w / v) protease peptone, 0.05% (w / v) yeast extract, 0.5% (w / v) nacl and 0.011% (w / v) bromothymol blue . The plates were then incubated at 37c and images of the plates were scanned after 16, 24 and 48 hours using an hp scanjet 4890 scanner . After the final scan, the phplate software (phpwin 4.2) was used to create the absorbance data (biochemical fingerprint) from the scanned images . The correlation coefficient using a pair - wise comparison of the biochemical fingerprints and clustered was determined according to the unweighted pair group method (upgma) with arithmetic averages . The mean similarity between the compared isolated minus 2sd was taken as the id - level of the system . Isolates showing similarity to each other above this level were considered as identical (common biochemical phenotypes: c - bpt) (13). Thirty food samples, each 10 from chicken, meat and cheese were collected from tehran local markets from april to september 2010 . The samples were sealed in a plastic bag, labeled immediately and transported to a microbiology laboratory in a cold cycle . Ten grams of each sample were suspended in 90 ml of saline and then heavily vortexed . The mixture was filtered using 0.45 filter membrane (millipore, sparks, md, usa). The filters were then transferred to m - enterococcus agar (becton dickinson and co., sparks, md, usa) supplemented with 4 g / ml vancomycin and incubated for 48 hours at 37c . Colonies suspected to be enterococcus were subjected to identification tests using the following characteristics: growth and hydrolysis of bile - esculin agar, growth in the presence of 6.5% nacl, absence of catalase, presence of pyrrolidonyle arylamidase, 0.04% tellurite reduction, arabinose utilization, arginine dehydrolase activity, methyl - a - d - glucopyranoside acidification, and motility and pigmentation . Minimum inhibitory concentration (mic) for vancomycin, teicoplanin and gentamicin were determined using the e test (biodisk ab, solna, sweden). Susceptibility tests to ampicillin (10 g), ciprofloxacin (5 g), gentamicin (120 g), erythromycin (15 g), tetracycline (30 g), chloramphenicol (30 g), linezolid (30 g), and quinupristin / dalfopristin (q - d) (15 g), (mast diagnostics ltd ., bootle, merseyside, uk) were performed by disc diffusion method according to the clinical laboratory standard institute (clsi) guidelines (12). All the vre isolates were investigated for vancomycin resistance genes . For dna extraction, one isolated colony from each plate the mixture was centrifuged and 10 l of the supernatant was used as the dna template in the pcr mix . Identification of van genotypes (vana, vanb) for each isolate of vre was performed by a separate pcr with specific primers as follows: vana, 5-catgaatagaataaaagttgcaata-3, 5-cccctttaacgctaatacgatcaa-3; vanb, 5-gtgacaaaccggaggcgagga-3, 5-ccgccatcctcctgcaaaaaa-3. The pcr assay was performed in a total volume of 25 l containing 10 mm tris - hcl (ph = 8.3), 1.5 mm mgcl2, 0.2 mm each dntps, 0.5 u taqdna polymerase (ht biotechnology, cambridge, uk), and each primer (40 pmol). The pcr cycle was as follows; initial denaturation at 94c for five minutes, 30 cycles of denaturation at 94c for one minute, annealing at 54c for one minute and extension at 72c for one minute, with a final extension at 72c for 10 minutes (13). The pcr was performed with an eppendorf mastercycler (eppendorf, hauppauge, ny). All the isolated vre were typed using a high - resolution biochemical fingerprinting method, phene plate (phplate) system, which is specifically developed for typing of enterococci strains (the phplate ab, stockholm, sweden). Briefly, a loopful of a fresh bacterial culture was inoculated in phplate growth media containing 0.2% (w / v) protease peptone, 0.05% (w / v) yeast extract, 0.5% (w / v) nacl and 0.011% (w / v) bromothymol blue . The plates were then incubated at 37c and images of the plates were scanned after 16, 24 and 48 hours using an hp scanjet 4890 scanner . After the final scan, the phplate software (phpwin 4.2) was used to create the absorbance data (biochemical fingerprint) from the scanned images . The correlation coefficient using a pair - wise comparison of the biochemical fingerprints and clustered was determined according to the unweighted pair group method (upgma) with arithmetic averages . The mean similarity between the compared isolated minus 2sd was taken as the id - level of the system . Isolates showing similarity to each other above this level were considered as identical (common biochemical phenotypes: c - bpt) (13). According to the biochemical tests, a total of 102 isolates belonged to enterococci, 48, 40 and 14 of which were from meat, chicken and cheese, respectively . Antibiotic susceptibility test revealed that 35, 27 and 8 isolates obtained from meat, chicken and cheese, respectively, were vancomycin - resistant . Conventional and molecular identification tests exhibited that all the isolates were e. faecium carrying vana . None of the isolates harbored vanb . Using the method of clsi, all the isolates were tested for their resistance against antimicrobial agents . All the vre isolates were also resistant to ciprofloxacin, erythromycin and ampicillin (100%). Almost the same antibiotic resistance was observed for gentamicin (95%), but only 5% of the isolates were resistant to chloramphenicol . The mic of the isolates for vancomycin and teicoplanin was 256 g / ml and for gentamicin - resistant isolates it was 1024 g / ml . The results of typing with phplate system showed a diversity of di = 0.78 for the e. faecium population . A total of 14 types with 10 common types (c - bpt) constituting 66 isolates and four single types (s - bpt) were seen (figure 1). Each common type comprised 2 - 31 strains . The vre from the meat samples showed 11 types comprising 35 isolates, while the chicken isolates contained only four types . Some of the isolates collected from different food samples on different sampling occasions were found to have the same c - bpt (table 1). Abbreviations: bpt, biochemical phenotype; ct: common type; and st: single type . According to the biochemical tests, a total of 102 isolates belonged to enterococci, 48, 40 and 14 of which were from meat, chicken and cheese, respectively . Antibiotic susceptibility test revealed that 35, 27 and 8 isolates obtained from meat, chicken and cheese, respectively, were vancomycin - resistant . Conventional and molecular identification tests exhibited that all the isolates were e. faecium carrying vana . Using the method of clsi, all the isolates were tested for their resistance against antimicrobial agents . All the vre isolates were also resistant to ciprofloxacin, erythromycin and ampicillin (100%). Almost the same antibiotic resistance was observed for gentamicin (95%), but only 5% of the isolates were resistant to chloramphenicol . The mic of the isolates for vancomycin and teicoplanin was 256 g / ml and for gentamicin - resistant isolates it was 1024 g / ml . The results of typing with phplate system showed a diversity of di = 0.78 for the e. faecium population . A total of 14 types with 10 common types (c - bpt) constituting 66 isolates and four single types (s - bpt) were seen (figure 1). The vre from the meat samples showed 11 types comprising 35 isolates, while the chicken isolates contained only four types . Some of the isolates collected from different food samples on different sampling occasions were found to have the same c - bpt (table 1). Abbreviations: bpt, biochemical phenotype; ct: common type; and st: single type . A high rate of vana containing enterococcus isolates was detected in food samples of chicken (9/10) and meat (10/10) in this study . Isolation of vana - containing enterococci strains in poultry and fresh slaughtered chicken samples has been reported in germany (14). Furthermore, high - level glycopeptide - resistant vana - type strains from supermarket - purchased chicken were detected in england (15). On the other hand, absence of vre from meats was reported in studies performed in the us (16, 17) which reflected the absence of vre isolation in animal food product . Elimination of vre, in part, may have been resulted during the food processing . On the contrary, in european countries, vre have been frequently isolated from meat products, which might be due to the usage of glycopeptides avoparcin antibiotic in food animal production environments before banning this antibiotic (18, 19). In this study, we obtained similar results to the european countries which may indicate the presence of similar diets in iran . In contrast, in a study by giraffa and sisto, there was no evidence of vre in dairy products (20). However, some years later, giraffa and colleagues in italy found that 50% of the cheeses examined were contaminated by vre (7). E. faecium was the only species isolated in all the samples in our study, while in most of the studies e. faecium and e. faecalis were simultaneously isolated from food samples . Klein et al . Reported a total of 34 vre strains isolated from raw minced beef and pork and 38% of vre isolates were identified as e. faecium, 35% were e. faecalis, and the remaining isolates were from the e. faecium group (21). Moreover, similar results were detected in the uk in fresh and frozen chicken, 58% and 40% of which were e. faecium and e. faecalis, respectively (15). Antibiotic susceptibility tests showed that in the present study, the vre isolates were resistant to at least four antibiotics including gentamicin, ciprofloxacin, erythromycin and ampicillin . This has been confirmed by other studies which have found the prevalence of antibiotic - resistant enterococci in farm animals and their meat to be higher than 60% (22, 23). These studies showed that extensive agricultural use of glycopeptides or other antibiotics has created animal reservoir of resistant enterococcal species to antibiotics which has complicated the control of infections caused by enterococci . Here we determined that resistance to gentamicin was very high among the animal products (95%). Using the synergism between aminoglycosides and b - lactams or glycopeptides eliminated owing to high - level aminoglycoside resistance, which is of vast clinical importance (24). Peters and colleagues have reported a very low gentamicin resistance in food from animal origin in germany (25). The difference in the reported antibiotic resistance among animal products could be due to geographical differences and the policy as well as production practices performed in these countries . All of our isolates were susceptible to tetracycline, in comparison with the results from peters and colleagues who found a high rate of tetracycline resistance in their strains (25). The reason for the absence of resistance to tetracycline in this study may be due to the fact that tetracycline is not used as a therapeutic antimicrobial in veterinary medicine in iran . In consistent with another study, here we reported that the prevalence of chloramphenicol resistance was very low among e. faecium strains (26).we found no resistant isolate to oxazolidinone, linezolid and q - d in this study (25). Although surveillance of enterococci from food sources for resistance to linezolid has not been reported extensively (26), there are some resistance reports to q - d in the usa, given the use of the analogue virginiamycin since 1974 (27, 28). In phplate analysis, we found that some of the c - bpt were found on different sampling occasions and food samples (i.e. C8, c9), indicating a high prevalence of certain e. faecium c - bpt in the food . In addition, in another study, typing with phplate exhibited a high diversity in a large number of enterococci isolated from different sources including food samples (29). On the other hand, genotyping with pulse - field gel electrophoresis profile (pfge) and random amplification of polymorphic dna (rapd) pcr revealed a marked heterogeneity in food isolates (30, 31). These results also showed that isolates with identical bpt pattern were found in different sampling occasions in the same food sample (i.e. Isolates in c10), suggesting that some of the strains persisted for a period of time . In conclusion, the high prevalence of multidrug resistance among enterococci isolated from food is a serious threat to public health . To the best of our knowledge, there is no use of antibiotic other than human use, in animal feeding in iran . The data presented here, on the other hand, suggested the presence of antibiotic pressure in food animals . The level of antibiotics in animal product, in turn, may be due to treatment regimens used for infections in animals.
The clinical effects of toxic alcohols are mainly due to depression of both the central nervous system and myocardial function.1 there are very few published reports of the magnetic resonance imaging (mri) features of central nervous system involvement in various types of toxic alcohol poisoning.15 all reports mention cerebral and/or cerebellar toxicity, also known as toxic encephalopathy . There have been no published reports of brain and spinal cord injury secondary to isopropanol (isopropyl alcohol) intoxication.15 a 60-year - old caucasian merchant ship captain presented to the emergency department of our hospital with headache, dizziness, and disorientation one day after ingestion of isopropanol along with ethanol (ethyl alcohol, drinking alcohol). He had celebrated his 60th birthday aboard ship by consuming ethanol and then isopropanol, because ethanol was not available later . His colleague accompanying him to the hospital brought an empty bottle of rubbing alcohol labelled as 70% volume per volume isopropyl alcohol with a capacity of 473 ml (one pint). The label on the bottle mentioned its indication for use as a first aid antiseptic and for rubbing and massaging . The patient s colleague mentioned that the patient had consumed most of the liquid in the bottle and that he (the colleague) had consumed a small amount . The patient s colleague did not give any recent or past history of substance abuse . An unenhanced computed tomography (ct) scan of the brain was performed immediately and was unremarkable . The patient collapsed in the hospital just 30 minutes after the ct scan was done . He became deeply comatose with a score of 3/15 on the glasgow coma scale (a scale to assess central nervous system status where a total score of 15 indicates best and normal outcome). He also developed hypotension, with a blood pressure of 80/40 mmhg, a pulse rate of 65 per minute, and a respiratory rate of 8 per minute . After administration of intravenous fluids and endotracheal intubation, the patient was admitted to the intensive care department . Blood investigations revealed leukocytosis of 26,000 (normal 4,00010,000) white blood cells per l, a hemoglobin of 15.7 (normal 1317) g / dl, and a mean corpuscular volume of 93 (normal 83101) fl . The initial arterial blood gas report indicated a ph of 6.731 (normal 7.357.45), a pco2 level of 28.2 (normal 3548) mmhg, a po2 level of 141 (normal 83108) mmhg, a bicarbonate level of 3.5 (normal 2128) mmol / l, and oxygen saturation of 95% (normal 95%98%). Other blood investigations revealed a blood urea nitrogen of 9.5 (normal 1.78.3) mmol / l, serum creatinine of 157 (normal 62124) mol / l, na of 141 (normal 134146) mmol / l, cl of 107 (normal 96110) mmol / l, hco3 of 5 (normal 2430) mmol / l, and capillary blood glucose of 7.2 (normal 3.35.5) mmol / l . His blood lactic acid level was significantly elevated at 8.7 (normal 0.52.2) mmol / ml . Blood levels of isopropanol and other toxic alcohols (like methanol, ethylene glycol, propylene glycol, and diethylene glycol) could not be obtained because the necessary investigations were not available . Investigations repeated at 2-hour intervals revealed increasing renal impairment, hyperglycemia, and electrolyte imbalance (low bicarbonate levels and hyperkalemia). No growth was detected on cultures of urine and blood, and no crystals were found in the urine on microscopic examination . The patient received hemodialysis because of severe acidosis and hyperkalemia, which led to gradual improvement in his blood ph and lactic acid levels . He was also treated with norepinephrine (as a vasopressor) and intravenous fluids, but there was no improvement in blood pressure . He received 500 ml of fractionated plasma protein stat over 30 minutes, 2,000 ml of normal saline over 2 hours, and was then kept on 200 ml / hour of normal saline . He did not respond to intravenous fluids and within the first 2 hours was started on norepinephrine 10 g per minute, which was gradually increased up to a maximum dose of 90 g per minute . Unenhanced mri scans of the brain and spine performed 6 days after hospital admission showed bilaterally symmetrical hyperintensities on t2-weighted, t1-weighted, t2 * -weighted, fluid attenuated inversion recovery (flair), and diffusion - weighted images in the cerebral and cerebellar cortex and white matter, basal ganglia, thalami, and brainstem (figures 14). A swollen and edematous cervical spinal cord was noted with t2-weighted and flair hyperintensities within it (figures 2 and 4). Cerebellar tonsillar herniation of 17 mm was noted to be compressing the proximal cervical cord (figures 2 and 4). Petechial hemorrhages were noted in the brainstem and the gangliocapsular regions bilaterally (figure 1). All the features described above are compatible with toxic brain and cervical spinal cord damage . Finally, the patient expired ten days after hospital admission, despite his improving blood picture . Chemicals involved in alcohol intoxication are ethanol, methanol, isopropanol, ethylene glycol, diethylene glycol, and propylene glycol . Ethanol is considered as a drinking alcohol and the rest as toxic alcohols . Among the toxic alcohols, isopropanol itself is more toxic than its metabolite (acetone), while the metabolites of the rest of the toxic alcohols are more toxic than the parent alcohol . Isopropanol poisoning is characterized by an increased osmolal gap in the setting of positive serum and urine ketones and does not cause metabolic acidosis, while the rest of the toxic alcohols cause mild to severe metabolic acidosis.1 when not mixed with ethanol or other intoxicants, the signs and symptoms of intoxication may start earlier (few hours) after isopropanol ingestion and may be delayed by up to a day or more after ingestion of other toxic alcohols . Unlike methanol and ethylene glycol, isopropanol is more toxic than its metabolites; hence, alcohol dehydrogenase inhibitors are not given.1 hemodialysis removes both isopropanol and its metabolite.1 in our case, the clinical and biochemical features were atypical for isopropanol toxicity likely due to coconsumption of ethanol and delayed patient presentation . Ingestion of approximately 200 ml of pure isopropanol can be lethal.1 the patient was managed symptomatically, mainly with hemodialysis . We wish to emphasize the atypical mri features in this case that have not been reported before in cases of toxic alcohol ingestion . In our case, these were likely due to reduced brain perfusion.6 these lesions were bilaterally symmetrical, and the distribution of the affected areas was highly indicative of a toxic injury.6 bilateral involvement of the basal ganglia and thalami has been reported in a few cases of ethylene glycol intoxication . However, cervical spinal cord involvement and cerebellar tonsillar herniation has not been reported before in cases of toxic alcohol ingestion and even in cases of other substance abuse . In humans, the opioid receptors are predominantly present in the cerebellum and the limbic systems, thus causing cerebellar - predominant toxicity in cases of opioid intoxication.6 involvement of the basal ganglia is also noticed in a few of these cases.6 a similar explanation may be given in cases of alcohol intoxication . To the best of our knowledge, this is the first reported case of cerebral, cerebellar, brainstem, and cervical spinal cord involvement on mri secondary to isopropanol intoxication . Here we present a very rare case of involvement of cerebrum, cerebellum, brainstem, and cervical spinal cord demonstrated on mri after coconsumption of isopropanol and ethanol . To our knowledge, involvement of the cervical spinal cord and cerebellar tonsillar herniation after toxic alcohol ingestion have not been reported before in the published literature.
Solitary true pancreatic cysts (stpcs), or epithelial cysts, are benign lesions that are extremely rare in adult patients . Advances in radiographic techniques have improved the ability to identify pancreatic cystic lesions . We report a case of a large and symptomatic stpc in a 47-year - old female patient who was treated successfully with spleen - preserving laparoscopic distal pancreatectomy . We also review the clinical and pathologic features of all reported stpcs within the past 25 years . To compose the review, we did a search of the international literature for stpcs that had occurred in adults . Clinical and pathologic information was collected for all of the reported pancreatic cysts, and a database was formed . The mean age of occurrence is 43.2 years, and the mean cyst size is 5.6 cm cystic lesions of the pancreas are relatively rare, although they are considered an increasingly important category with a challenging differential diagnosis . Cystic lesions of the pancreas are commonly classified as pseudocysts, parasitic cysts, neoplastic cysts, and various types of congenital or acquired developmental benign cystic lesions such as retention cysts, duplication cysts, epithelial or true cysts, and lymphoepithelial cysts . Based on the estimated relative frequency of pancreatic cystic lesions, pseudocysts are the most common . They are caused by alcoholic, biliary, or traumatic acute pancreatitis and are nonepithelialized and non - neoplastic cysts . Neoplastic cystic lesions are represented by cysts with true lining (mucinous and serous neoplasms) and cysts with degenerative / necrotic change in a neoplasm (solid pseudopapillary neoplasm, cystic ductal adenocarcinoma). Solitary true pancreatic cyst (stpc) is described as a simple thin - walled cyst with cuboidal lining and clear fluid content, whereas a lympoepithelial cyst is a uni- or multilocular cyst with well - differentiated squamous lining surrounded by a band of dense lymphoid tissue composed of mature t - lymphocytes with intervening germinal centers formed by b cells . Only a few cases of stpcs in adult patients are reported in the international literature . The aim of this study is to report a new case of stpc in an adult patient . We also review the literature on stpcs to determine the clinical features, management, pathological features, and prognosis of these lesions . A 47-year - old woman was referred to our department with a history of discomfort and pain in her upper abdomen . Upper abdominal ultrasonography (us) revealed the presence of a 5.0-cm cystic lesion located in the tail of the pancreas . Abdominal computed tomography (ct) and magnetic resonance imaging (mri) scans were subsequently performed and confirmed the presence of a low - attenuation and well - demarcated cystic area, 4.5 3.5 cm in diameter, located in the tail of the pancreas (figure 1). (b - d) mri images displaying a unilocular, homogeneous, low - attenuated and well - demarcated cystic mass . Although the imaging features of the lesion appeared to be benign, surgical excision was done to rule out the probability of malignancy . The cystic mass was excised en bloc with the tail of the pancreas, and the spleen was preserved (figure 2). Macroscopically viewed, the lesion was 6.6 cm in major diameter, with a fibrotic wall and an inner nodular appearance (figure 3). A sample was taken for cytological and biochemical examinations and the results showed normal levels of amylase, ca 199, and cea . Histopathologic studies showed that the cyst had a fibrotic wall and fibrosis in the inner surface foci of the cuboidal epithelium surface (figure 4a). Immunochemical staining results showed the epithelial surface of the cyst was positive for cea and cam 5.2 (figure 4b). (a) inner surface foci of the cuboidal epithelium (hematoxylin and eosin stain, original magnification 100). (b) positive stain for cam 5.2 (original magnification 400). Based on histopathologic and immunohistochemical findings, the diagnosis of an stpc was made . At the postoperative course, a small peripancreatic collection was detected 4 days after surgery and was conservatively treated . Postoperative imaging (us and ct) showed normal blood flow in the splenic vessels . Stpcs are typically diagnosed in childhood, predominantly in infants, and can be found in association with von hippel - lindau syndrome or polycystic disease of the kidneys . The occurrence of stpcs in adults is extremely rare . To date, only a few cases of stpcs in adults have been described . Table 1 shows in detail all of the reported stpcs within the past 25 years . Each article was carefully studied and a database was created that included the following stpc characteristics: gender, age, signs and symptoms, laboratory findings, diagnostic imaging modalities, localization, size, time of diagnosis, treatment, histopathology, fluid examination, and follow - up . It is believed that true cysts derive from abnormal segmentation of the primitive pancreatic ducts, with resultant sequestered endothelial cells . Well - documented solitary true pancreatic cysts f = female; m = male; n / r = not recorded; alp = alkaline phosphatase; us = ultrasound; ct = computed tomography; mri = magnetic resonance imaging; ercp= endoscopic retrograde cholangiopancreatography; dp = distal pancreatectomy; pd = pancreatoduodenectomy; spldp = spleen - preserving laparoscopic distal pancreatectomy; stpc = solitary true pancreatic cyst . Solitary true pancreatic cysts' features in the articles we studied, stpcs were found in 14 female and 4 male patients (f / m, 3.5:1). The mean diameter of the cysts was 5.6 cm, with range from 0.5 to 15.0 cm . Stpcs cause symptoms related to their size and location that include epigastric pain, nausea, vomiting, and biliary obstruction . Pain is described within the epigastrium and the back . A significant proportion (22.8%) of the stpcs were detected incidentally and diagnosed during imaging for an unrelated medical problem . The widespread use of abdominal imaging (us, ct, and mri) and the advances in quality of these technologies have increased the identification of asymptomatic pancreatic cysts . Typically, small (<2 cm), serous, cystic lesions of the pancreas are benign . Larger (> 2 cm), mucinous, multilocular cysts, or cysts with a solid component, carry the risk of malignancy . However, these diagnostic modalities usually fail to differentiate preoperatively among the histologic variants of pancreatic cystic lesions . With increasing use, endoscopic us plus fine - needle aspiration (eus fna) is currently becoming an indispensable tool in the diagnosis of cystic lesions of the pancreas . This combination provides more detailed anatomic information about the cyst than conventional us and allows the sampling of both cystic fluid and any solid component in smaller lesions . Nevertheless, in only one case of reported stpcs was an fna preoperatively performed, with uncertain results . The inability to accurately diagnose the pathologic lesions gives rise to a controversy in the management of these patients . Management of asymptomatic incidental pancreatic cysts of 2 cm includes a follow - up initially at 6 months and then yearly by performing an imaging test at intervals, whereas fluid analysis is unnecessary . Incidental cysts may enlarge over time, and an increase to 2.5 cm and/or a change in morphology is an indication for surgery . The size criterion for resecting an enlarging incidental cyst without a change in morphology, however, is uncertain because cysts of 3 to 4 cm usually have a low potential for malignancy . Surgical intervention is considered the appropriate treatment for pancreatic cysts when symptoms are presented or if the cystic mass enlarges and compresses adjacent organs . In these cases, the risk of malignancy is high and surgery is considered mandatory . Other factors that advocate for surgery are asymptomatic large lesions, a location in the tail of pancreas, and age (young patients 1735 years). Surgery includes cyst enucleation, cystoduodenostomy, cystogastrostomy, cystojejunostomy, and various types of pancreatectomies . Cyst enucleation is a safe and effective procedure for small lesions, especially if they are located in the pancreatic head . Benign and premalignant pancreatic body and tail cystic lesions can be treated with laparoscopic distal pancreatectomy (ldp). However, it is suggested the spleen be preserved if feasible, especially in young patients, because splenectomy can lead to life - threatening complications such as overwhelming postsplenectomy infection syndrome . There are two distinct approaches to perform a spleen - preserving laparoscopic distal pancreatectomy (spldp). The classic procedure is to identify, isolate, and preserve the splenic artery and vein . Alternatively, the splenic artery and vein are ligated with the pancreas, and perfusion of the spleen is maintained by the short gastric vessels . As with all pancreatic operations, wound infection, organ space infection, postpancreatectomy hemorrhage, and pancreatic fistula are all possible after ldp . He preoperative patency of the splenic vessels should be evaluated carefully, especially when spldp is attempted . Patients with poor vascular patency of the splenic vein had more postoperative pancreatic fistulas than did patients with normal vascular patency . Even after spldp, not all spleens can be salvaged because of postoperative hypoperfusion of the spleen, which may result in infarction and infection . After excision, the finding of a cuboidal epithelium layer is the key point in the diagnosis of an stpc . Stpcs usually contain clear fluid with normal amylase and lipase concentrations . In 83.3% of the reviewed cases, when it was performed, fluid analysis of stpcs showed normal amylase and lipase levels . Cyst fluid analysis may be helpful, but it is not always a sensitive and specific enough test . Immunostains included positive results for ca 199, cea, ca 125, and spain 1 . These are not specific, however, because they are also positive stain findings in neoplastic cysts, pseudocysts, and lymphoepithelial cysts . However, pseudocysts' lack of endothelial lining and lymphoepithelial cysts' lining is surrounded by a band of dense lymphoid tissue . To date, no case of malignancy has been reported in stpcs . Stpcs are extremely rare, benign pathologic entities, but the finding of a symptomatic or nonpancreatic cyst is still a diagnostic dilemma . Imaging, endoscopic techniques, and tumor marker assays may partially resolve the problem, but a final diagnosis is still made only by histopathologic examinations of the specimen after surgery.
Narcolepsy is an excessive sleepiness disorder which is characterized by excessive daytime sleepiness, sleep paralysis, cataplexy, nocturnal sleeping patterns, and hypnagogic hallucinations . Some of these symptoms, such as sleep paralysis and hypnagogic hallucinations, are thought to be the result of dissociated rem sleep . There is no cure for narcolepsy, but different medications can be used to offset symptoms experienced . Pharmacological treatment tries to stimulate the individual during the day and reduce sleep disturbances at night . Studies suggest that individuals with narcolepsy struggle with educational and occupational problems despite pharmacological treatment . It is believed that maintaining regular nocturnal sleep habits and attention to sleep hygiene can help to optimize daytime alertness . Massage therapy (mt) is a health care option that can aid in the prevention and treatment of pain and physical dysfunction through physical manipulation . Although massage therapy assessment and treatment often focus on physical outcomes, a well - recognized review of mt research suggests that massage therapy has a significant impact on psychological outcomes, such as reduction in anxiety and depression . No studies have specifically been conducted on the effect of massage therapy on individuals with narcolepsy . The effects of massage therapy on sleep have been studied in a number of different populations . One study investigated the effects of massage therapy and sleep in infants with low birth weight . This study showed that the group of infants with low birth rates that received the massage therapy intervention was more alert during the day . This study also showed that the massage therapy intervention facilitated positive sleep patterns in the infants, which included reducing breathing disorders such as sleep apnea and snoring, and increasing daytime alertness and improving the quality of sleep . A study investigating the effects of massage therapy on elders with sleep disorders in a nursing home showed that the intervention group which received the massage therapy treatments effectively produced a relaxation response in the elderly individuals as measured by their resting heart rate, systolic and diastolic blood pressure, and scores on the visual analogue scale (vas). In some of the cases, the intervention induced sleep, decreased sleep disruptions, and improved the overall sleep quality . This case report is the first to investigate the research question, does massage therapy have an effect on a patient with narcolepsy? It was hypothesized that massage therapy would improve the sleep patterns of a patient with narcolepsy . As the intervention would be conducted during the day, the treatment plan focused on providing a massage therapy intervention designed to be stimulating rather than sedative . Stimulating massage therapy techniques are thought to act as wake - promoting agents or stimulants to the body s sensory neurons . It was anticipated that this would promote proper sleep patterns by helping the participant stay alert during the day and possibly reduce sleep disturbances at night . This study is a prospective single - case design in which massage therapy was provided to a patient with narcolepsy . The participant was a 23-year - old teacher who was diagnosed with narcolepsy with cataplexy five years ago . Her symptoms include excessive daytime sleepiness, periodic leg movements (plm), and cataplexy . She experiences fatigue throughout the day which makes it more difficult to fall asleep at night . When she wakes up in the morning to go to work, she feels as though she has not had a restful sleep and continues to be tired throughout the day . When she comes home from work in the evenings, she feels exhausted and takes a nap, which sometimes turns into sleep for over 23 hours . She tries to take shorter, more frequent naps when possible, so that she does not fall asleep for long periods of time, but finds it difficult to do once she falls asleep . The intervention in this study was massage therapy and was administered by a student massage therapist who provided massage techniques within the scope of practice in ontario . At this point in her program, the student had completed about 1700 hours of her 2430-hour program . The massage therapy intervention was designed to positively impact the patient s sleep patterns by stimulating daytime wakefulness . The treatment primarily included rapid tapotement and petrissage applied to the anterior and posterior surfaces of legs, the back, and both arms . The protocol was pragmatic, meaning that the therapist could choose the exact techniques, the length of time each was administered, and the order . Each treatment was once a week for forty - five minutes, for five weeks . In the sixth week of the study, the questionnaire was completed with no intervention . The leeds sleep evaluation questionnaire (lseq) this tool uses ten visual analogue scales, each 100 mm, in four areas . These areas include the concerning ease of getting to sleep (gts), the perceived quality of sleep (qos), the ease of awakening from sleep (afs), and the integrity of early - morning behaviour following wakefulness (bfw). The questionnaire was completed in the week prior to the start of the massage therapy intervention and prior to the intervention in each of the following weeks . Each visual analog scale (vas) was measured and the resulting score recorded for analysis . The data were organized by grouping the visual analog scales by section (ease of getting to sleep, the perceived quality of sleep, the ease of awakening from sleep, and the integrity of early - morning behaviour following wakefulness). As such, ease of getting to sleep included the results of three vass, perceived quality of sleep included two vass, ease of awakening from sleep included two vass, and the integrity of early - morning behaviour following wakefulness included the results of three vass . The data were analyzed by calculating the percent change using the results of the final data collection point, subtracting the results from the first data collection point, and dividing it by the results of the first data collection point and multiplying by 100 ([data 6 - data 1]/data 1 100). The total change, found by combining the results of all of the visual analog scales into a composite score and using the same formula as above, was also calculated . The participant was a 23-year - old teacher who was diagnosed with narcolepsy with cataplexy five years ago . Her symptoms include excessive daytime sleepiness, periodic leg movements (plm), and cataplexy . She experiences fatigue throughout the day which makes it more difficult to fall asleep at night . When she wakes up in the morning to go to work, she feels as though she has not had a restful sleep and continues to be tired throughout the day . When she comes home from work in the evenings, she feels exhausted and takes a nap, which sometimes turns into sleep for over 23 hours . She tries to take shorter, more frequent naps when possible, so that she does not fall asleep for long periods of time, but finds it difficult to do once she falls asleep . The intervention in this study was massage therapy and was administered by a student massage therapist who provided massage techniques within the scope of practice in ontario . At this point in her program, the student had completed about 1700 hours of her 2430-hour program . The massage therapy intervention was designed to positively impact the patient s sleep patterns by stimulating daytime wakefulness . The treatment primarily included rapid tapotement and petrissage applied to the anterior and posterior surfaces of legs, the back, and both arms . The protocol was pragmatic, meaning that the therapist could choose the exact techniques, the length of time each was administered, and the order . Each treatment was once a week for forty - five minutes, for five weeks . In the sixth week of the study, this tool uses ten visual analogue scales, each 100 mm, in four areas . These areas include the concerning ease of getting to sleep (gts), the perceived quality of sleep (qos), the ease of awakening from sleep (afs), and the integrity of early - morning behaviour following wakefulness (bfw). The questionnaire was completed in the week prior to the start of the massage therapy intervention and prior to the intervention in each of the following weeks . Each visual analog scale (vas) was measured and the resulting score recorded for analysis . The data were organized by grouping the visual analog scales by section (ease of getting to sleep, the perceived quality of sleep, the ease of awakening from sleep, and the integrity of early - morning behaviour following wakefulness). As such, ease of getting to sleep included the results of three vass, perceived quality of sleep included two vass, ease of awakening from sleep included two vass, and the integrity of early - morning behaviour following wakefulness included the results of three vass . The data were analyzed by calculating the percent change using the results of the final data collection point, subtracting the results from the first data collection point, and dividing it by the results of the first data collection point and multiplying by 100 ([data 6 - data 1]/data 1 100). The total change, found by combining the results of all of the visual analog scales into a composite score and using the same formula as above, was also calculated . Overall, there was a 123% total change on the leeds sleep evaluation questionnaire between the initial and final use of the tool . Higher numbers indicate improvement on each of the visual analog scales, so the change is an improvement in symptoms (figure 1). Global leeds sleep evaluation questionnaire scores . In the first area, getting to sleep, a 148% improvement was seen following the six - week period (figure 2). For the questions about quality of sleep (qos), there was 1100% improvement from the beginning to end of the study (figure 3). The questions related to awake following sleep (afs) showed a 121% change (figure 4). Finally, behavior following wakening (bfw) improved by 28% (figure 5). The results of this study suggest that massage therapy had a positive effect on the sleep patterns of this participant with narcolepsy . In particular, substantial improvement was experienced by the participant in the area of quality of sleep . From figure 3, one can see that the data points suggest a steady improvement in quality of sleep over the study with the results tapering off in the last week of the study . Future studies might consider a longer time period to see if the impact of weekly treatments on quality of sleep plateaus following four treatments . The characteristic of getting to sleep also showed a fairly steady improvement over the study, although the percent change from beginning to end of the study was not as large . Awake following sleep shows an erratic pattern, and it is unclear whether the percent change seen was as a result of the massage therapy intervention or other factors . Behaviour following wakening did not show substantial improvement, and this would be interesting to explore further . It is worth considering whether this study was sufficiently long to capture any effect massage therapy might have on this characteristic . The results of this study suggest that these massage therapy treatments improved the participant s sleep patterns, in particular by helping her get to sleep and improving her quality of sleep . A study regarding the effects of massage therapy and sleep found similar results, whereby massage therapy improved sleep by reducing sleep latency due to less disturbed sleep . While massage therapy seemed to benefit the participant in this study, these results may not generalize to all patients with narcolepsy . In addition, this study cannot suggest a cause and effect relationship between massage therapy and narcolepsy; a more rigorous study is needed . It is recommended that future research focus on the population of individuals with narcolepsy and sleep behaviours in various populations . Based on the experience of this case study, the authors suggest the following considerations: timing of the intervention: does the timing of the intervention of massage therapy have an effect on the outcome? Timing the stimulating massage therapy treatment with times of fatigue may increase the effectiveness . Similarly, soothing massage therapy treatment closer to bedtime may also alter the impact of massage therapy on sleep behaviours . Dosage of the intervention: it is important to better understand the amount of massage therapy that is needed to create change in sleep patterns and to maintain that change . Using different lengths of treatment plans will help to better understand the appropriate dosage of massage therapy . Measurement tools: while the leeds sleep evaluation tool was easy to administer and analyze, future studies may choose to use additional or alternate measurement tools . Due to the importance of rem sleep patterns to individuals with narcolepsy, it would be interesting to investigate whether or not massage therapy treatments have an impact by altering the rem patterns during sleep . This single - case report was conducted to answer the question, does massage therapy have an effect on a patient with narcolepsy? Results of this study suggest that massage therapy can improve sleep patterns in a patient with narcolepsy . In particular, quality of sleep and the ability to get to sleep showed the most promising impact . Further clinical research on massage therapy and narcolepsy is needed to better understand the preliminary results of this case.
Recent explosive advances in high - throughput sequencing technologies have led to the identification of numerous somatic mutations in cancer genomes.16 however, only a few of them functionally contribute to tumorigenesis . Driver mutations that have a casual role in tumorigenesis from passenger mutations, which have no effect on tumorigenesis, is critical for our understanding of tumorigenesis . Yet, if the driver mutations are not required for tumor maintenance, those gene products cannot serve as anticancer drug targets.7,8 techniques used to assess whether the identified gene is associated with tumor maintenance in in vitro culture systems, include sirna - mediated gene expression knockdown or small chemical compound - mediated gene product activity inhibition.9 if these techniques abrogate gene product role in tumor cell growth, the gene may be required for tumor cell growth and therefore serves as a possible drug target for the inhibition of tumor growth.1013 several in vivo mouse systems have been used to verify these in vitro results . In particular, xenograft implantation systems have been extensively used to confirm in vitro data and to test the efficacies of small compounds for inhibiting tumor growth in mice.1416 in these systems, either tumor cells or tumor tissues are implanted into the immunocompromised mice to reproduce tumor growth . These xenograft implantation systems have several advantages over genetically - engineered mouse (gem) models, such as being easy to prepare and use in a large cohort of mice with synchronized tumor growth.14 however, tumor growth in xenograft implantation systems frequently fail to faithfully recapitulate the genetics and histology of corresponding human cancers, partially due to the lack of microenvironmental factors, including stromal cell components and an immune system.14 in this regard, conventional transgenic mouse models in which the transgene is expressed under the control of a tissue - specific promoter / enhancer regulatory elements are a more physiologically relevant system for determining whether the transgene expression is sufficient for tumor development and progression . Unfortunately, there is no means to regulate (either induce or suppress) transgene expression in a temporal manner, making it impossible to determine whether the transgene expression is required for the maintenance of tumor phenotypes . To overcome this limitation, the inducible transgenic mouse model was developed . In this system, if turning off the expression of the specific gene in tumor - bearing transgenic mice shows that transgene expression is required for tumor maintenance, the suppression of transgene expression will likely lead to tumor regression, validating the transgene protein as a target for future anticancer drug development . Given that tumor recurrence is always a concern in cancer therapy, understanding the molecular mechanisms underlying therapeutic resistance of tumor cells is critical for developing new therapeutic strategies against recurrent tumors . The majority of fully regressed tumors after turning off the expression of transgene by withdrawing doxycycline from the drinking water will reoccur in the original tumor site as transgene expression - independent.7,8,17 by analyzing these recurrent tumors, we can understand the molecular basis of how tumor cells escape from their dependence on doxycycline - induced transgene expression.1719 therefore, inducible mouse models enable us to extrapolate the collected in vivo data to predict therapeutic responses in clinical settings and develop new therapeutic strategies against recurrent tumors . In this review, we will highlight the importance of inducible mouse models in studying target validation and tumor recurrence by citing several previously developed inducible mouse models as examples . In the tet - on system, the expression of the transgene is turned on (induced) by administering doxycycline via drinking water and turned off (suppressed) by withdrawing doxycycline from the drinking water.20,21 two gem models are required for this system . The first model harbors a gene of interest fused to a modified tetracycline response promoter element (tre) that contains seven repeats of a 19-nucleotide tetracycline operator (teto) sequence.20,21 the second model expresses an artificial transcription factor of tetracycline - inducible transactivator (rtta) under the control of a tissue - specific promoter . To express the gene of interest in a specific tissue, these two mouse lines are crossed to generate compound mice harboring transgenes of both the gene of interest and rtta . In the presence of doxycycline (adding doxycycline into the drinking water), doxycycline binding to rtta enables the doxycycline - bound rtta to stably bind to the tre element, leading to the expression of the gene of interest in a specific tissue.20,21 in the absence of doxycycline (withdrawing doxycycline from the drinking water), doxycycline - free rtta cannot bind to the tre element, resulting in the suppression of the gene of interest . Mutations in exons 1821 of the human egfr (hegfr) gene encoding the atp - binding pocket of the receptor s tyrosine kinase domain are found in approximately 10% and 35% of patients with non - small cell lung cancer (nsclc) in the us and in east asia, respectively.2225 in particular, an l858r substitution in exon 21 (hegfr l858r) and an in - frame deletion in exon 19 (hegfr del) are the two most common mutations . However, the identification of these recurrent mutations at high frequencies in clinical specimens does not necessarily mean that these mutations functionally contribute to the initiation and progression of nsclc . Most importantly, for these mutations to serve as future therapeutic targets, these mutations should be required for the maintenance of nsclc . Therefore, to determine whether these two mutations are associated with initiation, progression, and tumor maintenance of nsclc, dr . Wong s group developed inducible transgenic mouse models.26 firstly, the group generated a transgenic mouse model in which either hegfr l858r or hegfr del was fused tre to generate two gem mouse models: tre - hegfr l858r and tre - hegfr del . Secondly, these mice were crossed with clara cell secretory protein promoter element - rtta (ccsp - rtta) mice to generate compound mice expressing either hegfr l858r or hegfr del in lung type 2 alveolar cells in a doxycycline - inducible manner 26: tre - hegfr l858r / ccsp - rtta and tre - hegfr del / ccsp - rtta . Administration of doxycycline resulted in tumor development in murine lungs, serially precancerous lesions and bronchioloalveolar carcinoma within a few weeks, and invasive adenocarcinoma with more than four weeks after induction.26 in addition, withdrawal of doxycycline to suppress the expression of hegfr l858r or hegfr del in tumor - bearing compound mice resulted in complete tumor regression without recurrences.26 these results suggest that lung tumor cells are dependent on the expression of their respective transgenes for their proliferation and survival, validating these hegfr mutant proteins as prospective drug targets for the treatment of lung cancers harboring these specific egfr mutations . These findings were immediately applied in preclinical trials of pharmaceutical inhibitors specific to the egfr mutants, such as gefitinib and erlotinib, to test their therapeutic efficacy against these inducible hegfr mutant - driven mouse lung tumors.26 these tumor cells showed dramatic responses to the egfr tyrosine kinase inhibitors, verifying that the egfr mutants serve as drug targets for the treatment of human lung cancers harboring these specific egfr mutations . In addition, these inducible hegfr mutant mouse models were validated as valuable tools for efficacy studies of newly developed egfr tyrosine kinase inhibitors . However, in clinical settings, the majority of primary egfr mutant non - small cell lung carcinomas that initially responded to egfr tyrosine kinase inhibitors became resistant to the inhibitors . 27,28 several genomic studies of recurrent patient tumor samples identified other genetic alterations, including the secondary mutation of egfr t790 m in the egfr gene and mutations in the k - ras gene.23,29,30 using another inducible mouse model that expresses a mutant egfr (egfr tl) containing both hegfr l858r from the primary tumor and egfr t790 m from the recurrent tumor in murine lungs in the same manner above, it was shown that egfr tl is oncogenic and essential for tumor maintenance.19 given that an irreversible egfr tyrosine kinase inhibitor, hki-272, previously showed high efficacy against gefitinib - insensitive egfr mutants, a therapeutic strategy combining hki-272 with gefitinib was proposed to treat primary tumors driven by egfr l858r and to prevent recurrent tumors driven by egfr t790 m . Preclinical trials of hki-272 alone in egfr tl - driven murine lung tumors showed suboptimal responses, consistent with unfavorable results from a phase i clinical trial of hki-272 for the treatment of previously treated nsclc patients.19 these studies suggest that results obtained from an inducible mouse model mirror clinical outcomes, validating inducible mouse models as promising tools for predicting clinical responses to cancer therapy . Her / neu protein overexpression and/or her / neu gene amplification were observed in approximately 20% to 30% primary human breast cancers, and were also associated with breast cancer progression and poor prognoses.31,32 to determine whether her / neu protein overexpression is associated with initiation, progression, and tumor maintenance of breast cancer, dr . Chodosh s group developed inducible transgenic mouse models.33 firstly, the group generated a transgenic mouse model (teto - neunt) in which a constitutively activate form of her2/neu with a substitution of valine for glutamic acid in the transmembrane domain (neunt) was fused to the minimal tet operator (teto). Secondly, these mice were crossed with mouse mammary tumor virus promoter - reverse tetracycline transactivator (mmtv - rtta) mice to express transgenes in the breast epithelia of the mammary ductal system . These mmtv - rtta / teto - neunt compound mice developed multiple invasive mammary carcinoma with pulmonary metastasis in the majority of tumor - bearing mice with doxycycline administration.33 after withdrawal of doxycycline, the tumors including pulmonary metastatic tumors, rapidly and fully regressed, suggesting that the activation of the her2/neu signaling pathway is required for tumor maintenance of both primary and metastatic tumors.18,33 since tumor recurrence is concern of breast cancer progression, they investigated the molecular mechanisms underlying recurrent tumors derived from fully regressed tumors after turning off the transgene expression in this mouse model.18 this study demonstrated that snail was upregulated in recurrent mammary tumors, and snail overexpression is sufficient for inducing rapid tumor recurrence after suppressing the expression of the transgene neunt in a xenograft model using tumor cell lines derived from the primary mouse tumors.18 therefore, snail may serve as a target for the treatment of recurrent breast cancer resulting from therapies against the her2/neu signaling pathway . C - myc gene amplification is also detected in up to 15% of human breast cancers, with a much higher frequency of approximately 50% in brca1-dysfunctional breast cancer.34 to investigate the functional contribution of c - myc overexpression in breast cancer development and progression, an inducible transgenic mouse model overexpressing c - myc (mmtv - rtta / teto - c - myc) was also generated by dr . Group.8 while c - myc overexpression is sufficient for the development of mammary adenocarcinoma, approximately half of the primary tumors failed to regress after turning off the expression of the c - myc transgene . In addition, half of the fully regressed tumors recurred at the site of original primary tumor.8 therefore, the majority of the initially c - myc - driven tumors eventually lost their dependence on the c - myc signaling pathway, suggesting that c - myc may not be appropriate as a therapeutic target for the treatment of human breast cancers harboring c - myc gene amplification . Many studies have reported the activation of the ras / raf / mek / erk pathway in the majority of androgen - depletion independent (adi) prostate cancers.3537 however, counter - intuitively, activating mutations in the ras / raf / mek / erk pathway are infrequent in human prostate cancers.7,9 to study the role of the activation of the ras / raf / mek / erk pathway in prostate cancer initiation and progression in vivo, dr . Chin s group generated an inducible mouse model (tyr - rtta / teto - braf), in which the expression of a potent activator of ras / raf / mek / erk signaling, braf, is targeted to the murine prostate epithelia by tyrosinase promoter / enhancer (tyr).7 although the tyrosinase promoter / enhancer has specific activity in melanocytes, it also showed activity in the prostate epithelia for unknown reasons.7 these mice developed invasive adenocarcinoma, and these tumors further progressed to indolent adi lesions after castration.7 however, as in the case of the inducible c - myc transgenic mouse model, transgene braf - driven prostate tumors constantly grew even after turning off the transgene expression.7 therefore, this study proposed that targeting braf for the treatment of adi prostate cancer does not have positive therapeutic potential . More than 95% of pancreatic ductal adenocarcinoma (pdac) harbor mutations in the kras gene.3840 the expression of kras in murine pancreatic progenitor cells at a physiologically relevant level induced the full spectrum of pancreatic cancers from pancreatic intraepithelial neoplasias to invasive pancreatic cancer.41,42 these results suggest that the mutant kras is an oncogenic driver for the development of pancreatic cancer . To determine whether the mutant kras is required for tumor maintenance, dr . Depinho s group generated an inducible mouse model wherein the mutant kras is expressed in murine pancreatic cells at a physiological level in a triple transgenic strain (teto - lox - stop - lox - kras / rosa26-lox - stop - lox - rtta - ires - gfp / p48-cre) in a doxycyline - inducible manner . In this mouse model, cre expression in murine pancreatic cells eliminates the lox - stop - lox cassette containing three repetitive transcriptional stop sequences from both tetolox - stop - lox - kras and rosa26-lox - stop - lox - rtta - ires - gfp to express rtta and gfp using the endogenous rosa26 promoter and to generate teto - kras.41 in the presence of doxycycline, the mutant kras is expressed in pancreatic cells of these triple transgenic mice . Leveraging this elaborate mouse model, they proved that mutant kras is required for the maintenance of mutant kras - driven pancreatic tumors.41 to understand the molecular mechanisms of kras - mediated pancreatic tumor maintenance in this mouse model, they analyzed changes in the transcriptome of mutant kras - driven tumor samples at 24 hours after doxycyline withdrawal . They demonstrated that multiple metabolic pathways are down - regulated in mutant kras - driven tumors upon the termination of transgene expression, suggesting that the mutant kras reprograms metabolism to enhance tumor growth.43 therefore, this study proposes the possibility that mutant kras itself and associated metabolic pathways serve as drug targets for the treatment of mutant kras - driven pancreatic cancers . Mutations in exons 1821 of the human egfr (hegfr) gene encoding the atp - binding pocket of the receptor s tyrosine kinase domain are found in approximately 10% and 35% of patients with non - small cell lung cancer (nsclc) in the us and in east asia, respectively.2225 in particular, an l858r substitution in exon 21 (hegfr l858r) and an in - frame deletion in exon 19 (hegfr del) are the two most common mutations . However, the identification of these recurrent mutations at high frequencies in clinical specimens does not necessarily mean that these mutations functionally contribute to the initiation and progression of nsclc . Most importantly, for these mutations to serve as future therapeutic targets, these mutations should be required for the maintenance of nsclc . Therefore, to determine whether these two mutations are associated with initiation, progression, and tumor maintenance of nsclc, dr . Wong s group developed inducible transgenic mouse models.26 firstly, the group generated a transgenic mouse model in which either hegfr l858r or hegfr del was fused tre to generate two gem mouse models: tre - hegfr l858r and tre - hegfr del . Secondly, these mice were crossed with clara cell secretory protein promoter element - rtta (ccsp - rtta) mice to generate compound mice expressing either hegfr l858r or hegfr del in lung type 2 alveolar cells in a doxycycline - inducible manner 26: tre - hegfr l858r / ccsp - rtta and tre - hegfr del / ccsp - rtta . Administration of doxycycline resulted in tumor development in murine lungs, serially precancerous lesions and bronchioloalveolar carcinoma within a few weeks, and invasive adenocarcinoma with more than four weeks after induction.26 in addition, withdrawal of doxycycline to suppress the expression of hegfr l858r or hegfr del in tumor - bearing compound mice resulted in complete tumor regression without recurrences.26 these results suggest that lung tumor cells are dependent on the expression of their respective transgenes for their proliferation and survival, validating these hegfr mutant proteins as prospective drug targets for the treatment of lung cancers harboring these specific egfr mutations . These findings were immediately applied in preclinical trials of pharmaceutical inhibitors specific to the egfr mutants, such as gefitinib and erlotinib, to test their therapeutic efficacy against these inducible hegfr mutant - driven mouse lung tumors.26 these tumor cells showed dramatic responses to the egfr tyrosine kinase inhibitors, verifying that the egfr mutants serve as drug targets for the treatment of human lung cancers harboring these specific egfr mutations . In addition, these inducible hegfr mutant mouse models were validated as valuable tools for efficacy studies of newly developed egfr tyrosine kinase inhibitors . However, in clinical settings, the majority of primary egfr mutant non - small cell lung carcinomas that initially responded to egfr tyrosine kinase inhibitors became resistant to the inhibitors . 27,28 several genomic studies of recurrent patient tumor samples identified other genetic alterations, including the secondary mutation of egfr t790 m in the egfr gene and mutations in the k - ras gene.23,29,30 using another inducible mouse model that expresses a mutant egfr (egfr tl) containing both hegfr l858r from the primary tumor and egfr t790 m from the recurrent tumor in murine lungs in the same manner above, it was shown that egfr tl is oncogenic and essential for tumor maintenance.19 given that an irreversible egfr tyrosine kinase inhibitor, hki-272, previously showed high efficacy against gefitinib - insensitive egfr mutants, a therapeutic strategy combining hki-272 with gefitinib was proposed to treat primary tumors driven by egfr l858r and to prevent recurrent tumors driven by egfr t790 m . Preclinical trials of hki-272 alone in egfr tl - driven murine lung tumors showed suboptimal responses, consistent with unfavorable results from a phase i clinical trial of hki-272 for the treatment of previously treated nsclc patients.19 these studies suggest that results obtained from an inducible mouse model mirror clinical outcomes, validating inducible mouse models as promising tools for predicting clinical responses to cancer therapy . Her / neu protein overexpression and/or her / neu gene amplification were observed in approximately 20% to 30% primary human breast cancers, and were also associated with breast cancer progression and poor prognoses.31,32 to determine whether her / neu protein overexpression is associated with initiation, progression, and tumor maintenance of breast cancer, dr . Chodosh s group developed inducible transgenic mouse models.33 firstly, the group generated a transgenic mouse model (teto - neunt) in which a constitutively activate form of her2/neu with a substitution of valine for glutamic acid in the transmembrane domain (neunt) was fused to the minimal tet operator (teto). Secondly, these mice were crossed with mouse mammary tumor virus promoter - reverse tetracycline transactivator (mmtv - rtta) mice to express transgenes in the breast epithelia of the mammary ductal system . These mmtv - rtta / teto - neunt compound mice developed multiple invasive mammary carcinoma with pulmonary metastasis in the majority of tumor - bearing mice with doxycycline administration.33 after withdrawal of doxycycline, the tumors including pulmonary metastatic tumors, rapidly and fully regressed, suggesting that the activation of the her2/neu signaling pathway is required for tumor maintenance of both primary and metastatic tumors.18,33 since tumor recurrence is concern of breast cancer progression, they investigated the molecular mechanisms underlying recurrent tumors derived from fully regressed tumors after turning off the transgene expression in this mouse model.18 this study demonstrated that snail was upregulated in recurrent mammary tumors, and snail overexpression is sufficient for inducing rapid tumor recurrence after suppressing the expression of the transgene neunt in a xenograft model using tumor cell lines derived from the primary mouse tumors.18 therefore, snail may serve as a target for the treatment of recurrent breast cancer resulting from therapies against the her2/neu signaling pathway . C - myc gene amplification is also detected in up to 15% of human breast cancers, with a much higher frequency of approximately 50% in brca1-dysfunctional breast cancer.34 to investigate the functional contribution of c - myc overexpression in breast cancer development and progression, an inducible transgenic mouse model overexpressing c - myc (mmtv - rtta / teto - c - myc) was also generated by dr . Chodosh s group.8 while c - myc overexpression is sufficient for the development of mammary adenocarcinoma, approximately half of the primary tumors failed to regress after turning off the expression of the c - myc transgene . In addition, half of the fully regressed tumors recurred at the site of original primary tumor.8 therefore, the majority of the initially c - myc - driven tumors eventually lost their dependence on the c - myc signaling pathway, suggesting that c - myc may not be appropriate as a therapeutic target for the treatment of human breast cancers harboring c - myc gene amplification . Many studies have reported the activation of the ras / raf / mek / erk pathway in the majority of androgen - depletion independent (adi) prostate cancers.3537 however, counter - intuitively, activating mutations in the ras / raf / mek / erk pathway are infrequent in human prostate cancers.7,9 to study the role of the activation of the ras / raf / mek / erk pathway in prostate cancer initiation and progression in vivo, dr . Chin s group generated an inducible mouse model (tyr - rtta / teto - braf), in which the expression of a potent activator of ras / raf / mek / erk signaling, braf, is targeted to the murine prostate epithelia by tyrosinase promoter / enhancer (tyr).7 although the tyrosinase promoter / enhancer has specific activity in melanocytes, it also showed activity in the prostate epithelia for unknown reasons.7 these mice developed invasive adenocarcinoma, and these tumors further progressed to indolent adi lesions after castration.7 however, as in the case of the inducible c - myc transgenic mouse model, transgene braf - driven prostate tumors constantly grew even after turning off the transgene expression.7 therefore, this study proposed that targeting braf for the treatment of adi prostate cancer does not have positive therapeutic potential . More than 95% of pancreatic ductal adenocarcinoma (pdac) harbor mutations in the kras gene.3840 the expression of kras in murine pancreatic progenitor cells at a physiologically relevant level induced the full spectrum of pancreatic cancers from pancreatic intraepithelial neoplasias to invasive pancreatic cancer.41,42 these results suggest that the mutant kras is an oncogenic driver for the development of pancreatic cancer . To determine whether the mutant kras is required for tumor maintenance, dr . Depinho s group generated an inducible mouse model wherein the mutant kras is expressed in murine pancreatic cells at a physiological level in a triple transgenic strain (teto - lox - stop - lox - kras / rosa26-lox - stop - lox - rtta - ires - gfp / p48-cre) in a doxycyline - inducible manner . In this mouse model, cre expression in murine pancreatic cells eliminates the lox - stop - lox cassette containing three repetitive transcriptional stop sequences from both tetolox - stop - lox - kras and rosa26-lox - stop - lox - rtta - ires - gfp to express rtta and gfp using the endogenous rosa26 promoter and to generate teto - kras.41 in the presence of doxycycline, the mutant kras is expressed in pancreatic cells of these triple transgenic mice . Leveraging this elaborate mouse model, they proved that mutant kras is required for the maintenance of mutant kras - driven pancreatic tumors.41 to understand the molecular mechanisms of kras - mediated pancreatic tumor maintenance in this mouse model, they analyzed changes in the transcriptome of mutant kras - driven tumor samples at 24 hours after doxycyline withdrawal . They demonstrated that multiple metabolic pathways are down - regulated in mutant kras - driven tumors upon the termination of transgene expression, suggesting that the mutant kras reprograms metabolism to enhance tumor growth.43 therefore, this study proposes the possibility that mutant kras itself and associated metabolic pathways serve as drug targets for the treatment of mutant kras - driven pancreatic cancers . This short review article provides an overview of the significance of doxycycline - inducible transgenic mouse models in studying target validation and tumor recurrence by summarizing studies using inducible mouse models for lung cancer (tre - hegfr l858r / ccsp - rtta, tre - hegfr del / ccsp - rtta, and tre - egfr tl / ccsp - rtta), inducible mouse models for breast cancer (mmtv - rtta / teto - neunt and mmtv - rtta / teto - c - myc), an inducible mouse model for adi prostate cancer (tyr - rtta / teto - braf), and an inducible mouse model for pdac (teto - lox - stop - lox - kras / rosa26-lox - stop - lox - rtta - ires - g fp / p48-cre). Despite investing much effort and time, many researchers have failed to identify driver genes that functionally contribute to tumor development . When a driver gene is successfully identified, the driver gene may later be found not to be required for tumor maintenance . Thus, this driver gene product is not useful as a future drug target for the treatment of related cancers . These failures can be partially attributed to the limitations of in vitro and in vivo systems in studying target validation and tumor recurrence.16 for example, promising results regarding target validation through loss - of function study using either sirna - mediated knockdown or small chemical inhibitor treatment with tumor cell lines are not frequently reproduced in in vivo systems . As mentioned above, conventional transgenic mouse models are limited for the study of target validation and tumor recurrence due to the inability to regulate transgene expression in a temporal manner . These limitations are overcome with an inducible transgenic mouse model . Despite the significance of inducible mouse models in studying target validation and tumor recurrence, many were wary about the extensive use of doxycycline due to its detrimental effects on mitochondria . In particular, doxycycline impairs mitochondrial proteostasis and induces some physiological changes associated with energy metabolism in mice.44 in addition, doxycycline is a tetracycline antibiotic, so it can change the gut microbiome, ultimately affecting mice immune system and metabolism.4547 these unwanted consequences might affect the tumorigenic potential of certain genes in this doxycycline - inducible mouse model . Therefore, the effects of doxycycline should be well controlled to study the tumorigenic potential of genes associated with immune responses and metabolic pathways in particular . The ultimate goal of cancer research is to develop therapeutic strategies against primary tumors and recurrent tumors through the study of target validation and tumor recurrence.
Laribacter hongkongensis is a facultative anaerobic, motile, non - sporulating gram - negative bacillus . It belongs to the family neisseriaceae of the subclass of proteobacteria (1). Since the first description of l. hongkongensis from the blood and empyema pus of a patient with alcoholic liver cirrhosis in 2001, the bacterium has subsequently been associated with community - acquired gastroenteritis (1, 2) however, accurate identification of l. hongkongensis is impossible using commercially - available phenotypic identification systems . Thus, the prevalence, pathogenic potential, and epidemiology of l. hongkongensis are unclear . The identification based on a molecular approach could permit more accurate determinations of incidence and evaluation of clinical relevance . Although l. hongkongensis has been reported in hong kong, china, and hungary (3), it has not hitherto been identified in korea . A 24-yr - old male was admitted to a hospital, on august 16, 2007, with a 15-day history of abdominal distension . The patient had been diagnosed with wilson's disease at 17-yr - of - age and had since undergone treatment for liver cirrhosis complicated by wilson's disease . The patient had no history of overseas travel and had no recall of ingesting undercooked freshwater fish for at least the previous year . On physical examination, blood pressure was 98/49 mmhg, pulse rate was 96 beats per min, and body temperature (36.5). Laboratory tests revealed a hemoglobin concentration of 8.3 g / dl, leukocyte count of 6,248/l, platelet count of 42,000/l, serum creatinine level of 0.94 mg / dl, serum bilirubin level of 6.7 mg / dl, and serum albumin level of 1.6 g / dl . The analysis of peritoneal fluid demonstrated an albumin level of 202 mg / dl and leukocyte count of 130/l (poly 15%). But, on day 6 following admission, fever developed, with a body temperature reaching 38.6. examination showed diffuse tenderness of the entire abdomen . The results of peritoneal fluid evaluation revealed a leukocyte count of 1,180/l (poly 74%). Empirical treatment with cefotaxime (2 g every 8 hr) was commenced with a presumptive diagnosis of spontaneous bacterial peritonitis . Two days later, growth on blood culture was identified as a gram - negative bacillus using the bact alert 3d culture system . The patient responded to cefotaxime and was discharged on post - admission day 12 . To identify isolate 07ac-292, biochemical characterization by vitek2 with an id - gnb card the isolate was identified as a. lwoffii at a confidence level of 94% . However, an acinetobacter differentiation method using the rpob gene (4) indicated that the isolate did not belong to the genus acinetobacter . The dna of the isolate was extracted and a portion of the 16s rrna gene was amplified and sequenced using the primer set as previously described (5). The determined sequences were compared with the genbank public database using the blastn program (http://blast.ncbi.nlm.nih.gov/blast.cgi). The 16s rrna sequence of the isolate 07ac-292 showed complete identity with several l. hongkongensis strains in the database . A phylogenetic tree constructed based on 16s rrna gene sequences also supported the identity of isolate 07ac-292 as l. hongkongensis (fig . Antibiotic susceptibility testing (ampicillin - sulbactam, cefepime, ceftriaxone, ceftazidime, amikacin, cefoperazone - sulbactam, imipenem, meropenem, doripenem, tetracycline, ciprofloxacin, rifampin, piperacillin - tazobactam, colistin, polymyxin b, and tigecycline) was conducted using the broth microdilution method according to clinical and laboratory standards institute (clsi) protocol (6). Minimum inhibitory concentration breakpoints and quality - control protocols were used according to non - fermenting gram - negative organism guidelines established by the clsi . Isolate 07ac-292 was resistant to piperacillin, intermediate resistant to ceftriaxon, and susceptible to the other tested antibiotics (table 1). L. hongkongensis was first isolated from a 54-yr - old chinese male with alcoholic cirrhosis and thoracic bacateremic empyema (1, 2). Subsequently, the bacterium has been isolated from patients in other parts of the world . The isolation of l. hongkogensis from patients who are residents in, or recent travelers to, asia, europe, america, and africa imply the global importance of the bacterium (7). Most reported cases were associated with recent travel and eating fish and symptoms are similar to salmonella and campylobacter gastroenteritis (2). Although freshwater fish is probably a major reservoir of l. hongkongensis, differences exist between isolates obtained from humans and fish using pulsed - field gel electrophoresis, suggesting that not all environmental clones are virulent (8). The presently - reported patient, who had underlying liver cirrhosis associated with wilson's disease, was suffering from neutrophilic ascites caused by l. hongkongensis . In contrast to previous described cases, the patient denied a history of ingestion of undercooked freshwater fish and overseas travel . It may be that the infection was hospital - acquired . Previous studies reported that this bacterium was associated with community - acquired gastroenteritis and traveler's diarrhea (2). It has been suggested that there was no carriage state of l. hongkogenesis (8), although the gastrointestinal tract has been suggested as a possible primary site of infection and enteric bacteria could enter the systemic circulation from the portal vein by passage through the liver in patients with portal hypertension (1, 2). The present l. hongkongensis infection may have originated from the intestinal flora . If so, it indicates that l. hongkongensis may exist in a carriage state in his intestine flora . In someone suffering from liver cirrhosis to date, all l. hongkongensis strains tested have been resistant to ampicillin and cephalosporins, but susceptible to the carbapenems, amoxicillin - clavulanate, aminoglycosides and fluoroquinolones (9). The isolate in this study showed a somewhat different antibiotic susceptibility profile, in that it was susceptible to ampicillin - sulbactam and several cephalosporins . A comparative study among l. hongkongensis isolates could more precisely determine the difference antibiotic susceptibility profiles, indicative of different origins . To our knowledge, this case is the first report of hospital - acquired l. hongkongensis bacteremia with neutrophilic ascites . Such misidentification might occur because l. honkingensis is not included in the database of commercial identification systems and biochemical tests are not enough to differentiate it from other species . Thus, it is conceivable that more l. hongkongensis infections have occurred, but have not been accurately identified due to the lack of proper identification tools . Since multidrug - resistant acinetobacter sp isolates are increasing in korea and l. hongkongensis is susceptible to most antimicrobial agents, correct identification of l. hongkongensis may reduce the inappropriate use of antimicrobial agents . Further clinical experiences with invasive diseases caused by l. hongkongensis are necessary to evaluate its potential pathogenesis . Extensive epidemiologic studies should be carried out to ascertain the etiologic association between l. hongkongensis and the intestinal normal flora, and to identify the host and the routes of transmission of l. hongkongensis.
The over emphasis of dental esthetics is increasing in daily life and concerns about the outward appearance also affect children . Anatomy, color and harmony of one's teeth are especially important to the appearance of the face . People who have well - positioned incisors are considered more attractive, intelligent and adjusted than others who have dental malocclusion and/or anomalies . Severe deformities of the face region cause sympathy and compassion in people . Paradoxically, more subtle deformities result in taunts and mockery, leading the individual to a situation of low self - esteem . A child's smile reveals important aspects of their quality - of - life and how the child interacts in his / her environment . A smile denotes a self - esteem, self - confidence and well - being . Low et al . Showed that children with concerns about their teeth show less smile security . Self - perception is a part of children psychological characteristics and it is essential to be aware of how much they like their smile and how happy they are with it . Oral disorders may expose an individual, particularly children of school age, to an embarrassing situation . Among the various health professions, dentistry commonly experiences situations in which children and adolescents have been subjected to bullying . In everyday clinical practice, the findings suggest that developmental dental anomalies have a deep impact on quality - of - life . Olweus describes bullying as an anti - social behavioral phenomenon that violates the rights of another person and reflects intentional and repeated aggression, verbal or physical, against any unable to defend him / herself and can occur in any social context . Their victims may have serious psychological consequences, isolation, depression, anxiety and can generate lower performance and learning . Bullying in schoolchildren is a global phenomenon and its effects can be short as long - term . The aim of this paper is to present three clinical reports in which children were discriminated in the school environment due to visible tooth anomalies involving anterior teeth . Ai is a hereditary disorder that affects the enamel in either quality or quantity, compromising teeth appearance . According to phenotype aspects, ai may be classified as hypoplastic, hypocalcified, hypomaturation or hypomaturation hypoplastic with taurodontism . The teeth esthetic and issues associated with the enamel such as sensitivity, staining and roughness may cause psychological and functional concerns to the patient . A 10-year - old female, sought dental care for extraction of all her teeth and dentures placement . According to her mother's report, all females and some males of their family had similar dental aspect . At the age of 14, the mother had all her teeth extracted and replaced by dentures . The patient was discriminated by the classmates at the break time and during group activities . The discriminatory behavior lasted for months and resulted in low learning performance, low self - esteem and introspectiveness . The school psychologist noticed that many embarrassing situations occurred due to the appearance of her teeth . During the clinical evaluation, the patient did not smile and avoided talking, trying to hide her mouth . In addition, it was clear that the family disapproved the child's appearance . Clinically, the child's presented yellow colored teeth with an irregular rough texture due to mineral deficiency and areas with an enamel loss . The condition was diagnosed as hypomaturation ai and the treatment performed was an intense remineralizing therapy followed by a composite resin veneers [figure 1a and b]. (a) clinical aspect before esthetic treatment of teeth stricken by hypomaturation amelogenesis imperfecta: rough - looking yellow teeth due to mineral deficiency and areas with loss of structure; (b) early treatment with esthetic veneers on permanent maxillary incisors eh is a developmental defect of enamel caused by a disturbance in the secretion or maturation of an enamel matrix . The severity of the defects depends on the phase of amelogenesis involved and the duration of the stimulus on the ameloblasts . It may be related either to hereditary causes, affecting all the teeth on both dentitions or acquired ones, involving one or more teeth . When eh is related to a hereditary cause a 10-year - old female patient presented a malformation of the permanent maxillary left canine (hypoplasia) [figure 2a] that affected masticatory function and social life . (a) permanent maxillary left canine with dental hypoplasia as a result of disturbance during the apposition of enamel; (b) aspect after the cosmetic restorative treatment during early childhood, the child was outgoing and communicative . However, at age of 9, your social behavior started changing . According to the family report a similar behavior changing occurred at school, the child stopped talking and did not participate in collective activities . With family consent, the child was evaluated by a psychologist and she reported being constantly called a vampire by classmates because of her canine tooth . The child was referred for dental treatment with a direct cosmetic restorative restoration [figure 2b] to improve her self - esteem and self - image, associated with counseling to minimize the effects of bullying . The term mih defines a hypomineralization of systemic origin that affects one to four permanent first molars . Mih has been linked to environmental changes such as pre - and perinatal problems, respiratory diseases, high fever diseases such as chicken pox and the frequent use of antibiotics in early childhood . Recently, studies showed that genetic variation in some enamel formation genes is associated with mih . The prevalence of mih varies considerably throughout the world, ranging from 2.5% to 40% . An 8-year - old girl, after a careful anamnestic and clinical evaluation, was diagnosed with mih [figure 3a]. According to her mother, the family was concerned about the permanent incisors opacities . Due to partial eruption of the teeth, however, the child still suffered with a verbal mocking by schoolmates, who said that she had not brushed her teeth and had rotten teeth . Family members, including aunts and cousins began to talk about the girl's appearance . The dental treatment was performed with a glass ionomer cement to remineralize the structure before placing veneers [figure 3b]. (a) molar - incisor hypomineralization: mineral and structural deficiency in permanent incisors and molars; (b) initial stage of esthetics treatment on maxillary incisors in all three cases, patients had suffered direct or indirect discrimination, exposing them to bullying in school and in their family environments . The children did not feel emotionally supported since they were always subject of comments about how different their teeth were . On physical and clinical examination and according to the reports, it was observed that there were no other factors beyond the tooth, which contributes to bullying . After temporary treatment, they all reported higher self - esteem, greater happiness, freedom and social acceptance . Ai is a hereditary disorder that affects the enamel in either quality or quantity, compromising teeth appearance . According to phenotype aspects, ai may be classified as hypoplastic, hypocalcified, hypomaturation or hypomaturation hypoplastic with taurodontism . The teeth esthetic and issues associated with the enamel such as sensitivity, staining and roughness may cause psychological and functional concerns to the patient . A 10-year - old female, sought dental care for extraction of all her teeth and dentures placement . According to her mother's report, all females and some males of their family had similar dental aspect . At the age of 14, the mother had all her teeth extracted and replaced by dentures . The patient was discriminated by the classmates at the break time and during group activities . The discriminatory behavior lasted for months and resulted in low learning performance, low self - esteem and introspectiveness . The school psychologist noticed that many embarrassing situations occurred due to the appearance of her teeth . During the clinical evaluation, the patient did not smile and avoided talking, trying to hide her mouth . In addition, it was clear that the family disapproved the child's appearance . Clinically, the child's presented yellow colored teeth with an irregular rough texture due to mineral deficiency and areas with an enamel loss . The condition was diagnosed as hypomaturation ai and the treatment performed was an intense remineralizing therapy followed by a composite resin veneers [figure 1a and b]. (a) clinical aspect before esthetic treatment of teeth stricken by hypomaturation amelogenesis imperfecta: rough - looking yellow teeth due to mineral deficiency and areas with loss of structure; (b) early treatment with esthetic veneers on permanent maxillary incisors eh is a developmental defect of enamel caused by a disturbance in the secretion or maturation of an enamel matrix . The severity of the defects depends on the phase of amelogenesis involved and the duration of the stimulus on the ameloblasts . It may be related either to hereditary causes, affecting all the teeth on both dentitions or acquired ones, involving one or more teeth . When eh is related to a hereditary cause a 10-year - old female patient presented a malformation of the permanent maxillary left canine (hypoplasia) [figure 2a] that affected masticatory function and social life . (a) permanent maxillary left canine with dental hypoplasia as a result of disturbance during the apposition of enamel; (b) aspect after the cosmetic restorative treatment during early childhood, the child was outgoing and communicative . However, at age of 9, your social behavior started changing . According to the family report a similar behavior changing occurred at school, the child stopped talking and did not participate in collective activities . With family consent, the child was evaluated by a psychologist and she reported being constantly called a vampire by classmates because of her canine tooth . The child was referred for dental treatment with a direct cosmetic restorative restoration [figure 2b] to improve her self - esteem and self - image, associated with counseling to minimize the effects of bullying . The term mih defines a hypomineralization of systemic origin that affects one to four permanent first molars . Mih has been linked to environmental changes such as pre - and perinatal problems, respiratory diseases, high fever diseases such as chicken pox and the frequent use of antibiotics in early childhood . Recently, studies showed that genetic variation in some enamel formation genes is associated with mih . The prevalence of mih varies considerably throughout the world, ranging from 2.5% to 40% . An 8-year - old girl, after a careful anamnestic and clinical evaluation, was diagnosed with mih [figure 3a]. According to her mother, the family was concerned about the permanent incisors opacities . Due to partial eruption of the teeth, a preventive treatment was performed . However, the child still suffered with a verbal mocking by schoolmates, who said that she had not brushed her teeth and had rotten teeth . Unfortunately, the stained surface fractured, what increases the bullying frequency . Family members, including aunts and cousins began to talk about the girl's appearance . The dental treatment was performed with a glass ionomer cement to remineralize the structure before placing veneers [figure 3b]. (a) molar - incisor hypomineralization: mineral and structural deficiency in permanent incisors and molars; (b) initial stage of esthetics treatment on maxillary incisors in all three cases, patients had suffered direct or indirect discrimination, exposing them to bullying in school and in their family environments . The children did not feel emotionally supported since they were always subject of comments about how different their teeth were . On physical and clinical examination and according to the reports, it was observed that there were no other factors beyond the tooth, which contributes to bullying . After temporary treatment, they all reported higher self - esteem, greater happiness, freedom and social acceptance . In recent decades, bullying has been reported as one of the most prevalent forms of violence in schools and as a precursor to other more serious forms of violence . The negative effects of bullying affect not only the victim but also the family, school and even society, since longitudinal studies have showed that children who are bullied are more likely to develop antisocial behavior such as vandalism, drug abuse and violent attacks in adulthood, as well as low self - esteem and lower sense of empathy toward others . The bullying prevalence in school - aged children varies greatly, under several factors as gender, age and culture of victims and perpetrators . In addition, the study design, cultural differences, bullying understanding, the time frame used to determine the frequency of bullying and the criteria used to differentiate between victims and non - victims are also factors that may explain these variation . In general, the most common form of direct aggression caused by bullying is verbal (30.9%), followed by spreading rumors against the victim (24.8%) and physical aggression (14.7%). The prevalence of indirect aggression is possibly underestimated due to the failure of both individuals and teachers to recognize it as a form of bullying . In the cases reported here, fortunately discriminatory act was limited to verbal abuse, there was a clear discriminatory attitude from family members that declared that such changes were not normal and that the teeth were ugly, without being careful not to embarrass the child . Although, we know all the consequences of such behavior, we believe that such conduct was a result of the lack of clarification about the causes and possible treatments for enamel defects . The facial pattern appears to be more influential than the individual's dental appearance . However, in the cases described, the bullying was due to dental problems that influenced the individual's personality . In this context, it is reasonable that clinicians may encounter children who are experiencing bullying at school . According lyznicki et al ., the clinician's role involves to identify the children at risk, counsel families, screening for psychiatric co - morbidities and prevention . However, to seehra et al ., a clear guidance to dental care practitioners is lacking, especially because this situation may involve other specialties . The esthetic procedures performed in young children were temporary, since the child is still growing . They are not very expensive at this point and minimally or not invasive, giving the opportunity for a permanent treatment later . After completion of dental treatment and the involvement of family and school psycho - pedagogical monitoring, patients reported they no longer were victims of verbal abuse . Significant improvements in self - esteem, self - confidence and socialization of all patients, began within a few days after completion of treatment . In addition, the parental satisfaction regarding the appearance of their children was much improved . In this context, we highlight the importance of dental care and cosmetic dentistry to improve the quality - of - life of patients and to combat this discriminatory behavior that has been growing in recent years, especially in school environments . The cases reported here demonstrate the need for a new teaching approach in dental schools, to sensitize dental professions to the negative social consequences of poor oral hygiene, rampant caries, developmental orofacial defects, including bullying . We believe that prompt dental treatment can corroborate to social integration, well - being and self - esteem of individuals and may reduce even prevent their exposure to bullying . However, psychological distress is not automatically reversed after dental treatment, which may have a negative impact for life . Thus, even after dental care, the child must be monitored by a psychologist . By providing a satisfactory esthetic condition to patients and parents, the cosmetic dental treatment was capable of restoring self - esteem and self - confidence, culminating in a greater socialization in school and in their own home environment, reducing the exposure of these individuals to bullying and its consequences.
A 37-year - old man of english descent presented with long - standing photophobia, reduced vision, and retinal pigmentary changes . He subsequently developed lymphoma in the left kidney, treated by nephrectomy and rituximab, and two bowel lymphomas treated with chemotherapy and rituximab . At age 29, he developed peripheral myopathy when dd was diagnosed on muscle biopsy . He took cyclosporin 100 mg bd, mycophenolate 500 mg bd, ramipril 10 mg daily, amlodipine 10 mg daily, pravastatin 20 mg daily, ezetimibe 10 mg daily, prednisolone 5 mg daily, and aspirin 100 mg daily . On examination, he was highly myopic; best - corrected snellen visual acuity (va) was 20/30 in the right eye and 20/200 in the left eye . Fundoscopy revealed midperipheral pigmentary changes, with near - complete loss of rpe in the remaining retina . Cirrus spectral - domain optical coherence tomography (sd - oct) (zeiss, germany) scanning demonstrated asymmetric macular cysts in the inner retinal layers and patchy photoreceptor loss in both maculae [fig . 1, table 1]. Full - field erg testing was performed using a gold foil electrode according to the international society for clinical electrophysiology of vision standards (lkc technologies, inc . ). This showed non - specific abnormalities: scotopic responses were of reduced amplitude and normal implicit time; photopic responses were of normal amplitude but increased implicit time . Fundus photographs right eye (a), left eye (b), infrared images right eye (c), left eye (d), and horizontal spectral - domain macular oct scans at baseline right eye (e), left eye (f), and after treatment right eye (g), left eye (h) with oral acetazolamide of a patient with histologically confirmed danon disease . These demonstrate the peppered pigmentary mottling and cystic - appearing lesions within the macula with photoreceptor loss and minimal response to treatment retinal thickness and visual function in response to treatment treatment with the carbonic anhydrase inhibitor (cai) dorzolamide 2% topically bd to both eyes was initiated . Following 30 weeks of treatment, the patient reported no change in central vision or glare symptoms . There was no significant change in vision, central subfield thickness, or macular cube volume . He was then treated with acetazolamide 500 mg daily while cyclosporine levels and renal function were monitored . Following 18 weeks of treatment, he reported no change in central vision or glare symptoms . Best - corrected va was 20/40 in the right eye and cf in the left eye . Central subfield thickness, macular cube volume, and repeat erg testing (roland, germany) showed no significant changes . Danon retinopathy is a rare x - linked disorder related to mutations in the lamp2 gene . The patient showed characteristic signs of danon retinopathy and his erg results were typical of the disease . The patient was myopic, but the macular changes are not typical of pathologic myopic foveoschisis, typically a thickened retina at the posterior pole, with hyporeflective splitting between the outer and inner retina, or of progressive myopic maculopathy, both of which are usually detected in patients with a posterior staphyloma . Patients with retinal dystrophies have been reported to have both cystoid macular edema (cme) and non - cme macular cysts . The patient refused fluorescein angiography, which is the diagnostic test to differentiate between cme and non - cme cysts, but the asymmetry of cystic changes seen on oct is typical of cme associated with late hyperfluorescence on intravenous fluoresce in angiography and strongly suggests that the cysts are cme rather than non - cme cysts . The cme is probably due to leakage of fluid through the rpe consequent to failure of the rpe pump . The reason for abnormal lamp2 protein resulting in presumed rpe pump failure is not known . To our knowledge, this is the first reported case of cme in histologically confirmed dd . In our case, topical dorzolamide 2% eye drops and oral acetazomide were not effective in the treatment of cme associated with dd . The reasons for lack of effect may have included low dose of acetazolamide, limited by renal disease; pre - existing photoreceptor atrophy and long - standing visual loss, particularly in the left eye; and pre - existing rpe atrophy, given the mechanism of action of cai via rpe acidification which helps to enhance chloride transport, and consequently water transport . Also, any non - cme macular cysts may not respond to cais . Lack of effect of cai is in contrast to previous studies which have demonstrated a beneficial effect of cais in patients with retinal dystrophy and cme such as retinitis pigmentosa, and patients with macular cysts, including x - linked retinoschisis and enhanced s - cone syndrome, although some patients fail this treatment . Despite lack of response in our patient, we recommend consideration be given to carbonic anhydrase inhibition for patients with cme associated with danon retinopathy, given the response seen in other retinal conditions with associated cme and macular cysts with this treatment . A further treatment option to consider for similar patients is intravitreal bevacizumab, which this patient refused . In conclusion, to our knowledge our patient is the first reported case of danon retinopathy with cme.
Patients with hematologic disorders are at a greater risk for prolonged bleeding; therefore, it is important to reduce opportunities for bleeding during surgical implant procedures . Prolonged bleeding can interfere with the initial process of osseointegration, and recurrent bleeding from a wound site leads to persistence of iron in the tissue . Excessive tissue iron is associated with fibrosis, poor wound healing, and excessive angiogenesis1 . If bone apposition does not occur, fibrous - scar tissue is formed between the implant surface and the surrounding bone2, which results in the absence of an anchoring function of the endosseous implant . Additionally, a lack of white blood cells (wbcs) increases the amount of oral pathologic organisms, which might play a role as an etiological factor in late implant complications . Aplastic anemia is a rare hematological disease characterized by pancytopenia and bone marrow hypoplasia, where normal hematopoietic tissue is replaced by fatty tissue . The incidence rate of aplastic anemia is two per one million persons per year, as reported by young3 . Though the etiology of aplastic anemia is typically unknown, it can be inherited, idiopathic, or acquired . Acquired aplastic anemia can be caused by intake of certain drugs and chemicals45, radiation therapy6, viral infection78, or pregnancy . This characterization indicates that the immune system attacks the bone marrow, resulting in insufficient blood cell production . Patients that suffer from aplastic anemia complain of general fatigue, shortness of breath, and pale appearance due to low red blood cell (rbc) count . In addition, thrombocytopenia increases the risk of severe bleeding at various sites and is associated with oral manifestations such as petechia, ecchymosis, hematoma, gingival bleeding, and bleeding after tooth extraction9 . In patients with severe aplastic anemia, infection and bleeding can be life threatening, and such patients have demonstrated a high mortality rate . Although patients with moderate aplastic anemia might not require treatment, frequent and regular blood cell counting is important . Recent therapeutic advances, such as immunosuppressive therapy with bone marrow transplantation, use of growth factor alone or in conjunction with immunesuppressive therapy after bone marrow transplantation, blood component transfusion, and improved infection control, have increased survival rates1011 of such patients . This study evaluated the following parameters; 1) the amount of peri - implant bone resorption, 2) level of osseointegration as measured by the amount of fine trabecular bone in close contact with the fixture, 3) probing depth, and 4) periabutment gingival connective tissue contour . The purpose of this study was to demonstrate the effectiveness of implant - supported restoration in aplastic anemic patients by evaluating radiographic and clinical features . A 44-year - old woman presented to the department of oral and maxillofacial surgery of chonnam national university hospital (gwangju, korea) to evaluate missing teeth in the maxilla and the mandible and possible implant - supported restoration . The patient had been diagnosed with severe aplastic anemia at the department of hemato - oncology of chonnam national university hospital in july 1995 and had received immunosuppressive therapy . She has been under regular follow - up and had taken 1 mg of folin (folic acid) and 50 mg of pyridoxine (vitamin b6) from may 2004 to june 2005 . A general assessment was performed based on panoramic radiograph and cone - beam computed tomography, and we planned to install 14 endosseous implants . Ten implants were inserted in the mandible in the first stage, and four implants were inserted in the maxilla in the second stage, with elevation of both sinuses and autogenous bone grafts . During the preoperative evaluation, the department of hemato - oncology confirmed that the patient would be able to maintain a greater than 1,000 absolute neutrophil count and have lower chances of postoperative bleeding if she was able to maintain a greater than 80,000 platelet count during the surgery . A complete blood cell count investigation revealed that the patient had an rbc of 3.51 million, a wbc count of 3,000, and a count of 84,000 platelets per milliliter of blood . These results indicated that both extraction of teeth #17, #16, #26, and #27 with poor prognosis and implant surgery could be performed without any special consideration . After five months of healing, the full mucoperiosteal flap was elevated with a midcrestal incision and two vertical releasing incisions under intravenous sedation with midazolam (0.05 mg / kg) and the local anesthesia lidocaine plus with epinephrine 1:100,000 . Ten osstem us ii (osstem implant co., busan, korea) implants were installed in the spaces left by teeth #32, #33, #34, #36, #37, #42, #43, #44, #46, and #47 (fig . A), and then provisional implants were placed in the sockets of teeth #31, #35, #41, #45. (fig . D) primary closure of the flap involved periosteal releasing incisions, and 3 - 0 vicryl (ethicon inc ., cornelia, ga, usa) was used as suture material . Three months after implant installation, a second surgery was performed with an apically repositioned flap in order to produce keratinized gingiva, which helps maintain a healthier gingival environment. (fig . B) four months postoperatively, the provisional prostheses were replaced with the final prostheses. (fig . C) we obtained panoramic radiographs using the kodak 8000c system (carestream health inc ., atlanta, ga, usa) and evaluated all panoramic and periapical radiographs with piview star (infinitt healthcare co., ltd ., seoul, korea). Marginal bone change was estimated from the border between the implant platform and implant abutment to the lowermost part of the absorbed marginal bone around the implant fixture, based on radiographs performed immediately after the operation . The magnification ratio on the radiographs was corrected by comparing the actual length of an implant fixture with that on the radiographic image . After two months of loading on the final mandible prostheses, bilateral sinus elevation was performed with ramal bone graft . The oval - shaped bony windows were removed from the lateral walls of the sinuses, and sinus membranes were carefully elevated, followed by bone grafting . Particulated ramal bone graft material was used in combination with bio - oss (geistlich pharma ag, wolhusen, switzerland), and four osstem us ii implants were simultaneously placed at sites #16, #17, #26, and #27. (fig . E) fibrin glue was injected into the bone graft site to enhance the bone healing process . All of the procedures were performed under intravenous sedation and local anesthesia with a vasoconstrictor, and the medications were the same as listed above . Seven months after implant installation in the sinus elevation site, prosthetic loading was applied, and peri - implant bone change was evaluated in panoramic and periapical radiographs . After one year and three months of prosthetic loading on the lower jaw, marginal bone change was evaluated around the implants using panoramic and periapical radiographs (fig . F), which revealed peri - implant bone resorption of 1.37 mm on #16, 1.73 mm on #17, 1.30 mm on #26, 1.11 mm on #27, 1.32 mm on #32, 1.70 mm on #33, 1.89 mm on #34, 1.0 mm on #36, 0.86 mm on #37, 0.50 mm on #42, 0.72 mm on #43, 1.0 mm on #44, 2.52 mm on #46, and 2.12 mm on #47 . Tranexamic acid at a concentration of 500 mg/5 ml was available for perioperative bleeding . However, no severe bleeding occurred intraoperatively or postoperatively, and no postoperative complications were noted . On follow - up, the marginal bone had not changed significantly one year and three months after prosthetic loading . In periapical radiographs, fine trabecular bone was observed around each fixture, which indicates that osseointegration was successful for all of the implants . We measured the probing depth, which is the distance from the gingival margin to the periodontal pocket, on the mesiobuccal and distobuccal sides of each tooth . The average of the measured values for each tooth was calculated, and a mean probing depth of 1.66 mm was obtained using the williams colorvue probekit (hu - friedy, chicago, il, usa). All procedures, from surgery to restoration, were uneventful over a follow - up period of seven years. (fig . F) no implants showed mobility, and the patient did not complain of pain or discomfort during the seven - year follow - up period . In addition, there was no evidence of peri - implant radiolucency seven years after implant installation . Finally, only two implants (#46, #47) showed marginal bone resorption greater than 2 mm after seven years of follow - up . Implant - supported restoration has become common practice in dentistry and is associated with long - term successful outcomes . This could increase the popularity of implant - supported restoration among dentists and the general public even though its effect in medically compromised patients, especially those with bleeding disorders, has not been thoroughly demonstrated . Special considerations for these medically compromised patients include bleeding control during operative and postoperative periods in order to avoid life - threatening situations, achieving successful osseointegration, and long - term implant maintenance . An extended period of adequate hemostatic function is necessary to accommodate the normal healing process, probably because the risk of hemorrhage is increased by vascular remodeling and angiogenesis during the healing process12 . Furthermore, patients with aplastic anemia are susceptible to peri - implant infection as aplastic anemia is characterized by marked hypoplastic bone marrow and pancytopenia (anemia, leucopenia, and thrombocytopenia). We were not able to collect data regarding specific blood management strategies for successful implant placement in cases of aplastic anemia, so we followed the general guidelines . We prepared a blood transfusion to maintain platelet count during surgery, since a platelet count less than 25,000/ml indicates a high risk of spontaneous oral bleeding9 . Tranexamic acid was also prepared for prolonged retention of intact weak blood clots and to prevent bleeding in plasminrich areas such as the oral cavity . Tranexamic acid can significantly reduce blood loss after oral surgery in patients with hemophilia and can be used topically or systemically13 . Radiographic examinations with implant mobility tests are the most reliable for assessing osseointegration . Although two of the present implants showed moderate peri - implant bone resorption, observed while loading dental prostheses, other implants were well - maintained, fulfilling the success criteria cited by albrektsson et al.14 . The first two implants were categorized into group ii (satisfactory survival), and the others into group i (success, optimum health), according to the health scale developed by the international congress of oral implantologists consensus conference for implant success in pisa in 200715 . Fine trabecular bone structure was observed around all of the implant fixtures, which created sufficient anchorage necessary for long - term implant maintenance . Stable, rigid, fixated implants have been reported with pocket depths ranging from 2 to 6 mm15; in the presented case, the mean probing depth was 1.66 mm after 21.4 months of prosthetic loading . Thus, based on these results, implant surgery can be performed safely even for patients with hematologic disorders by applying three principles of implant installation; induction of initial hemostasis, maintenance of hemostasis during the healing period, and immobilization of the implant . These can be achieved through appropriate systemic treatment and precise management of local soft tissue.
Individuals with a hematologic malignancy may have an immunosuppressive condition and develop cutaneous or invasive bacterial infections . The infections may be a first sign of a malignancy or a sign of a relapse . Acute promyelocytic leukemia (apl; acute myeloid leukemia m3) is caused by balanced reciprocal chromosomal translocation t(15;17), which produces an oncogenic protein pml - rara by fusion of the promyelocytic leukemia gene (pml) and the retinoic acid receptor a gene (rara). Apl is created by a blockage of differentiation, resulting in overproduction of immature myeloid cells of promyelocytes in the bone marrow . We herein report a 71-year - old man with cellulitis as a first sign of apl . A 71-year - old man was referred to us on october 2011 with a painful eruption on the right thigh that had appeared 12 days earlier . A physical examination revealed a swollen and painful erythematous lesion with an elevated temperature (38.6c) on the right thigh (fig . Laboratory blood examination results were as follows: white blood cell count 1,300/l with neutrophils 18.4% (band neutrophils 10.0% and segmented neutrophils 8.4%), lymphocytes 56.0%, monocytes 0.3%, eosinophils 0%, basophils 0%, metamyelocytes 1.3% and leukemic cells 24.0%; red blood cells 3.29 10/l; hemoglobin 11.5 g / dl; platelets 11.9 10/l; c - reactive protein 18.526 mg / dl; soluble interleukin-2 1,249 u / ml; ferritin 607 ng / ml; c3 170 mg / dl; c4 36 mg / dl; ch50 73.8 u / ml; fibrinogen 632 mg / dl; fibrin degradation product 24.1 g / ml; antithrombin iii 109%; thrombin - antithrombin iii complex 6.3 ng / ml; plasmin - a2 plasmin inhibitor complex 3.1 g / ml; d - dimer 15.8/g / ml, and endotoxin less than 5.0 pg / ml . Blood cultures showed no bacterial growth . G - band testing of twenty bone marrow cells showed 46,xy, t(15;17)(q22;q12) in 19 cells and 46,xy in 1 cell . All - trans retinoic acid (atra), idarubicin hydrochloride, and cytarabine were administered after the bacterial infection had been eliminated . One month after the administration of atra, a sample of bone marrow cells contained the following: myeloblasts 2.1%; promyelocytes 3.5%; myelocytes 21.8%; metamyelocytes 20.2%; band neutrophils 12.0%, and segmented neutrophils 5.6% . Apl cells can be forced to differentiate in the presence of atra . In this case, a bone marrow examination indicated that the apl cells differentiated into maturing cells after the administration of atra . Patients with leukemia are predisposed to pancytopenia, and may show infectious disorders resulting from leukocytopenia, purpura resulting from thrombocytopenia, and shortness of breath resulting from erythrocytopenia . Cellulitis may develop secondary to a bacterial infection in patients with leukemia, including apl . Cellulitis is an inflammation of loose connective tissue of dermal and subcutaneous tissue, and is caused by bacterial infection . However, cellulitis without leukocytosis may be present in patients with malignant hematologic disorders as shown here, or in persons with cold cellulitis by cutaneous leishmaniasis or leprosy . Girmenia et al . Reported that their analysis of septicemias in apl patients receiving atra and idarubicin showed a significantly lower rate of bloodstream infections, even though patients receiving atra occasionally suffered life - threatening and lethal infections . The authors noted that 6 of 89 patients with apl withdrew from antileukemic treatment due to infections . Infection control before and during the treatment is indispensable for individuals with apl, even though septicemias are less frequent in apl than in other leukemias . Dermatologists and hematologists should keep in mind that patients with a hematologic malignancy, such as apl, can develop cellulitis with leukocytopenia.
Bioluminescence imaging (bli) using luciferase reporters has been indispensible for noninvasive monitoring of different biological processes such as tumor volume and transcriptional activation during tumor development / therapy, as well as immune cell infiltration into the tumor environment . Unlike end - point analysis, bli provides real - time, noninvasive assessment of in situ biological events, thereby giving a better picture of the kinetics of an entire process . For example, as few as 10 cells expressing firefly luciferase (fluc) can be detected in deep tissue in some animal models . Additional progress has been made by the discovery and molecular construction of different luciferases with a multitude of properties, including secreted reporters such as gaussia luciferase (gluc), multicolor light emission spectra for better tissue penetrance in vivo and spectral deconvolution, increased thermostability, and light output . One limitation to current bioluminescence imaging is that typically only one and at most two luciferase reporters are used to measure one or two parameters . As tumor formation is a complex process, concurrent measurement of several events will be important for the development of novel therapeutics and their transition to the clinic . In this study, we have characterized a codon - optimized vargula hilgendorfii luciferase (vluc) for mammalian gene expression, and showed that this luciferase can be multiplexed with gluc and fluc for sequential imaging of three different biological processes in the same biological system . We then applied this triple imaging system to monitor the effect of adeno - associated virus (aav)-mediated soluble tumor necrosis factor - related apoptosis - inducing ligand (strail) therapy against intracranial glioma tumors . We first cloned vluc cdna, codon - optimized for mammalian gene expression, into a lentivirus vector under the control of the cytomegalovirus promoter (lenti - vluc). This vector also expresses the mcherry fluorescent protein separated from vluc by an internal ribosomal entry site, used to monitor transduction efficiency . Since vluc cdna carries a natural signal sequence, it is secreted to the conditioned medium once expressed in mammalian cells . We first observed the level of vluc secretion by transducing 293 t cells with lenti - vluc and evaluating the vluc levels in cell lysates and conditioned medium using the vargulin substrate . We observed that the majority of vluc activity (78%) was contained in the medium fraction showing efficient secretion (figure 1a). Next, we evaluated the light emission kinetics of vluc over time and observed a slow decay in light emission, with 44% of the initial signal remaining 5 minutes after substrate addition (figure 1b). To further characterize vluc as a mammalian cell reporter, we measured the stability of the enzyme over time at 37 c . Conditioned medium from cells expressing vluc were incubated at 37 c in a humidified cell incubator . We observed that vluc levels retained full activity over 12 days indicating high enzyme stability in conditioned medium (figure 1c), similar to that reported for the secreted gaussia luciferase . We next evaluated vluc as a reporter to monitor cell viability and proliferation over time . U87 glioma cells transduced with lenti - vluc were seeded in a culture well and aliquots of conditioned media were collected at different time points and analyzed for vluc activity . In parallel, we observed a high correlation (r = 0.98) between the vluc assay and the established viability assay (figure 1d). To validate vluc as a reporter for in vivo imaging nude mice were injected subcutaneously with 2 10 u87 cells stably expressing vluc (through transduction with lenti - vluc). Ten days later, mice were injected intravenously (iv; through retro - orbital route) or intraperitoneally (ip) with vargulin (4 mg / kg body weight) and imaged using a cooled charge - coupled device camera at different time points . For iv injected mice, we observed the peak luminescent signal immediately upon injection of vargulin, which rapidly declined to 25% of initial signal by 6 minutes and down to 10% by 26 minutes (figure 2a). For ip injected vargulin, the peak occurred 14 minutes after substrate injection (figure 2a). We then evaluated the possibility of multiplexing vluc with gluc and fluc for triple bioluminescence imaging by measuring the specificity of each luciferase for its substrate in vivo . U87 cells stably expressing vluc, gluc, or fluc (under control of the cytomegalovirus promoter) were implanted subcutaneously in nude mice at three different sites . Ten days later, mice were imaged first for fluc - mediated bioluminescence imaging after ip injection of d - luciferin (200 mg / kg body weight) and acquiring photon counts 10 minutes after injection . Twenty - four hours later, mice were imaged immediately after iv injection of coelenterazine (5 mg / kg body weight), and again signal was obtained only from tumor - expressing gluc (figure 2b). Finally, after another 24 hours, mice were iv injected with vargulin (4 mg / kg body weight) and imaged immediately, which showed a signal only in tumor - expressing vluc (figure 2b). Quantification of the luciferase signal from each tumor revealed that fluc and gluc mediated similar bioluminescence signal, whereas vluc had a significantly lower signal (~100-fold lower; n = 3 mice; p = 0.016; figure 2c). We applied the triple luciferase reporter system to monitor glioma response to a gene therapeutic approach using the secreted soluble variant of the anticancer agent trail . We first cloned strail under control of the constitutively active chicken -actin (cba) promoter into an aav2 inverted terminal repeat - flanked transgene cassette and pseudotyped it with an aavrh.8 capsid (aav - strail; figure 3a). As a control, we packaged a similar vector expressing green fluorescent protein (gfp) driven by the cba promoter . We next cloned vluc cdna under control of cba promoter in another aav2 vector pseudotyped with aavrh.8 capsid (aav - vluc; figure 3a). We then engineered u87 glioma cells to stably express fluc (u87-fluc) under control of cytomegalovirus promoter using a lentivirus vector (figure 3a). Since trail is known to activate a series of events including the nuclear factor-b (nf-b) pathway, we engineered a lentivirus vector expressing gluc under the control of five tandem repeats of nf-b responsive elements (figure 3a) as described, and used it to transduce the u87-fluc cells generating u87-fluc / nf - gluc cells . To determine the functionality of the aav - strail construct, we transduced u87 cells with 10 genome copy (gc)/cell of aav - strail or aav - gfp control vector . Three days later, conditioned media from these cells were harvested and analyzed for trail expression using an elisa kit . We observed a trail concentration of 124 ng / ml from conditioned medium of aav - strail infected cells, while it was undetectable in the media from aav - gfp control cells, displaying the proper expression and secretion of strail into the conditioned medium of cells (data not shown). Transfer of conditioned media from u87 cells transduced with aav - strail onto fresh u87 cells provided a modest (~25%) yet significant (p = 0.0035) killing effect (supplementary figure s1). We then stereotactically implanted 10 u87-fluc / nf - gluc cells into the striatum of nude mice and allowed tumor formation . Nineteen days later, mice were randomly divided into two groups (n = 5/group). The first group was infused into the same coordinates used for tumor implantation with 10 gc of both aav - vluc + aav - gfp (aav - vluc / gfp; serves as a negative control for therapy). The second group of mice was infused with 10 gc of both aav - vluc (to monitor successful gene delivery) and aav - strail (antitumor therapy; aav - vluc / strail). As a control, ten days after vector injection, we monitored aav gene delivery by injecting mice (iv) with vargulin substrate and immediately imaging using a cooled charge - coupled device camera . Evident bioluminescent signal (average radiance of 5 10 p / second / cm / sr 2.3 10) was seen at the injection site of all aav - vluc injected mice, and not the aav - gfp controls, showing successful gene delivery (figure 3b). Mice in both groups were monitored for tumor growth (fluc imaging) at week 2 post - vector injection (corresponding to week 4 posttumor injection), which showed a robust antitumor response in mice treated with aav - vluc / strail as compared with the control group (aav - vluc / gfp; figure 3c). Trail binding to its death receptors recruits tnfr1-associated death domain protein (tradd) leading to nf-b activation . We therefore sought to detect nf-b induction and therefore trail binding to glioma cells in our model . We first confirmed the functionality of the nf - gluc construct by incubating u87-fluc / nf - gluc cells with tumor necrosis factor-, a known activator of nf-b, and showed a specific induction of gluc expression 48 hours after treatment (figure 3d). Next, mice in both the aav - vluc / gfp (control) and aav - vluc / strail groups were imaged 22 days and 23 days after aav injection for fluc and gluc, respectively . An increase in fluc signal was observed at this time point, showing that tumors regained resistance to strail therapy (data not shown). As expected, specific fluc bioluminescence at the tumor location was observed in both groups of mice, but visible gluc signal was detected only in mice injected with aav - vluc / strail and not aav - vluc / gfp control animals, indicating binding of strail to glioma cells and induction of the downstream nf-b pathway (figure 3e, f). All together, these results show that the triple luciferase system developed here could be used to image three independent cellular processes sequentially in the same animal model . We next performed an extensive analysis of aav - mediated strail therapy in mice bearing intracranial u87 tumors . Ten mice were injected with 5 10 u87-fluc / nf - gluc cells into the striatum as above, and 14 days later, mice were randomized into two groups (n = 5/group) where the first group was infused into the same tumor implanted site with 10 gc of a combination of aav - vluc / gfp and the second group with the same amount of aav - vluc / strail . Mice were imaged at day 2, 7, 14, and 21 after vector injection for tumor - associated fluc signal . Similar to the first experiment, aav - mediated strail expression slowed the tumor growth as compared with control mice . At day 2 after vector injection, the aav - vluc / gfp control group had a twofold higher signal (p = 0.46) as compared with the aav - vluc / strail - treated mice (figure 4a, b). This difference increased to 6.13- (p = 0.048) and 33.7-fold (p = 0.0008) on day 7 and 14, respectively . All mice in the aav - vluc / gfp group were sacrificed between day 14 and 21 after vector injection due to tumor burden, while the entire group in aav - vluc / strail remained alive . Interestingly, the aav - vluc / strail group showed a 193-fold increase in the fluc signal (and therefore tumor growth) between days 14 and 21, presumably due to u87-gained resistance to strail therapy (figure 4a, b). We also performed an experiment with a higher input of u87 cells and obtained similar results (supplementary figure s2). We confirmed the luminescence data with a survival analysis, which revealed a significant survival increase (p = 0.0088) for mice injected with aav - vluc / strail compared with aav - vluc / gfp controls (figure 4c). The median survival time for the aav - vluc / gfp group was 35 days while it was 80 days for the aav - vluc / strail group . We first cloned vluc cdna, codon - optimized for mammalian gene expression, into a lentivirus vector under the control of the cytomegalovirus promoter (lenti - vluc). This vector also expresses the mcherry fluorescent protein separated from vluc by an internal ribosomal entry site, used to monitor transduction efficiency . Since vluc cdna carries a natural signal sequence, it is secreted to the conditioned medium once expressed in mammalian cells . We first observed the level of vluc secretion by transducing 293 t cells with lenti - vluc and evaluating the vluc levels in cell lysates and conditioned medium using the vargulin substrate . We observed that the majority of vluc activity (78%) was contained in the medium fraction showing efficient secretion (figure 1a). Next, we evaluated the light emission kinetics of vluc over time and observed a slow decay in light emission, with 44% of the initial signal remaining 5 minutes after substrate addition (figure 1b). To further characterize vluc as a mammalian cell reporter, we measured the stability of the enzyme over time at 37 c . Conditioned medium from cells expressing vluc were incubated at 37 c in a humidified cell incubator . We observed that vluc levels retained full activity over 12 days indicating high enzyme stability in conditioned medium (figure 1c), similar to that reported for the secreted gaussia luciferase . We next evaluated vluc as a reporter to monitor cell viability and proliferation over time . U87 glioma cells transduced with lenti - vluc were seeded in a culture well and aliquots of conditioned media were collected at different time points and analyzed for vluc activity . In parallel, we observed a high correlation (r = 0.98) between the vluc assay and the established viability assay (figure 1d). To validate vluc as a reporter for in vivo imaging, we first characterized its bioluminescence reaction properties in a quantitative tumor model . Nude mice were injected subcutaneously with 2 10 u87 cells stably expressing vluc (through transduction with lenti - vluc). Ten days later, mice were injected intravenously (iv; through retro - orbital route) or intraperitoneally (ip) with vargulin (4 mg / kg body weight) and imaged using a cooled charge - coupled device camera at different time points . For iv injected mice, we observed the peak luminescent signal immediately upon injection of vargulin, which rapidly declined to 25% of initial signal by 6 minutes and down to 10% by 26 minutes (figure 2a). For ip injected vargulin, we then evaluated the possibility of multiplexing vluc with gluc and fluc for triple bioluminescence imaging by measuring the specificity of each luciferase for its substrate in vivo . U87 cells stably expressing vluc, gluc, or fluc (under control of the cytomegalovirus promoter) were implanted subcutaneously in nude mice at three different sites . Ten days later, mice were imaged first for fluc - mediated bioluminescence imaging after ip injection of d - luciferin (200 mg / kg body weight) and acquiring photon counts 10 minutes after injection . Twenty - four hours later, mice were imaged immediately after iv injection of coelenterazine (5 mg / kg body weight), and again signal was obtained only from tumor - expressing gluc (figure 2b). Finally, after another 24 hours, mice were iv injected with vargulin (4 mg / kg body weight) and imaged immediately, which showed a signal only in tumor - expressing vluc (figure 2b). Quantification of the luciferase signal from each tumor revealed that fluc and gluc mediated similar bioluminescence signal, whereas vluc had a significantly lower signal (~100-fold lower; n = 3 mice; p = 0.016; figure 2c). We applied the triple luciferase reporter system to monitor glioma response to a gene therapeutic approach using the secreted soluble variant of the anticancer agent trail . We first cloned strail under control of the constitutively active chicken -actin (cba) promoter into an aav2 inverted terminal repeat - flanked transgene cassette and pseudotyped it with an aavrh.8 capsid (aav - strail; figure 3a). As a control, we packaged a similar vector expressing green fluorescent protein (gfp) driven by the cba promoter . We next cloned vluc cdna under control of cba promoter in another aav2 vector pseudotyped with aavrh.8 capsid (aav - vluc; figure 3a). We then engineered u87 glioma cells to stably express fluc (u87-fluc) under control of cytomegalovirus promoter using a lentivirus vector (figure 3a). Fluc was used as a marker for tumor volume . Since trail is known to activate a series of events including the nuclear factor-b (nf-b) pathway, we engineered a lentivirus vector expressing gluc under the control of five tandem repeats of nf-b responsive elements (figure 3a) as described, and used it to transduce the u87-fluc cells generating u87-fluc / nf - gluc cells . To determine the functionality of the aav - strail construct, we transduced u87 cells with 10 genome copy (gc)/cell of aav - strail or aav - gfp control vector . Three days later, conditioned media from these cells were harvested and analyzed for trail expression using an elisa kit . We observed a trail concentration of 124 ng / ml from conditioned medium of aav - strail infected cells, while it was undetectable in the media from aav - gfp control cells, displaying the proper expression and secretion of strail into the conditioned medium of cells (data not shown). Transfer of conditioned media from u87 cells transduced with aav - strail onto fresh u87 cells provided a modest (~25%) yet significant (p = 0.0035) killing effect (supplementary figure s1). We then stereotactically implanted 10 u87-fluc / nf - gluc cells into the striatum of nude mice and allowed tumor formation . Nineteen days later, mice were randomly divided into two groups (n = 5/group). The first group was infused into the same coordinates used for tumor implantation with 10 gc of both aav - vluc + aav - gfp (aav - vluc / gfp; serves as a negative control for therapy). The second group of mice was infused with 10 gc of both aav - vluc (to monitor successful gene delivery) and aav - strail (antitumor therapy; aav - vluc / strail). As a control, ten days after vector injection, we monitored aav gene delivery by injecting mice (iv) with vargulin substrate and immediately imaging using a cooled charge - coupled device camera . Evident bioluminescent signal (average radiance of 5 10 p / second / cm / sr 2.3 10) was seen at the injection site of all aav - vluc injected mice, and not the aav - gfp controls, showing successful gene delivery (figure 3b). Mice in both groups were monitored for tumor growth (fluc imaging) at week 2 post - vector injection (corresponding to week 4 posttumor injection), which showed a robust antitumor response in mice treated with aav - vluc / strail as compared with the control group (aav - vluc / gfp; figure 3c). Trail binding to its death receptors recruits tnfr1-associated death domain protein (tradd) leading to nf-b activation . We therefore sought to detect nf-b induction and therefore trail binding to glioma cells in our model . We first confirmed the functionality of the nf - gluc construct by incubating u87-fluc / nf - gluc cells with tumor necrosis factor-, a known activator of nf-b, and showed a specific induction of gluc expression 48 hours after treatment (figure 3d). Next, mice in both the aav - vluc / gfp (control) and aav - vluc / strail groups were imaged 22 days and 23 days after aav injection for fluc and gluc, respectively . An increase in fluc signal was observed at this time point, showing that tumors regained resistance to strail therapy (data not shown). As expected, specific fluc bioluminescence at the tumor location was observed in both groups of mice, but visible gluc signal was detected only in mice injected with aav - vluc / strail and not aav - vluc / gfp control animals, indicating binding of strail to glioma cells and induction of the downstream nf-b pathway (figure 3e, f). All together, these results show that the triple luciferase system developed here could be used to image three independent cellular processes sequentially in the same animal model . We next performed an extensive analysis of aav - mediated strail therapy in mice bearing intracranial u87 tumors . Ten mice were injected with 5 10 u87-fluc / nf - gluc cells into the striatum as above, and 14 days later, mice were randomized into two groups (n = 5/group) where the first group was infused into the same tumor implanted site with 10 gc of a combination of aav - vluc / gfp and the second group with the same amount of aav - vluc / strail . Mice were imaged at day 2, 7, 14, and 21 after vector injection for tumor - associated fluc signal . Similar to the first experiment, aav - mediated strail expression slowed the tumor growth as compared with control mice . At day 2 after vector injection, the aav - vluc / gfp control group had a twofold higher signal (p = 0.46) as compared with the aav - vluc / strail - treated mice (figure 4a, b). This difference increased to 6.13- (p = 0.048) and 33.7-fold (p = 0.0008) on day 7 and 14, respectively . All mice in the aav - vluc / gfp group were sacrificed between day 14 and 21 after vector injection due to tumor burden, while the entire group in aav - vluc / strail remained alive . Interestingly, the aav - vluc / strail group showed a 193-fold increase in the fluc signal (and therefore tumor growth) between days 14 and 21, presumably due to u87-gained resistance to strail therapy (figure 4a, b). We also performed an experiment with a higher input of u87 cells and obtained similar results (supplementary figure s2). We confirmed the luminescence data with a survival analysis, which revealed a significant survival increase (p = 0.0088) for mice injected with aav - vluc / strail compared with aav - vluc / gfp controls (figure 4c). The median survival time for the aav - vluc / gfp group was 35 days while it was 80 days for the aav - vluc / strail group . Luciferases have played a major role in advancing our understanding of biological processes . A broader array of biocompatible, nontoxic, and efficiently expressed reporters that can be used together with existing luciferases can serve to expand this potential . The present study demonstrates for the first time a triple luciferase reporter system for in vivo bioluminescence imaging . We showed that vargula luciferase could be multiplexed with gaussia and firefly luciferases for sequential monitoring of three distinct biological phenomena . We also showed that this triple bioluminescence imaging system yield specific, and detectable bioluminescence signal in deep tissues such as the brain (with intact skull, ~3.5 mm) of mice . Finally, we applied these reporters to monitor three cellular processes in an orthotopic brain tumor model in response to aav - strail therapy, thus validating the system for different applications . Since the cloning and sequencing of vargula (formerly cypridina) hilgendorfii cdna in 1989 however, to our knowledge, no reports have applied it for in vivo imaging in mammals, probably owing to the unavailability of its substrate vargulin, which is now commercially available . Vluc cdna possess a signal sequence and therefore it is naturally secreted from cells allowing real time, multi time point analysis from the same well . In this study, we characterized a codon - optimized vluc cdna for mammalian gene expression and showed vluc to be very stable with no significant decline in activity over 12 days in cell - free conditioned media at 37 c . Despite that the majority of vluc was found in the conditioned media of cells due to its native signal sequence, the intracellular vluc level was efficient for in vivo imaging . In applications where higher sensitivity is required, a membrane - bound variant of vluc could be used, which should yield higher cellular activity similar to recent reports for gaussia luciferase . Several systems for multimodal imaging exist, which incorporate different technologies such as fluorescence, bioluminescence, positron emission tomography, and magnetic resonance imaging . While substantial multiparameter information can be gained by these systems, they have several drawbacks, including higher cost (e.g., magnetic resonance imaging), logistical concerns such as short half - life of some positron emission tomography probes, and broad technical expertise . While fluorescent reporter - based imaging as well as luciferases emitting at different wavelengths can be used for multimodal applications, they require expensive and complicated instrumentation with spectral deconvolution to visualize each biological parameter . Furthermore, fluorescence - based imaging is limited by a high background noise due to tissue autofluorescence as well as single animal analysis . On the other hand, bli has low - to - no background and is thus more sensitive for deep tissue applications . The triple in vivo bioluminescence imaging system described here yields multiparameter information distinguished by sequential imaging using different specific substrates, while remaining both cost - effective and highly sensitive, as well as being user friendly; only a simple charge - coupled device camera is required with no need for sophisticated instrument hardware / software . Moreover, this same system can be extended and applied to any field to monitor three distinct biological phenomena in small animals . It is important to note, however, that we have tested vluc expression in the brain of mice bearing intracranial tumors . Since tumors may have comprised the integrity of the blood brain barrier, it would be important to evaluate vargulin in a model with intact blood brain barrier to validate the ability of this substrate in crossing this biological barrier . Another drawback of the triple - reporter system is the need of methanol to dissolve the vargulin and coelenterazine substrates, limiting imaging frequency due to potential toxicity of the alcohol . The use of water - soluble coelenterazine or the development of similar vargulin substrate would alleviate this issue . Trail has been regarded as an anticancer agent; however, significant cancer types, including gliomas, are resistant to trail - induced cell death . Mechanisms of resistance include downregulation of death receptors, decoy receptor expression, as well as overexpression of the caspase-8 inhibitor, c - flip, due to deregulation of the mtor signaling pathway . In addition, nf-b induction by trail has been reported as a tumor cell resistance mechanism . Another disadvantage of using trail for brain tumor therapy is its inability in crossing the blood brain barrier . In this study, we circumvented this problem by delivering strail directly to brain tumor environment using aav - mediated gene delivery . We chose to use aavrh.8 as we have previously shown that this serotype yields excellent transduction efficiency of murine normal brain . Injection of aavrh.8 into the tumor results in transduction of primarily neuronal cells surrounding the tumor . The transduced neurons serve as a therapeutic reservoir surrounding the tumor by synthesizing and secreting strail, which in turn will find and bind its death receptor present specifically on glioma cells . Despite this continuous release of the anticancer agent by the normal brain, we observed that tumors acquired resistance to strail therapy; however, a significant increase in survival was observed using aav - mediated expression of strail compared with the control group . The exact mechanism of trail resistance in this model is currently not known, but may involve nf-b signaling, as mice injected with aav - vluc / strail showed activation of the nf-b transcription factor, as monitored by gluc imaging . It is noteworthy to mention that u87 cells showed low sensitivity to strail - mediated death in culture, as compared with the initial high sensitivity in vivo . The culture experiment used conditioned medium from cells secreting strail (124 ng / ml as determined by elisa) and therefore the cells will only get a one - shot delivery, given that toxicity of trail to glioma cells is dose - dependent . (25% killing) using a dose of 100 ng / ml of strail in culture . Although the level of aav - mediated strail expression in the brain is unknown, the transduction efficiency of the vector serotype used (aavrh.8) is very high in the murine brain . Reported that despite this apparent low trail - sensitivity in vitro, an increase in survival rate in vivo was observed when using mesenchymal stem cell to deliver strail to u87 gliomas . Finally, resistance factors that allow glioma cell survival in culture may not suffice for in vivo tumor growth in a complex multicellular environment . In conclusion, we showed that each of these luciferases (vluc, gluc, and fluc) is specific to its own substrate and can be multiplexed together to monitor three distinct biological events in the same biological system . This reporter system could be extended to different fields where simultaneous monitoring of multiple parameters is required . U87 human glioblastoma cell line and 293 t human kidney fibroblast cells were obtained from the american type culture collection (manassas, va). Both cell lines were cultured in high glucose dulbecco's modified eagle's medium (invitrogen, carlsbad, ca) supplemented with 10% fetal bovine serum (sigma, st louis, mo) and 100 u / ml penicillin, 100 g / ml streptomycin (invitrogen) in a humidified atmosphere supplemented with 5% co2 at 37 c . The aav - cba - vluc vector was constructed by replacing egfp in aav - cba - egfp with the human codon - optimized vluc cdna (a kind gift from dr rampyari walia; targeting systems, el cajon, ca). Aav - cba - strail vector consists of a transgene cassette for soluble, secreted trail carrying amino acid (aa) 1150 from human flt3l, an isoleucine zipper domain, and the extracellular domain (aa 114281) of the human trail designed based on previously reported h - flex - zipper - trail . In all aav vectors, all vectors carry a woodchuck hepatitis virus post - trancriptional regulatory element (wpre) downstream of the transgene . Aav vector stocks were produced by cotransfection of 293 t cells by calcium phosphate precipitation of vector plasmid, a mini - adenovirus helper plasmid pf6 (from dr weidong xiao, university of pennsylvania medical center, philadelphia, pa), and aavrh.8 helper plasmid par8 as described . Aav2/rh.8 vectors were purified and titered as described, yielding typical titers of 10 genome copies (gc) per milliliter . For strail expression in culture, aav vectors were packaged as aav2 since this serotype is known to transduce cells in culture much more efficiently as compared with aav2/rh.8 . U87fluc - mcherry cells stably and constitutively expressing both firefly luciferase (fluc) and mcherry were generated by transduction with lentiviral vector cscw2-fluc - imcherry, which has been previously described . U87fluc - mcherry cells were subsequently transduced with a lentivirus previously described as lenti - nf - gluc, which encodes a gluc transgene cassette driven by five tandem repeats of nf-b responsive elements . This double - transduced u87 cells is referred to as u87fluc / nf - gluc cells . The lentivirus vector encoding vluc was constructed by replacing the fluc insert in cscw2-fluc - imcherry with the human codon - optimized vluc cdna . We achieve> 95% transduction efficiency of u87 cells at a multiplicity of infection of 10 with the different lentivirus constructs . We do not find it necessary to sort the cells owing to this high transduction rate . In vivo tumor models . All animal experiments were approved by the massachusetts general hospital subcommittee on research animal care following guidelines set forth by the national institutes of health guide for the care and use of laboratory animals . Six to 8 weeks old athymic nude mice were anesthetized with a mixture of ketamine (100 mg / kg) and xylazine (5 mg / kg) in 0.9% sterile saline . For subcutaneous tumors, mice were injected with 100 l of a 50:50 mixture of matrigel basement membrane matrix (bd biosciences, franklin lakes, nj) and 12 million u87 cells expressing gluc, vluc, or fluc resuspended in opti - mem . For the brain tumor model, 10 u87fluc/5nf - gluc cells (in 2 l opti - mem) were intracranially injected in the left midstriatum of nude mice using the following coordinates from bregma in millimeters: anteroposterior + 0.5, mediolateral + 2.0, and dorsoventral 2.5 . These injections were performed using a micro 4 microsyringe pump controller (world precision instruments, sarasota, fl) attached to a hamilton syringe with a 33-gauge needle (hamilton, reno, nv) at a rate of 0.2 l / min . Before aav vector injection, mice were randomized into separate groups by placing all mice into a single cage by one operator and selected for each group by a different operator . For aav vector injections, mice were anesthetized as above and injected intracranially with 10 gc of each vector using the same coordinates as for tumor injections . Aav vectors were infused at a rate of 0.2 l / min using a micro 4 microsyringe pump controller attached to a hamilton syringe with a 33-g needle . In vitro and in vivo luciferase imaging . D - luciferin was purchased from gold biotechnology (st louis, mo) and resuspended at 25 mg / ml in phosphate - buffered saline . For fluc imaging, mice were injected ip with 200 mg / kg body weight of d - luciferin solution, and imaging was performed 10 minutes later . Coelenterazine was obtained from nanolight technology (pinetop, az) and resuspended at 5 mg / ml in acidified methanol . For gluc imaging, mice were injected iv (through retro - orbital route) with 5 mg / kg body weight of coelenterazine solution diluted in phosphate - buffered saline and imaging was performed immediately . Vargulin substrate was obtained from nanolight technology or from targeting systems and was resuspended at 5 mg / ml in acidified methanol . For monitoring cell proliferation with vluc, we refreshed the media of cells 4 hours before measurements to avoid accumulation of the reporter . For vluc in vivo imaging, mice were injected iv (unless otherwise noted) with 4 mg / kg body weight (diluted in phosphate - buffered saline), and imaging was performed immediately . For sequential imaging of all three reporters, we imaged fluc on day 1, gluc on day 2, and vluc on day 3, allowing 24 hours waiting period between the different imaging sessions . Imaging was performed using an ivis spectrum optical imaging system fitted with an xgi-8 gas anesthesia system (caliper life sciences, hopkinton, ma). Data analysis for signal intensities, and image comparisons were performed using living image software (caliper life sciences). To calculate radiance for each animal, regions of interest were carefully drawn around each signal, which is expressed as radiance (photons / second / cm / steradian). The functionality of the aav - strail vector was tested by transducing u87 cells with 10 gc / cell with aav2-strail or a negative control vector aav2-gfp . Three days later, media was harvested from all wells and a quantikine human trail elisa (r&d systems, minneapolis, mn) was performed as per the manufacturer's instructions . Data presentation and calculations . For experiment in figure 1a, we calculated total relative light unit (rlu) in media and cell lysate as follows: (rlu / volume assayed) total volume in cell lysate or media . To calculate the percentage of secreted vluc, we used the following equation: (total rlu in media)/(total rlu in media + total rlu in cell lysate) 100 . To calculate gluc flux in figure 3f, we first divided fluc values (tumor size) for each mouse to the lowest fluc value of the three mice (arbitrarily set to one). Next the gluc values were divided by each of the adjusted values to get the gluc flux, which was adjusted for tumor size . Statistical analysis . Statistical analysis was performed using graphpad prism version 5.01 software (la jolla, ca). For comparisons between two samples, an unpaired two - tailed t - test was performed . A p value of <0.05 was considered to be statistically significant . For analysis between multiple groups, a one - way analysis of variance was performed followed by a bonferroni's multiple comparison test to compare two groups . U87 glioma cells are moderately sensitive to killing by conditioned media from donor cells transduced with aav - strail u87 glioma cells are moderately sensitive to killing by conditioned media from donor cells transduced with aav - strail . Click here for additional data file.
We recently proposed pathological parameters of renal lesions observed in anti - neutrophil cytoplasmic antibody (anca)-associated vasculitis (aav) patients . The purpose of this proposal was (1) standardization of pathological findings in aav should be authorized in japan; (2) comparison with the european vasculitis study group (euvas) standardization should be available; and (3) pathological parameters correlated with specific clinical findings should be evaluated (table 1). As a result, the pathological parameters selected were almost compatible with those selected by euvas except for the collapse of glomeruli as the chronicity parameter; however, further evaluation using these parameters to investigate potential markers for the probability of end - stage renal disease (esrd) is needed.table 1pathological parameters nominated for evaluation of active and chronic lesion in anca - related vasculitis in japan (comparable with euvas) glomerular lesion no . Of normal glomeruliactive lesionchronicity lesion mesangial proliferation sclerotic lesion endocapillary hypercellularity global sclerosis tuft necrosis segmental sclerosis cellular, fibrocellular crescent formation fibrous crescent <50% <50%> 50%> 50% rupture of bowman s capsule adhesion collapse tubulointerstitial lesion active lesionchronicity lesion tubulitis atrophic tubule disruption of tubular basement membrane interstitial fibrosis interstitial cell infiltration granulomatous lesion peritubular capillaritis vascular lesions active lesionchronicity lesion necrotizing arteriosclerosis endoarteritis cell infiltration thromboembolism granulomatous lesion parameter not nominated in euvas pathological parameters nominated for evaluation of active and chronic lesion in anca - related vasculitis in japan (comparable with euvas) parameter not nominated in euvas among the parameters listed above, the number of normal or sclerotic glomeruli was proved substantially to be a prognostic indicator of renal outcome in accordance with basal renal function [24]; however, no sufficient consensus exists regarding the pathological classification . Recently, using some of the glomerular parameters, an international working group of renal pathologists proposed a new histopathological classification of glomerulonephritis (gn) in aav with four categories (focal, crescentic, mixed and sclerotic), corresponding to the severity of renal function loss in this order during a 5-year follow - up . As the evaluation was performed in 100 cases, consisting of 39 cases of granulomatosis with polyangiitis (gpa) and 61 cases of microscopic polyangiitis (mpa) in 32 centers in 9 european counties, the influence of the relatively mixed races and disease types could not be excluded . In japan,> 90% of anca - positive gn is diagnosed as mpa, in which renal involvement is more frequent than in gpa, as previously reported . In this study, we evaluated the predictive potential of this newly proposed categorization in myeloperoxidase (mpo)-anca - dominant mpa patients in japan . Eighty - seven patients with primary systemic vasculitis, in accordance with the chapel hill consensus criteria, diagnosed and treated from 2001 to 2010 in three centers (kitano hospital in osaka, tokyo women medical college in tokyo and shimoshizu national hospital in chiba) were analyzed . In all cases, hematoxylin and eosin, methenamine silver, periodic acid - schiff, and masson trichrome staining were used for evaluation . The histological categorization based on glomerular lesion was performed following berden s group focal 50% normal glomeruli, crescent 50% of glomeruli with cellular crescents, sclerotic 50% of glomeruli with global sclerosis, and mixed <50% normal, <50% crescentic, <50% globally sclerotic glomeruli . Renal and life survivals were analyzed at onset, 6 months, 1 year and 5 years after renal biopsy in available patients (87 at onset and 6 months, 84 at 1 year, 78 at 5 years). Median age was almost identical to the european study; however, males were dominant in japan in contrast to a slight female dominance in europe (table 2).table 2comparison among evaluations of gn histological categories with clinical background in europe, china and japaneuropean japanchina patients (number)10087121centers (number)3231median age (range)62.6 (2080)63.0 (1785)57.2 (1581)male to female (number)54:4637:5064:57clinical diagnosis (%) gpa39 (39)049 (40.5) mpa61 (61)87 (100)68 (56.2) renal - limited vasculitis004 (3.3)anca test (indirect immunofluorescence or elisa) pr3-anca45013 mpo - anca4776108 anca()200 missing3110median number of glomeruli per biopsy (range)14.8 (1049)26.5 (1098)25.7 (ns)pathological classification number (%) focal16 (16)40 (46.0)33 (27.3) crescentic55 (55)7 (8.0)53 (43.8) mixed16 (16)26 (29.9)24 (19.8) sclerotic13 (13)14 (16.1)11 (9.1)serum creatinine (mg / dl) focalns1.51 1.492.22 1.90 crescentic2.42 1.675.01 2.73 mixed3.37 3.173.86 2.69 sclerotic7.52 4.928.51 3.42death at 1-year follow - up25/10011/84nsrenal survival at 1-year follow - up focal, crescentic, mixed, sclerotic (%) 93, 84, 69, 50100, 86, 96, 35100, 73, 83, 29renal survival at 5-year follow - up focal, crescentic, mixed, sclerotic (%) 93, 76, 61, 50100, 86, 96, 29nsdata of three patients were lost due to transfer to different hospitals before 1-year follow - up ns not shown in the report comparison among evaluations of gn histological categories with clinical background in europe, china and japan data of three patients were lost due to transfer to different hospitals before 1-year follow - up ns not shown in the report all cases in japan had mpa; mpo - anca was positive in 76/87 (87.3%). The median glomerular number was 26.5 in japanese samples . At 6 months follow - up, 11 patients reached esrd and a further 8 patients had died . At 1-year follow - up, no more patients had reached esrd and a total of 11 patients had died . At 5-year follow - up, 18 patients had died and another 12 patients had reached esrd . In japanese patients, almost half of the cases were categorized as focal (40/87; 46.0%) with 14/87 (16.1%) as sclerotic . Of the other 32 cases, only 7 (8.0%) were categorized as crescentic, with the remaining 26 cases (29.9%) being classed as mixed . As shown in fig . 1, the kaplanmeier curve at the 5-year follow - up showed no increase of probability to esrd in focal cases and a low increase in mixed cases; however, this increased with the ascending categories of crescentic and sclerotic gn.fig . 1renal survival (no development of end - stage renal failure) according to the four histologic categories in japanese cohorts renal survival (no development of end - stage renal failure) according to the four histologic categories in japanese cohorts the predictive value and reproducibility of this new classification from japan, europe and china were compared in a recent report . As shown in table 2, among the 100 respective patients (32 centers; europe), 121 (1; china) and 87 (3; japan), the gpa: mpa ratio was similar between europe and china (39:61 and 49:64) in contrast to all mpa (0:87) in japan . On the other hand, for serum anca positivity, mpo - anca positivity was dominant in china (89.1%) and japan (87.4%) compared to europe (45%), where there was relatively high pr3-anca positivity (47%) compared with china and japan (10.7 and 0%, respectively). The average numbers of glomeruli per case were significantly higher both in japan (26.5) and china (25.7) than in europe (14.8). The distribution of the four histological categories of gn were similar in europe and china with crescentic cases being dominant (55 and 47%, respectively), whereas in japan, the number in this category was significantly lower (8.0%). The probability of developing esrd increased with the ascending categories of focal, crescentic, mixed, and sclerotic in europe, and focal, mixed, crescentic and sclerotic in china . In japan, as mentioned above, there was no increase of probability to esrd in focal and mixed, but there was a high increased in sclerotic, as in europe and china . Median age was almost identical to the european study; however, males were dominant in japan in contrast to a slight female dominance in europe (table 2).table 2comparison among evaluations of gn histological categories with clinical background in europe, china and japaneuropean japanchina patients (number)10087121centers (number)3231median age (range)62.6 (2080)63.0 (1785)57.2 (1581)male to female (number)54:4637:5064:57clinical diagnosis (%) gpa39 (39)049 (40.5) mpa61 (61)87 (100)68 (56.2) renal - limited vasculitis004 (3.3)anca test (indirect immunofluorescence or elisa) pr3-anca45013 mpo - anca4776108 anca()200 missing3110median number of glomeruli per biopsy (range)14.8 (1049)26.5 (1098)25.7 (ns)pathological classification number (%) focal16 (16)40 (46.0)33 (27.3) crescentic55 (55)7 (8.0)53 (43.8) mixed16 (16)26 (29.9)24 (19.8) sclerotic13 (13)14 (16.1)11 (9.1)serum creatinine (mg / dl) focalns1.51 1.492.22 1.90 crescentic2.42 1.675.01 2.73 mixed3.37 3.173.86 2.69 sclerotic7.52 4.928.51 3.42death at 1-year follow - up25/10011/84nsrenal survival at 1-year follow - up focal, crescentic, mixed, sclerotic (%) 93, 84, 69, 50100, 86, 96, 35100, 73, 83, 29renal survival at 5-year follow - up focal, crescentic, mixed, sclerotic (%) 93, 76, 61, 50100, 86, 96, 29nsdata of three patients were lost due to transfer to different hospitals before 1-year follow - up ns not shown in the report comparison among evaluations of gn histological categories with clinical background in europe, china and japan data of three patients were lost due to transfer to different hospitals before 1-year follow - up ns not shown in the report all cases in japan had mpa; mpo - anca was positive in 76/87 (87.3%). The median glomerular number was 26.5 in japanese samples . At 6 months follow - up, 11 patients reached esrd and a further 8 patients had died . At 1-year follow - up, no more patients had reached esrd and a total of 11 patients had died . At 5-year follow - up, in japanese patients, almost half of the cases were categorized as focal (40/87; 46.0%) with 14/87 (16.1%) as sclerotic . Of the other 32 cases, only 7 (8.0%) were categorized as crescentic, with the remaining 26 cases (29.9%) being classed as mixed . As shown in fig . 1, the kaplanmeier curve at the 5-year follow - up showed no increase of probability to esrd in focal cases and a low increase in mixed cases; however, this increased with the ascending categories of crescentic and sclerotic gn.fig . 1renal survival (no development of end - stage renal failure) according to the four histologic categories in japanese cohorts renal survival (no development of end - stage renal failure) according to the four histologic categories in japanese cohorts the predictive value and reproducibility of this new classification from japan, europe and china were compared in a recent report . As shown in table 2, among the 100 respective patients (32 centers; europe), 121 (1; china) and 87 (3; japan), the gpa: mpa ratio was similar between europe and china (39:61 and 49:64) in contrast to all mpa (0:87) in japan . On the other hand, for serum anca positivity, mpo - anca positivity was dominant in china (89.1%) and japan (87.4%) compared to europe (45%), where there was relatively high pr3-anca positivity (47%) compared with china and japan (10.7 and 0%, respectively). The average numbers of glomeruli per case were significantly higher both in japan (26.5) and china (25.7) than in europe (14.8). The distribution of the four histological categories of gn were similar in europe and china with crescentic cases being dominant (55 and 47%, respectively), whereas in japan, the number in this category was significantly lower (8.0%). The probability of developing esrd increased with the ascending categories of focal, crescentic, mixed, and sclerotic in europe, and focal, mixed, crescentic and sclerotic in china . In japan, as mentioned above, there was no increase of probability to esrd in focal and mixed, but there was a high increased in sclerotic, as in europe and china . The histopathological findings of aav in the kidney are considered to show a variety of lesions, of which crescentic and/or focal necrotizing gn as well as small - vessel arteritis are the most prominent . In addition to the baseline laboratory data concerning renal lesions such as hematuria, proteinuria and decreased estimated glomerular filtration rate with systemic inflammatory signs such as c - reactive protein and organ involvement symptoms such as hemoptysis, renal histological findings have been expected to give highly reliable information not only to select the treatment protocol but to predict the outcome at baseline . Trials for the global standardization of active and chronic pathological parameters specifically in aav have been performed not only in euvas but also in japan, where a higher prevalence of mpa than euvas has been recognized, although the aav prevalence itself is almost the same . As shown in table 1, almost all parameters are common in euvas selection, so our japanese standardization of clinicopathologically critical parameters in aav seems to be globally fulfilled . The new classification of gn into four categories (focal, crescentic, mixed, sclerotic) by selecting some of the parameters of berden et al . The significantly lower frequency of crescentic and relatively higher frequency of focal cases were noted; this might be partly attributed to the earlier intervention of renal biopsy after discovering a urinary or renal function abnormality in japan . The relatively low creatinine level of the focal group in japan compared with that of the same group in china might support this tendency . As the progression of renal injury tends to be different between mpa and gpa, comparisons should be performed only between mpa in europe and in japan . This was not possible in this classification study because there were no data on the ratio of mpa in the crescentic group in europe . In this study, the kaplan this indicates that the prognosis of this group is attributed to additional pathological parameter such as tubulointerstitial or vascular lesions nominated previously in europe and japan . At present, at least for mpa - oriented cohorts in japan, this classification only by glomerular parameters might be insufficient to predict the probability of progressing to esrd . The similarity of the gpa / mpa ratio between europe and china in contrast to that of mpo - anca dominancy between japan and china indicates that many gpa are mpo - anca - positive in china, as chinese authors have stated . The gpa dominancy might be attributed partly to the localization of the center at a high latitude, which has been reported to be related to the high prevalence of gpa . Although the numbers in the four categories were similar between europe and china, there was a difference in the order of the increase of probability of progressing to esrd between mixed and crescentic . The significantly more favorable prognosis of mixed than crescentic in china is similar to japan, where both focal and mixed rarely showed progress to esrd . In conclusion, the mixed group in the new classification has high heterogenicity of histological activity and chronicity, which shows the insufficiency of this classification for prediction of the probability of progressing to esrd . Re - evaluation of the predictive value by adding other parameters such as interstitial or vascular lesions for mpa - oriented cohorts is expected.
Intraluminal papillary tumors of extra- or intrahepatic bile ducts generally show high- or low - grade dysplasia or are well - differentiated adenocarcinomas . These types of tumors are not infrequently associated with superficial spread of carcinoma cells along the biliary mucosa without invasiveness . Approximately 10% of hilar bile duct carcinomas are of the papillary type while approximately 15% of intrahepatic cholangiocarcinomas are of the intraductal growth type . This type of biliary papillary tumor shows less invasiveness and more favorable outcome compared with other types of intrahepatic cholangiocarcinomas and not infrequently presents as multifocal papillary epithelial lesions in the bile duct with or without mucin production . Surveyed such cases in taiwan and also in japan, and they proposed the collective term of intraductal papillary neoplasm of the bile duct (ipnb) for such biliary papillary tumors . Interestingly, the clinicopathological features of ipnb resemble those of intraductal papillary mucin - producing neoplasia (ipmn) of the pancreas [8, 9, 10]. Although ipnb is considered to be of relatively low - grade malignancy [8, 11], the clinical spectrum and characteristics of ipnb itself including postsurgical prognosis have not been fully evaluated . In this report, we present a case of ipnb with subserosal invasion by the component of poorly differentiated carcinoma . Here we describe a case of ipnb in a 77-year - old male with liver dysfunction and diabetes . The patient had no history of liver diseases such as viral hepatitis or alcoholic liver disease . Increased alkaline phosphatase level was found during the follow - up of diabetes . When examining abdominal images, biliary tumor was observed and, therefore laboratory data showed mild obstructive jaundice as 1.5 mg / dl of total bilirubin, 49 iu / l of alanine aminotransferase, and 663 indocyanine green retention rate at 15 min was increased with 27.4% (normal range <l10%) and the liver uptake ratio by tc - galactosyl human serum albumin liver scintigraphy was decreased with 0.88 (normal range> 0.9), which indicated injured liver function by biliary obstruction . Serum level of ca19 - 9 was highly increased with 124 u / ml (normal range <37 u / ml). Computed tomography (ct) showed a tumor of the bile duct 3 cm in size extending to the hepatic hilum (fig . The bile duct in the right lateral sector was obstructed by tumor invasion and the portal branch in the same sector was stenotic . We performed right hepatectomy and combined resection of the common hepatic and bile duct and a regional lymphadenectomy was added . Papillary growing soft tumor was observed in the upper and middle bile duct, and the right lateral glisson's pedicle and the cystic duct were obstructed by the tumor invasion (fig . The hepatic duct in the right paramedian sector and the left hepatic duct were remarkably dilated without obstruction and the intraductal lumen showed normal appearance in the resected specimen . Histologic finding showed remarkably papillary adenocarcinoma in the layer of the intraluminal side (fig . 3a), which was superficially extending (2 cm from the main tumor) to the epithelium of the intrahepatic duct . In the subserosal layer, the tumor transformed to poorly differentiated adenocarcinoma which obstructed the bile ducts in the right lateral sector (fig . Slight perineural, lymphatic and venous infiltrations were observed; however, there was no lymph node metastasis . Immunohistochemical analysis showed positive expression of muc1, muc4 and muc5ac, negative expression of muc2 and p53 overexpression . By pathologic consultation, the tumor was diagnosed as an invasive bile duct carcinoma arising from ipnb, pacreatobiliary type . Ipnb may evolve through a common pathological process and shares clinicopathological features with pancreatic cystic neoplasms such as ipmn . Helical ct and mr imaging can identify dilation and cystic changes in biliary and pancreatic ducts in asymptomatic patients [9, 12], enabling diagnosis of malignancy at an earlier stage and providing reliable patient follow - up . Since the mechanism of carcinogenesis of pancreatic cystic neoplasms has been clarified by advances in histological and molecular diagnostic techniques, it is now clear that intraepithelial pancreatic neoplasia is the microscopic precursor lesion of pancreatic carcinoma, and ipmn or mucinous cystic neoplasm are macroscopic precursor lesions . The diagnostic algorithm for pancreatic cystic tumors is designed to distinguish benign from malignant lesions [9, 13]. In bile duct carcinomas, on the other hand, intraepithelial biliary neoplasia or ipnb are thought to be similar precursor lesions, and the molecular characteristics of carcinogenesis have been clarified as well [14, 15]. However, in our experience, most ipnb contained carcinomatous lesions, even the small cystic lesions, and mucin production was observed in half of the cases (unpublished data), which was different from ipmn . Since the similarities and differences between ipnb and ipmn have not been fully clarified yet, the diagnostic or treatment algorithm for ipnb remains to be established at this stage . When ipnb is detected, most cases should be scheduled for surgical resection based on their malignant potential . We have experienced 7 patients of ipnb including the present case; 4 presented abdominal pain or discomfort due to biliary obstruction . However, there has been an increase in the number of reported cases of biliary neoplasms of the hepatobiliary system characterized by marked dilatation of the bile ducts or cystic biliary lesions with or without mucin secretion . The number of patients who undergo surgical treatment for such lesions has also increased due to diagnostic and surgical improvements along with development of imaging modalities . Advanced imaging modality can identify dilation and cystic changes in the biliary duct at an early stage in such cases . Yeh et al . Reported the reliable classification of cholangiographic types according to the histopathological types of ipnb . According to this imaging classification, the cholangiographic pattern of the present case was type iiia (intrahepatic polypoid or cystic neoplasia with operable concomitant malignancy), in which the prevalence of microinvasive adenocarcinoma and papillary adenocarcinoma with stromal invasion wase dominant by yeh's classification . The present case showed a papillary adenocarcinoma with stromal invasion . Therefore, their morphological classification using an image diagnostic tool may be reliable to identify malignancy of ipnb . The onset of finding in the present case was elevated alkaline phosphatase level, which might be reliable to find development of biliary carcinoma . The previous report recommended hemihepatectomy or extended hemihepatectomy for cases with extending tumor or diffusely dilated bile ducts . In the present case, we also selected right hepatectomy for complete curable resection because the tumor seemed to extend to the confluent of hepatic duct in the right paramedian sector by ct and superficial spreading of the tumor along the biliary epithelium is also a characteristic feature of ipnb . In the present case, 2 cm of superficial extension along the epithelium to the hepatic duct was observed as expected . It is important to decide on major hepatic resection in such cases because of diffuse dilatation of the bile duct extending in a segment or lobe and multifocal carcinogenesis . However, in the present case, the infiltrative portion showed poorly differentiated adenocarcinoma and, therefore, it is necessary to consider the malignant potential even in ipnb with papillary growth . Since similarity between ipmn and ipnb has been considered and prognosis of patients with invasive ipmn is very poor, similar to those with pancreatic ductal adenocarcinoma, patients with invasive ipnb should be carefully monitored and considered to have similar prognosis to those with cholangiocellular carcinoma . The expression of muc1, muc2, or muc5a might correlate with malignant transformation from ipnb to invasive or mucinous carcinoma of the bile duct . In our case, the expression of muc2 and p53 was negative, and this finding might be according to the previous report [23, 24]. Further studies of cellular characteristics such as protein expression or genetic alterations will be necessary to clarify the pathogenesis of ipnb . Ipnb or intraductal papillary growth type of intrahepatic cholangiocarcinoma is a good indication for surgical resection, and complete resection is associated with better prognosis compared with other cholangiocarcinomas . In the present case, lymph node metastasis was not observed despite deep invasion with the microscopic infiltration of cancer cells in the surrounding tissues . The recent report by yeh et al . Revealed poor prognosis in patients with lymph node metastasis (the 5-year survival rate was 14%). To accomplish good prognosis after treatment for ipnb, diagnosis at the earliest stage followed by curative resection is necessary . In conclusion, we report the clinicopathological findings in a case of ipnb with stromal invasion who underwent surgical resection . The malignant potential of ipnb is usually lower than that of other bile duct carcinomas; however, it is noted that invasive transformation might sometimes occur even in ipnb . Surgical complete resection is a better therapeutic choice for ipnb to obtain the good prognosis.
Spare parts surgery means use of parts from non - salvageable digits in replantation or cross replantation (amputated non - replantable digits used to reconstruct thumb). It is a well - established procedure in microsurgery . Here, we present a case of harvesting double free flaps from an amputated non - replantable lower limb for the contralateral limb . This is based on the gillies principle, which states -do not throw away any tissue . This opportunity does not come very often in the clinical practice and one must be ready to grab it when it does! To the best of our knowledge, there are a very few reported cases, where double free flaps from the amputated limb or digit has been used for spare parts surgery . A 45 year old male patient who was run over by a train resulting in a right leg amputation at the level of the knee and a crush injury of the left foot . The right lower limb had a severe comminution and bone loss at the knee joint, with the loss of skin and soft- tissue and crushing of muscle above and below the knee [figures 1 and 2]. The left forefoot was completely degloved and all the toes were crushed and degloved as well [figures 3 and 4]. Amputated right lower limb right lower limb amputation stump crushed left foot - dorsal aspect crushed left foot - plantar aspect the right lower limb was deemed not replantable as the knee joint was severely damaged and not salvageable, in addition, debridement of crushed and devitalized tissues would result in a 15 - 20 cm shortening and a limb that was at least 15 cm short with fused knee joint would not be functionally useful and primary insertion of prosthetic knee joint was not considered to be feasible by the attending orthopaedic surgeon . Focus was then shifted to the crushed left foot with a view to perform immediate debridement and early soft - tissue cover, to salvage as much of the foot as possible and get it fully healed and weight bearing at the earliest . Stable and sensate skin cover were vital, as this would be the only surviving foot . A large defect such as this would require a large distant flap or even two flaps . Best replacement for skin on the foot was anatomically identical skin from the opposite foot, which in this case was provided by the well - preserved amputated limb . The general condition of the patient was stable and he had no other life - threatening injuries . Immediate double free tissue transfer from the amputated limb was done . A plantar flap based on the posterior tibial vessels [figure 5] and a dorsal flap based on the dorsalis pedis vessels [figure 6], with the communication between the dorsal and plantar systems left intact through the deep branch of the 1 dorsal metatarsal artery . Satisfactory inset of dorsal flap [figure 7], and of plantar flap [figure 8] was achieved . Post - operatively the patient developed a hematoma below the dorsal flap . Since flaps were harvested from the amputated limb, small blood vessels that could not be seen were probably left unligated . Re - exploration and evacuation of hematoma was performed . Harvest of flap from plantar aspect harvest of flap from dorsum inset of plantar flap long - term follow - up - dorsal flap long - term follow - up - plantar flap for years surgeons have been amputating limbs at a higher level in limb crush injuries, to ensure good skin and soft - tissue cover for the stump and to make the stump more appropriate for prosthesis fitting . Covering the stump with vascularised tissue using the microvascular techniques from another part of the body has now become common . In crush injuries of the foot, very often, early free tissue cover to replace the lost skin and soft - tissue can avoid an amputation . This will however require surgery on another part of the body, with additional trauma and scarring . Spare parts surgery, transferring tissue from the amputated limb using microvascular techniques, is one step ahead because it uses tissue that would otherwise have been discarded, without any additional scarring and other such sequelae . Since such opportunities are rare and have to be grasped immediately, we need to constantly be aware of this possibility and ready to use it to maximise patient benefit . If the patient is not haemodynamically stable, the harvested flap can be stored by wrapping in a saline soaked gauze in a sterile container and placing the container in a box of ice or cold water as per the same guidelines as for an amputated limb for replantation . Recommended ischaemia times for reliable success with spare part surgery are parallel to that recommended in replantation i.e., 12 h of warm and 24 h of cold ischaemia for harvesting flaps from amputated digits and 6 h of warm and 12 h of cold ischaemia for harvesting flaps from major amputations . Use of skin preservation solutions to preserve the skin flaps has been tried in an animal model . Furthermore occurrence of haematoma below the flap in spare part surgery is a realistic complication resulting from inability to achieve haemostasis during the harvest of the flap from an amputated achieving haemostasis is then a must after perfusion of the flap lest the flap bleeds after perfusion resulting in a haematoma post - operatively.
The most important goal of rehabilitation for a lower limb amputee is the independent performance of daily activities through restoration of functional walking ability . Miller et al . Reported that 52% of prosthesis users experienced falls and 49% of amputees have a fear of falling . It also said that the fear of falling limits the daily movement of amputees and causes further deterioration of the balance ability required for walking1 . Therefore, physical therapy for lower limb amputees should be focused on the prevention of falls during movement / walking for the improvement of everyday activities . For this purpose, it is necessary to properly evaluate the balance ability of amputees and to perform training to improve the balance ability based on the results of that evaluation . The objectives of the present study were to elucidate what type of compensatory movement and postural strategy are used by lower limb amputees for posture control during the period from prosthesis fitting to hospital discharge by analyzing the muscle activity patterns of the lower limb muscles and to elucidate the changes over time in the muscle activity pattern during the course of physical therapy . Elderly patients also have a higher risk of falling than younger patients because of biomechanical, physiological, and psychological differences . However, no study has yet focused on comparing time - dependant changes in muscle activity patterns between younger and elderly patients . Therefore, the other purpose of this study was to reveal the differences in muscle activity patterns during standing between younger and elderly patients . A young male, 34 years old, and an elderly female, 84 years old, who had a lower limb amputated were the subjects . Neither of the patients had a history of orthopedic or neurological disease in the unaffected lower limb or the trunk . A patient who undergoes below - knee amputation due to diabetes has a normal deep sensation in the unaffected ankle joint . In both of our cases, the fitting between the socket and the stump on wearing was well adjusted with no pain in the stump . The prosthesis used by the lower limb amputees was a total surface bearing liner type and a sach foot (table 1table 1 . Characteristics of the subjectssubjectssexage (year)height (cm)weight (kg)amputation sitesocketankle jointcauseweek to start pt after amputationweek to wear prosthesis after amputationweek to discharge after wearing prosthesis for the first timeyoungm3317572right bkatsbsachtumor174elderf8515045left bkatsbsachdiabetes1514 m: male, f: female, bka: below knee amputee, tsb: total surface bearing, sach: solid ankle cushion heel; j - foot m1170, pt: physical therapy). Surface electromyography (emg) measurements were performed of the gastrocnemius medial head (ga) of the unaffected lower limb . The electrodes were placed on the most prominent bulge of the ga muscle following seniam guidelines . After sufficient treatment with skinpure to reduce the inter - electrode resistance to no more than 10 k on the skin, disposable electrodes (blue sensor, nf-50-k / w, ambu) were attached at an inter - electrode distance of 2 cm . The emg signals were amplified (to 1.0 mv) and band - pass filtered between 201,000 hz using a synact mt-11 (nec corp ., tokyo, japan). The emg signals were digitized using a waveform analysis system, power lab 16s (ad instruments ltd, hasting, uk), at a sampling frequency of 1,000 hz, and stored on a personal computer for off - line analysis . After data input, waveform processing was performed using waveform analysis software, chart36.8, ad instruments . Two different tasks of maintaining the static standing position were performed on the floor . The test subjects were asked to stand on the floor with their feet shoulder - width apart and look at a point set at 1 m distance to the front at the height of the eye for 10 seconds . The first task, reference task, was to maintain upright standing position on the prosthesis while holding parallel bars . The second task, experimental task, was forward weight shift while maintaining the standing position without grasping the parallel bars . The recorded data were smoothed using the root mean square (rms) value of every 100 msec using waveform analysis software . The reference contraction is judged to be reliable by being reproducible, giving approximately equal results within subjects over trial . Therefore, we used the emg of another task as a reference contraction for emg normalization . The normalized level of% rmsemg was determined using the following formula . M: male, f: female, bka: below knee amputee, tsb: total surface bearing, sach: solid ankle cushion heel; j - foot m1170, pt: physical therapy% rmsemg = experimental task rmsemg (second task) / reference task rmsemg (first task) 100% maximum walking speed was measured in 10 m walking . Depending on the condition at the time of the measurements, subjects were allowed to use a crutch, or a single cane on the unaffected side while walking . The surface muscle activity and the walking speed were measured once a week, a total of four occasions, starting on the day of prosthesis fitting (first week). This study was conducted after approval was obtained from the ethics committee of tohoku university graduate school of medicine . The study patients were sufficiently informed about the purpose and methods of the study, and their consent to participation was obtained . When the time - dependent changes in muscular activity of ga, which is the most active muscle in the standing position, were investigated in the forward weight shift task, two unique patterns were observed . For the younger subject, no muscle activity of ga was observed in the standing position tasks in the first week, but the amount of muscular activity of ga gradually increased over time . On the other hand, for the elderly subject, increased activity of ga was observed in the forward weight shift task in the first week, when the prosthesis was worn for the first time, followed by a gradual decrease in activity over time (table 2table 2 . Time shift of activity in ga (% rmsemg) and walking speed (m / min)week%rmsemg (%) mws (m / min)youngerelderlyyoungerelderlyfirst0.6618.4348.5815.12second4.7116.4873.8924.57third5.9812.6580.3224.33fourth8.855.4110043.17%rmsemg:% root mean square electromyography, mws: maximum walking speed). The maximum walking speed gradually increased over the period from when the prosthesis was worn for the first time to when the subjects were discharged from the hospital (table 2). The basic elements for the smoothest and most energy efficient walk are proper balance ability and posture control function2 . In lower limb amputation, the contralateral lower limb function influences subsequent ability to perform daily living and transfer activities, and standing balance on the unaffected side is an important determinant of whether walking with a prosthesis is possible or not3 . Reported that the muscle of the unaffected lower limb of prosthesis wearers tended to adopt a balancing strategy to compensate for the loss of the ankle joint and muscles due to amputation4 . Therefore, lower limb amputees need to adapt to asymmetrical body weight and muscular strength when they exert effort to maintain static standing while wearing a prosthesis . In the present study, we evaluated the changes in surface emg activity of the gastrocnemius during standing, and the walking speed with a prosthesis . The muscular activity of ga is most conspicuous when the amputee is standing with the prosthesis for the first time . When the static standing position is maintained, the ga is activated in preparation for the forward movement of the center of pressure5, 6 . We found two unique muscle activity patterns were exhibited by the two below - knee amputees . When the prosthesis was first worn, the younger subject was so careful about maintaining the standing posture that adequate forward movement of the center of gravity could not be achieved . For the elderly subject, however, marked activity of the ga was found, and ga activity at the final assessment was lower than that of the initial measurement, especially during the forward weight shift task . These time - dependent changes in the muscular activity of ga indicate how younger and elderly amputees adapt muscular force to maintain standing posture, and it might also be as a cause of falls by elderly patients in the early stage of standing or gait training . The subjects learned the method for shifting the center of gravity forward in a stable manner, resulting in the gradual reduction or increase of ga muscle activity . There are many factors known to contribute to the improvement of the walking speed of amputees . As far as the results of this study are concerned, it can be concluded that one important factor is regaining the ability to be able to shift the center of gravity in the static standing position through the efficient muscle activity of the unaffected lower limb . In conclusion, the trend of muscle activities over time should contribute to the development of effective rehabilitation programs . Due to the limited number of subjects included in this study, a statistical analysis of sufficient power could not be performed . Furthermore, there were obvious differences in biomechanical, physiological, and psychological attributes because of age differences . Biomechanical or physiological perspective approaches are very important . In this study, however, there was no history of disorders of the trunk, such as lordosis or kyphosis, so we only focused on the differences in emg . Further studies with larger numbers of subjects stratified by the causes and types of amputation are needed to clarify the specific factors associated with the acquisition of the ability to walk by amputees, and the development of efficient methods for physical therapy and programs.
The most common location in the alimentary tract is the oesophagus, where the tumour is responsible for most diagnoses of benign tumors of the organ . The frequency of occurrence ranges between 0.005% and 7.9% of the population [24]. It is often symptomless and is accidentally diagnosed in tests . If symptoms occur, usually it is dysphagia and pain in the epigastrium and chest [5, 6]. Depending on the size and location of the lesion patients are qualified for a particular type of surgery . At present a wide range of operations are available, from minimally invasive submucosal resections carried out during endoscopy, through surgery involving opening of the thorax, to subtotal oesophageal resection with the transhiatal method or thoracotomy . Although oesophageal leiomyoma is a benign neoplasm, its diagnostics and treatment are often a serious challenge for the whole team of physicians . Below we present a case study of a patient whose considerable oesophageal leiomyoma was both a diagnostic and therapeutic problem . A patient, aged 54, was admitted to the department of general surgery and surgical oncology, wielkopolska cancer centre, where she was to be treated for a posterior mediastinal tumour, which filled the left half of her chest . The lesion was diagnosed in routine periodic examinations at her place of work in march that year (chest radiograph). The patient had not reported any ailments which may have been related to the presence of the lesion . The patient was referred to the thoracic surgery outpatient clinic and then admitted to the thoracic surgery department, wielkopolska center of lung disease and thoracosurgery in june that year to undergo diagnostics and treatment . The patient underwent computed tomography of her thorax, where a pressed left lung lower lobe modelled on an enormous mass was described . The tumour came out of the stomach and caused concentric thickening of its wall, mainly around the body . The lesion was adjacent to the pericardium, modelling the left lower pulmonary vein, lifting the left main bronchus and pressing the basal segmental bronchi of the left lung lower lobe . The maximum size of the infiltration was 12 13 18 cm (fig . In the conclusions from the examination the radiologist suggested that the diagnosis should take a lymphoma or myosarcoma into consideration, gist being less likely . In order to obtain a histopathological diagnosis, no neoplastic cells were found; the smears had an extremely low cell count . Due to the results a transthoracic core - needle biopsy of the lesion was carried out, which brought no diagnosis, either . Its result only corresponded to relatively numerous very fine fimbriae of low cell connective tissue with individual fine vessels . Due to the lack of histopathological diagnosis and unequivocal image of ct examination the patient was qualified for exploratory thoracotomy with biopsy of the lesion . During the surgery the lesion was found to be more advanced than the ct examination indicated, because in practice, the tumour occupied the entire thoracic segment of the oesophagus and the gastric cardia, which was moved to the thorax . Part of the tumour seemed to enter the abdominal cavity through the oesophageal hiatus . A specimen collected in the thoracotomy produced the diagnosis of mesenchymal spindle cell carcinoma without distinct traits of malignancy . Later, in august 2012, the patient was admitted to the surgical department, greates poland cancer centre, pozna, poland . The patient had diagnostics supplemented with computed tomography of the abdominal cavity, which confirmed the presence of an oesophageal tumour filling the left pleural cavity . Apart from that, the examination did not reveal pathology within the organs and structures beneath the diaphragm . The patient underwent gastroscopy, which did not reveal any lesions in the oesophageal or gastric mucosa . The patient was qualified for surgery carried out by a team of surgeons and thoracic surgeons . Next, the thorax was opened on the left, in the scar after the previous thoracic surgery . The encapsulated tumour was resected by cutting off the oesophagus proximally in the upper thoracic segment and distally within the region of the gastric cardia relocated to the thorax . As was described in the ct examination of the thorax, the tumour was adjacent to the pericardium and the lower lung lobe base, which required careful dissection of the lesion . The resected thoracic segment of the oesophagus passed right through the middle of the tumour . The alimentary tract was reconstructed by means of a gastric prosthesis, anastomosing it on the left side of the neck to the stump of the cervical segment of the oesophagus . On the 11 day after the surgery the patient was discharged from hospital with a recommendation of a follow - up examination at the surgical oncology outpatient clinic . The histopathological image based on the results of immunohistochemical examinations (sma, ki67, calp, cd 117, s100, cd 34, dog 1) corresponded to oesophageal leiomyoma . The patient remains under control of the surgical oncology outpatient clinic, where she has check - ups . Three months after the surgery no pathological lesions were found in computed tomography of the thorax ., postoperative photograph of the tumour the analysis of a group of 63 patients with the tumour, who were treated in one centre, revealed that the location in the central part of the oesophagus concerned 47.6% of the patients and the distal location 37.1% of the patients . Similarly, in another study the most frequent location in the distal segment of the oesophagus concerned 46% of the patients . In the case under study the main part of the lesion was also located in the distal segment of the oesophagus . Due to the slow growth of these tumours the vast majority of them (59%) some authors report a correlation between the size of the lesion and the occurrence of clinical symptoms . The average size of the tumour in patients with symptoms was 5.2 cm (0.8 - 14 cm), whereas in patients without symptoms the average size of the lesion was much smaller 0.4 cm (0.1 - 1.0 cm). The literature describes different diameters of resected tumours, ranging from 0.1 cm up to the maximum size of 29 cm [1, 9, 10]. In the case under study, in the final histopathological examination the lesion was 22 cm . In spite of this size the tumour did not cause any clinical ailments and was diagnosed during routine periodic examinations at work, where the patient had her chest x - rayed . The diagnosis of leiomyoma is based on imaging examinations and biopsy in order to obtain a histopathological diagnosis . By choice endoscopy and endoscopic ultrasound are the examinations where the lesions can be seen as submucosal movable masses with untouched mucosa, which enables exclusion of the malignant process . In most cases the tumours did not cause lesions in the oesophageal mucosa, which also did not occur in the case under discussion . Unfortunately, the centre treating the patient does not have a possibility to carry out endoscopic ultrasound (eus). . Apart from the eus, computed tomography and magnetic resonance are the examinations that enable diagnosis . The computed tomography image shows a mass bound to the oesophagus, without traits of lymphadenopathy, which enables the diagnosis of a benign lesion . In the case under discussion there was a relatively large discrepancy between the computed tomography description and the actual image observed in exploratory thoracotomy . The collection of specimens for histopathological diagnosis is a subject of discussion [6, 11]. Some authors stress that multiple biopsies may cause adhesions in the thorax, which later make minimally invasive resection of the lesion much more difficult and which cause weakening of the mucosa, which is of key importance in an attempt to enucleate the tumour without resection of the oesophagus [7, 12]. Besides, the collected material is usually insufficient to make a final diagnosis, especially if it is only cytological smear . At present histopathological diagnostics also includes the immunohistochemical examination, but it is necessary to have an appropriate amount of material to make the examination . In the case under study neither the attempt of a fine - needle aspiration biopsy nor core - needle biopsy resulted in diagnosis . Even when a specimen was collected during exploratory thoracotomy, the final histopathological diagnosis was made when the entire lesion was resected and a panel of immunohistochemical examinations was carried out . The choice of surgical technique depends on the size and location of the lesion and on the occurrence of clinical symptoms . Usually these lesions are accidentally diagnosed and they do not cause any clinical symptoms . Some authors do not recommend resection of the lesions . Larger lesions, over 5 cm in size, which cause clinical symptoms, have a visible growing tendency, cause ulceration and their final histopathological diagnosis is uncertain, need to be radically resected [5, 13]. So far four cases of malignant transformation of leiomyoma have been described . The tumour located in the oesophagus can be resected with a minimally invasive method during thoracoscopy . Patients whose tumours do not exceed 5 cm are qualified for minimally invasive surgery, although some authors suggest that during thoracoscopy it is possible to resect lesions sized even up to 8 cm . It may be difficult to resect smaller lesions than 1 cm, because precise location of the lesion may cause problems . Jiang et al . Described a group of 40 patients with oesophageal leiomyoma, where in two out of five patients whose tumours were smaller than 1 cm in diameter it was impossible to locate the lesions in thoracoscopy and it was necessary to apply conversion . In as many as 10% of cases with the diagnosis of leiomyoma it is necessary to resect the oesophagus . Usually this concerns larger lesions than 8 cm, which occupy the entire oesophagus and strongly adhere to the mucosa [5, 14]. By expanding growth there is a report of a case of oesophageal leiomyoma pressing the left atrium of the heart and causing symptoms of dyspnoea and fatigue . The ailments were caused by obstructed flow of blood to the left atrium and the left ventricle . Similarly, in the case under study the tumour closely adhered to the pericardium and the left lobe base, which required careful dissection of the lesion . Resection of the oesophagus with the lesion causes elimination of all ailments and prevents the occurrence of reflux, which is sometimes described in the literature after operations where only the tumour was resected . Besides, many authors note that a long - lasting pressure of the tumour causes thinning and impairment of the muscularis mucosae, which may cause healing difficulties and result in the formation of a fistula . When large oesophageal leiomyomas are treated, a possible transformation into leiomyosarcoma should always be taken into consideration . Therefore, the best procedure with large lesions is resection of the oesophagus with the lesion . Oesophageal leiomyoma, which is a benign neoplasm, is a serious diagnostic and therapeutic challenge . Smaller lesions sized 5 - 8 cm may be resected with minimally invasive methods, but lesions sized over 8 - 10 cm require precise preparation for the surgery, because resection of the oesophagus may be necessary . Combining the skills of a team of surgeons and thoracic surgeons provides an optimal therapeutic procedure to a patient with a large tumour . A complete resection of the lesion combined with resection of the oesophagus was the optimal therapeutic procedure, which led to the patient's recovery.
Hemochromatosis is a hereditary or secondary disorder resulting from iron - overload and iron - impregnation in the organs, leading to organ damage . Hereditary hemochromatosis is an autosomal recessive disease common in caucasians and caused by a gene mutation related to iron absorption in the bowels . Some case reports from korea suggest that end - stage renal disease patients with hemodialysis or patients with aplastic anemia had secondary hemochromatosis caused by repeated transfusions . Unlike hereditary hemochromatosis where iron is impregnated in parenchymal cells, repeated transfusion induced - iron - overload mainly impregnates iron into reticuloendothelial cells which has been considered to be less harmful organ failure than parenchymal iron accumulation . The authors report a case of a patient with aplastic anemia who received a total of approximately 1,400 units of red blood cells (rbcs) over 15 years . She was admitted for asymptomatic hypocalcaemia and diagnosed with secondary hemochromatosis accompanied by hypoparathyroidism, hypothyroidism, hepatic dysfunction, and cardiac dysfunction . To the best of the author's knowledge, fifteen years ago she was diagnosed with aplastic anemia, treated with antilymphocyte globulin and antithymocyte globulin, and had routine transfusions of rbcs of 2 units per week (about 1,400 units over 15 years). In addition, starting 10 years ago, selective iron - chelating agent (deferasirox 1,500 mg / day) was administered due to increased serum ferritin levels . Continued hypocalcaemia was detected in blood tests at regular follow - up visits and the patient visited the endocrinologic department as an outpatient for additional examinations and treatment . She had a blood pressure of 120/80 mm hg, pulse rate of 78 beats per minute, temperature of 36.8, and a respiratory rate of 20 breaths per minute . Chest examination showed that breathing was clear, heartbeat was regular and no unusual sounds were detected . Her abdomen was flat and soft with no unusual sounds, and the liver and spleen were not palpated . Neurological tests showed no local neurological symptoms, chvostek sign or trousseau sign . In the laboratory findings, peripheral blood test indicated a white blood cell count of 700/mm (neutrophil 150), hemoglobin level of 8.0 g / dl, and platelet count of 37,000/mm . A serum biochemical test reported blood urea nitrogen levels of 10.1 mg / dl, serum creatinine of 0.7 mg / dl, albumin levels of 3.9 g / dl, aspartate aminotransferase levels of 53 iu / l, alanine aminotransferase levels of 60 iu / l, alkaline phosphatase of 94 iu / l, and total bilirubin of 0.5 mg / dl . In a serum electrolyte test, however, total calcium was 6.7 meq / dl (albumin corrected calcium 6.78 meq / dl; normal range, 8.4 to 10.2), and ionized calcium was 1.78 serum ferritin levels were 27,583.03 ng / ml (normal range, 4.63 to 274.6), iron levels were 291 g / dl (normal range, 65 to 157), and total iron binding capacity was 389 g / dl (normal range, 256 to 426) which showed an increase . The transferrin saturation level was high to 74.8% (normal range, 22% to 46%). She was diagnosed with hypocalcaemia induced by hypoparathyroidism based on test results showing intact parathyroid hormone (ipth) levels of 31 pg / ml (normal range, 9 to 55), 25(oh)vitd of 7.03 ng / ml (normal range, 4.8 to 52.8), and 1,25(oh)2vitd levels of 36.2 pg / ml (normal range, 25.1 to 66.1). Although 1,000 mg of calcium carbonate and 0.5 g of alfacalcidol were given twice a day, hypocalcaemia continued with increased levels of 24 hours urine calcium (700 mg / day) and 24 hours urine creatinine (0.99 g / day); thiazide 12.5 mg was also administered to maintain the total calcium at 7.5 to 8.0 meq / dl and ionized calcium at 1.9 to 2.2 tests were performed to determine if the hemochromatosis had spread to other organs; chest x - rays showed no signs of cardiomegaly, but computed tomography (ct) before contrast enhancement showed thyroid (hounsfield unit 157.0) and liver (hounsfield unit 136.0), as well as a increased signal intensity associated with the cardiac muscle and enlarged inferior vena cava and hepatic veins (fig . 1). The t2-weighted magnetic resonance imaging (mri) of the abdomen revealed a dark signal intensity in the liver, spleen, bone marrow, and pancreas, leading to the secondary diagnosis of hemochromatosis (fig . Organs showing signs of iron impregnation were tested for function; the thyroid function test showed elevated levels of thyroid stimulating hormone (tsh, 17.18 iu / ml; normal range, 0.4 to 4.8) with free thyroxine levels of 1.28 ng / dl (normal range, 0.8 to 1.71), and triiodothyronine levels of 1.43 ng / ml (normal range, 0.6 to 1.6). Thyroid peroxidase antibody and thyroglobulin antibody were both negative which resulted in apparent hypothyroidism caused by hemochromatosis . The patient was receiving low - dose of thyroid hormone (levothyroxine 50 g / day), but her tsh levels were still over 10 iu / ml at follow - up . The most recent test result showed tsh levels of 8.12 iu / ml, and the dose of thyroid hormone was increased to 150 g / day for follow - up observation . Echocardiography revealed dilated left ventricular chamber size (left ventricular end diastolic diameter 54 mm) with global hypokinesia, and a moderate to severe left ventricular systolic dysfunction (ejection fraction 23%), which suggests congestive heart failure with dilated cardiomyopathy and restrictive diastolic dysfunction induced by hemochromatosis . The patient is medicated with digoxin 0.1875 mg, candesartan 8 mg, bisoprolol 2.5 mg, and spiractone 25 mg without any significant symptoms and is on follow - up observation as an outpatient . With this patient, secondary hemochromatosis occurred, induced by iron over - absorption due to repeated transfusion for aplastic anemia . While on follow - up observation, she was diagnosed with hypocalcaemia caused by hypoparathyroidism, subclinical hypothyroidism, hepatic failure, and cardiac dysfunction . Excessive iron absorption is known to cause various organ failures due to iron accumulation of the multiple organs, but it is rare to find clinically severe organ failure cases that involve secondary hemochromatosis induced by transfusion . Hemochromatosis is a genetic or secondary disorder caused by damaged cells and organ failure after iron overload within the parenchyma of the organ . Patients with symptoms and signs such as increased levels of iron concentration in the blood, iron - binding capacity, and serum ferritin concentration, hepatomegaly, pigmented skin, diabetes mellitus, heart disease, arthritis, and lower urinary tract symptoms can be diagnosed if the patient has a secondary cause that induces iron overload or if iron impregnation is confirmed by tissue biopsy - with hfe genetic mutation . Normally the human body maintains iron levels at 3 to 4 g. one milligram of iron for males and 1.5 mg for menstruating females are lost every day, but equal amounts of iron are absorbed by intestinal mucosa . However, in the case of hemochromatosis, 4 mg of iron are absorbed per day, which increases iron levels in the blood, leading to increased blood ferritin levels and iron - impregnation of the organs . Hereditary hemochromatosis was transmitted as an autosomal recessive trait associated with the hla - a locus on the short arm of chromosome 6 . Secondary factors include over - absorption of iron due to inadequate production of rbcs in cases of mediterranean anemia and sideroblastic anaemia . When 1 unit of rbc is transfused it contains 200 to 250 mg of iron . If rbc production is inadequate because of bone marrow failure such as aplastic anemia, infused iron cannot be used to produce rbcs and instead is captured by macrophages . When this iron - capturing behavior of macrophages reaches its limit, overloaded iron is impregnated into multiple organs such as the liver, heart, spleen, pancreas, and bone marrow, which results in multiple organ failures . In the early stages of hemochromatosis, fatigue or weakness can be accompanied by arthritis or skin pigmentation . Increased levels of iron impregnation in organs such as the liver, heart, pancreas, thyroid, parathyroid, and pituitary gland can cause functional failures . One of the organs commonly impregnated is the liver, and impregnation of iron in the parenchyma of the liver can cause hepatomegaly, which is associated with increased levels of hepatic enzyme and can cause of liver cirrhosis and hepatocellular carcinoma . Hepatocellular carcinoma, cardiomyopathy, conduction disturbance, and congestive heart failure can increase mortality in patients with hemochromatosis . Regular check - ups with follow - up visits are necessary to prevent these issues . Diabetes mellitus is the most common disease associated with endocrine system abnormalities, and is due to reduced insulin resistance and secretion induced by siderosis in liver and pancreas -cells . Gonadotropin deficiency commonly includes the entire pituitary hormone, resulting in impotency, menstrual irregularities, and decreased libido . Siderosis can also cause functional failures in the adrenal, parathyroid, and thyroid, although these issues are less common than diabetes or gonadotropin deficiency . There are not a sufficient number of studies on the correlation between multiple organ damage and degree and period of siderosis . In 2001, kwon et al . Reported a correlation between total volume of blood transfusion and increased serum ferritin, but there was no correlation between total volume of blood transfusions and endocrine system complications in 14 pediatric patients with repeated transfusions . Chern and lin reported that 10.7% (3/28) of patients who received blood transfusions over an extended period acquired hypoparathyroidism, which is often accompanied by other issues such as hypogonadism and diabetes mellitus . In the case study being presented here, the patient was diagnosed with hypoparathyroidism from hypocalcaemia without markedly increased ipth, and organ examinations confirmed iron accumulation to the thyroid, liver, and cardiac muscle . This indicates that hemochromatosis patients with the rare complication of hypothyroidism and hypoparathyroidism need to be tested for other organ functions and treated accordingly . In the future, larger - scale prospective studies are necessary to determine correlations between symptoms and the volume and period of blood transfusions that accompany multiple organ failures . Hemochromatosis can be correctly diagnosed if siderosis is confirmed from a liver tissue biopsy . However, as shown in this case, for a patient with low platelet levels, even with repeated platelet transfusions, an invasive examination is difficult to perform . Instead of a liver tissue biopsy, reported a correlation between blood ferritin levels and signal intensity of the liver in ct, and suggested that if serum ferritin levels are above 1,000 ng / ml . However, although ct has a high specificity (96%), its sensitivity is relatively low (63%) for diagnosing secondary hemochromatosis . Meanwhile, mri has both high specificity and sensitivity for diagnosing hemochromatosis and evaluating the treatment progress . In this case study, ct before contrast enhancement showed markedly elevated hounsfield units in the liver (136.0) compared to the normal range (61.09.0), and a t2-weighted liver mri revealed darker sections of liver, spleen, and bone marrow, which also suggest hemochromatosis . In the case presented here, hypothyroidism related to other factors was excluded since the patient had multiple functional failures of the organs caused by hemochromatosis . The ct results included elevated hounsfield units of the thyroid (157) compared to normal range (118.112.2), graves disease level of 69.517.6, and hashimoto thyroiditis of 61.49.1 . The elevated hounsfield units led to the patient's diagnosis of siderosis - induced hypothyroidism . In terms of hypoparathyroidism, previous studies also reported hemochromatosis in the study subjects . Due to difficulties with imaging and biopsy examinations, hypoparathyroidism induced by hemochromatosis was diagnosed although the patients presented with hypocalcemia without increased ipth . Patients with secondary hemochromatosis induced from repeated transfusions may have irreversible functional disorders with severe impacts or consequences on their daily life . Therefore, it is critical to restrict transfusions and iron chelate agents as needed, and schedule patients for regular follow - up visits . Furthermore, it is necessary to establish screening examinations each period, with a larger - scale study requiring repeated transfusions to clarify the effects of the amount and period of blood transfusions on endocrine failure.
Fixed implant - supported prostheses for edentulous mandibles are proved the best way to overcome the problems of traditional dentures, which enable patients to get similar bite force and comfort as natural teeth.1 however, a variety of prosthetic designs associated with implant fixed prostheses can be observed . The treatment protocol often adopted and favored by dentists is to place six parallel implants anterior to the bilateral mental foramen that are connected to one intact piece by a fixed bridge.2 several retrospective studies have demonstrated that by changing the design of the fixed restoration, the implant number can be reduced to four, and the completed mandibular fixed prosthesis can achieve the same success rate as a fixed bridge containing six implants.345 for edentulous patients, four - implant - supported fixed protocol not only can simplify the operation and reduce trauma, but also can save one - third of the cost, which is desirable for elderly patients . This specific treatment modality is called " all - on - four",6 featured by two anterior implants parallel and two terminal implants tilting distally, and a 10-tooth or 12-tooth prosthesis is built on the full - ach framework . In the all - on - four protocol, the cantilever extension is primarily determined by doctor's experience and patient's oral condition.7 santiago suggested that the minimum number of implants was four and the cantilever length of 10 mm was the safest.8 chiara9 found that if four implants were placed anterior to the mental foramen, a complete restoration of 14 teeth led to significantly increased stress levels on each implant, compared with the restoration of 12 teeth ., the safety to restore the full 14 teeth mainly depended on the length of cantilever, which could be kept in safe ranges by changing the position tilting angle and the length of the terminal implants . Experiments showed that it was possible to restore missing teeth to two teeth distal to the terminal implants,2 which was safe as long as the ratio of cantilever length to the distance between the anterior and terminal implants was equal or less than 2.10 to reduce the cantilever length, the terminal implants were usually tilted by a certain degree distally, which might be 17, 30, 34, or 45, and different tilting angles had different effects on the stress distribution.91112 however, another factor that may also influence the cantilever length has been neglected, that is the implant length . Experiments show that if the length of implant is reduced by 1/3, then the maximum length of cantilever should be shortened by 1/2.13 if the tilting angle of terminal implants is set, to ensure the implant shoulder stays at the level of alveolar crest, the practitioner must either maintain the apex of terminal implants unchanged and extend the implant length, or maintain the implant length unchanged and move the apex upward . The two methods can bring differences in the lengths of cantilevers which can exercise influence on stress distributions of the implants, periimplant bone, and framework . Whether there are some matching relations between the length and the tilting angle of the terminal implants has not yet been noticed in the literature . In present study, the terminal implants were placed in the second premolar, three - dimensional (3d) finite element analysis was used to explore the effects of different tilt angles (0, 30 and 45) and different lengths (s for standard length and l for long length) of the terminal implants on the stress distributions of implants, bone, and superstructure when a full arch fixed prosthesis was used to restore the complete set of 14 teeth in edentulous mandibles . Primary mandibular impression of an edentulous patient was obtained, and an individual tray was made . Two plaster casts were poured sequentially, which were randomly named cast a and cast b. in cast a, a lower full resin denture without buccal and lingual base was completed and this had a complete set of 14 mandibular artificial teeth, set up based on the dental arch morphology, requiring the second molar to be at least 2 mm anterior to the retromolar pad . When the denture was fitted back into cast b, it fitted closely with the cast . The lower full denture and cast b were scanned with a ct scanner (toshiba aquilion one - volume ct, with a slice thickness of 0.5 mm) to obtain dicom data . The data were imported into the itk - snap 3d medical image processing software (version 2.2.0). Based on the pixel gray value, the images were divided into two different regions: the mandible and the prosthetic upper framework . After triangular surface mesh reconstruction, the files were exported . Then, the mesh model files were imported into hypermesh version 10.0 to establish the implant - surface mesh model, according to the experimental design . The material properties used in the finite element analysis were provided in table 1 . The tetrahedral element (solid45) for the four implants to be embedded completely in the bone, implants (3.5 mm diameter and 10 mm long) were employed with the shoulder located at the same level as the alveolar crest . To simplify the model, the abutment and implant were set as an integral component and were ascribed the same parameters . The abutment was a cone with the diameter of upper surface and lower surface was 2.16 mm and 3.5 mm respectively, and the height was 3.5 mm . The framework was ascribed the gold - palladium alloy parameters,8 with the assumption that it had a complete and passive fit on the implants . The tetrahedral finite element mesh model files of the bone, framework, and implants were imported into ansys 9.0 and given boundary conditions . The mandible, framework, and implants were assumed to be continuous, homogeneous, isotropic, and linear elastic materials.91415 a 240-n load was applied to the mesiobuccal cusp of the right second molar in a direction perpendicular to the lingual slope (approximately 30 to the occlusal plane).8 four implants were employed and five 3d model groups were established . The two anterior implants (10 mm long) were parallel placed at the bilateral lateral incisor . The five groups were established based on the distal tilt angle and the length of terminal implants with its apex kept on the long axis of the second premolar . For tilt0-s group, for the other four groups, terminal implants were tilted distally by 30 and 45 relative to the long axis of anterior implants . To ensure the implant shoulder was at the level of the alveolar crest and the apex was on the long axis of the second premolar one was the same as the anterior implants, 10 mm, and the implant apex was approximately 9 mm (tilt of 30) or 7 mm (tilt of 45) distance from the alveolar crest . In the other condition, the implant apex was 10 mm distance from the alveolar crest, and the implant length was 12 mm (tilted 30) or 14 mm (tilted 45). 1 . The left side was the loading side, and from left to right, the implants were consecutively named #1, #2, #3, and #4 . 2 . The #1 implant in all of the model groups showed the greatest level of stress, followed by the order of #2> #3> #4 . The stress value of the #1 implant was approximately 3 times greater than that of #2 and 11 - 20 times greater than that of #3 and #4, and was concentrated near the neck (fig . 3). Compared with tilt0-s, the tilt30-s group and tilt30-l group exhibited similar patterns of stress variation on the four implants, with the stress value of the #1 implant reduced by 15.9% respectively; that of the #2 implant reduced by 13.6% and 16.1%, respectively; that of the #3 implant reduced by 21.8% and 26.4%, respectively; that of the #4 implant, in the tilt30-s group displayed no significant changes, in the tilt30-l group reduced by 10.3% . However, the stress changes on the implants were significantly different between the tilt45-s group and tilt45-l group . Compared with the tilt0-s, the maximum stress levels of the #1 implant in the tilt45-s and the tilt45-l were reduced by 7.6% and 32.6%, respectively; those of the #2 implants were reduced by 18.5% and 28.8%, respectively; those of the #3 implants showed the greatest reduction of 26.4% and 33%, respectively; those of the #4 implant, in the tilt45-s increased by 2.8%, in the tilt45-l reduced by 27.9% . 4 revealed that the maximum von mises stress of implant - bone interface were all located around the distal neck of the #1 implants on the loading side . . Compared with tilt0-s, the stress levels of the rest groups were reduced by 26.3%, 28.7%, 33.5%, and 36.3%, respectively . Fig . 6 indicated that the maximum stress distribution of the upper framework was entirely located in the distal cantilevers of the #1 implants . Compared with the tilt0-s, the stress levels of the tilt30-l and the tilt45-l (have extended posterior implants) were increased by 6.5% and 2.6%, respectively; by contrast, the stress levels of the tilt30-s and the tilt45-s, in which the posterior implants were the same long as the anterior implants, were reduced by 8.4% and 11.0%, respectively (fig . In order to extend the fixed implant - supported restoration success rate, four sites of the prosthesis need more attention for their stress level: the implant - bone interface,16 the implant - abutment connection, the abutment - framework connection, and the upper framework . Excessive stress on any of the four sites will cause frequent repairs, even failure of the prosthesis . To date, most of the research has focused on the implant - bone interface.9121517 excessive stress on this site would cause absorption of the peri - implant bone, leading to implant loss . Excessive stress on the implantabutment connection would cause abutment screw loosening or fracture.1819 if excessively high stress was concentrated at the connection between the abutment and the upper framework, the results would be a loosened prosthesis or a loosened or fractured screw that connected the abutment and the framework . Excessive stress on the framework (beyond the maximum yield stress of the material) would lead to the frame's break.202122 the incidence of all the biological, mechanical, and technical complications was more frequent in cantilever bridge than in single crown and splinted crowns.23 therefore, a comprehensive analysis and evaluation of stress levels at these positions in different implant configurations could provide a meaningful reference for design optimization . In the present study, under the same loading conditions, the #1 implant (which was closest to the loading point) in each group had the greatest stress . There were significant differences in the stress levels among the four implants in the same model . The maximum stress level of the #1 implant was approximately 2 - 3 times greater than that of the #2 implant and 11 - 20 times greater than the level of the #3 and #4 implants, suggesting that the differences in the stress received by the four implants were not related to implant configurations . In all five model groups, the maximum stress was located near the neck of the terminal implant on the loading side, which was consistent with previous findings.5 the position of the implant neck clinically corresponded to the implant - abutment connection and abutment - framework connection . This fact suggested that if all - on - four scheme was employed, the terminal implant abutment screw, the occlusal retention screw (when screw used for retention), or the cement (when cement was used for retention) must withstand greater stress, which should be paid more attention when prosthetic scheme was determined . The inclination angle of terminal implant and the length of cantilever have significant effects on stress distributions in implant - supported fixed prostheses.152425 fazi et al.11 found that the stress level of the implant, the bone, and the framework were related to the tilt angle, which was in the order of 34 tilt <17 tilt <0 tilt . The present study revealed that the inclination of terminal implant indeed resulted in the decrease of stress level of the implants on the loading side to various degrees . The tilt45-l group (terminal implants with the largest length and tilt angle, meanwhile the shortest cantilever length), exhibited the greatest reduction in stress on the four implants . However, the tilt45-s group, which had the largest tilting angle (greater than the tilt0-s group, the tilt30-s group and tilt30-l group) but a little shorter cantilever (10 mm) than the tilt30-s group (12 mm) and tilt30-l group (11 mm), the #1 implant exhibited less stress reduction (7.6%) compared with the tilt30-s group and tilt30-l group, whereas the stress on the #4 implant increased slightly . Therefore, the stress on implant was not reducing accordingly with the tilt angle increasing and the stress variation was not completely explained by the change of tilt angle or the cantilever length . The length of the terminal implant also affected the stress level on implants . Because the upper framework was an integral - arc symmetrical structure that connected all implants, the four implants exhibited mutual restraint in the presence of a unilateral force . In the tilt45-l group, an isosceles right triangle embedded in the mandible was formed by the implant shoulder, the implant apex, and the intersection between the second premolar' long axis and the alveolar crest . In addition, the loading direction was at a 30 angle with occlusal plane . Whether this unique stress performance was related to the unique stress interaction caused by the 45 angle and the symmetrical arc framework was unclear and worthy of further study . Based on the stress influence on the implants of the five model groups, the tilt45-l was the preferred configuration, followed by the tilt30-s . The tilt45-l group, in which terminal implant tilted distally by 45 and the length was extended to 2 times that of the anterior implant, caused a maximum reduction in the stress on all the four implants . The preservation of vertical bone around the implant was considered the key to success for implant - supported restorations.16 many factors can affect the stress distribution of the surrounding bone, including implant number and position, cantilever length, tilt angle of the terminal implants, the occlusal surface morphology of artificial teeth, and the mandibular morphology.9111526 even loading direction can change the stress distribution.15 the stress variation in peri - implant bone after distal tilting of terminal implants remained controversial . Some studies suggested that although the inclination of the terminal implant might shorter the cantilever, the implant shoulder was closer to the loading point, and greater stress was delivered to the surrounding bone.122728 roshanak12 demonstrated that configuration with the terminal implant tilted resulted in 9% increased stress at its surrounding bone . However, others reported that four - implant configurations with the terminal implant tilted distally can resulted in reduction in stress on the implants, the bone, and the prosthetic components . 112425 kim et al.17 demonstrated that distal tilting of the posterior implant by 30 reduced the bone stress by 17%, whereas bevilacqua et al.27 suggested that the stress levels in the cortical bone and the cancellous bone could be reduced by 52% and 47.6%, respectively . In addition to the tilt angle, the cantilever length could also affect the stress on the bone.15 chiara9 compared the stress in two all - on - four configurations that employed a 5 mm cantilever versus a 15 mm cantilever, and found that the maximum values of compressive stress and tensile stress in 15 mm cantilever group were significantly higher than 5 mm cantilever group . The results of the present study demonstrated that the stress at the terminal implant - bone interface exhibited various reductions after tilting distally . The tilt30-l group, the tilt45-s group, the tilt30-s group, and the tilt45-l group exhibited reductions of 26.3%, 28.7%, 33.5%, and 36.3%, respectively, compared with the tilt0-s group . This pattern of changes was not fully consistent with the cantilever length (11 mm, 10 mm, 12 mm, and 7 mm, respectively) or the implant length (12 mm, 10 mm, 10 mm, and 14 mm, respectively) of the model groups listed in table 2 . Because oblique loading was applied to the occlusal surface of the framework, we speculated that the reduction in stress level would be related not only to the reduced cantilever but also to the direction of loading and the morphology of the upper framework; moreover, a best - match relation might exist between the tilt angle and the length of the terminal implants . Of the five groups, we could clearly identify the tilt45-l group exhibited the greatest reduction in bone stress, followed by the tilt30-s group . Fazi et al.11 demonstrated that, after the terminal implants were distally tilted by 17 and 34, the maximum stress in the framework was decreased by 11% and 18%, respectively . The results of the present study indicated that the length of the terminal implants might also have an effect on the stress in the framework . Compared with the tilt0-s group, the framework stress levels of the tilt30-s and tilt45-s, which had four equal - length implants, were reduced by 8.4% and 11%, respectively, but in the tilt30-l and tilt45-l, which had extended terminal implants, it was slightly increased (by 6.5% and 2.6%, respectively). The maximum stress in the framework was located at the cantilever distal to the terminal implants, suggesting that the strength of framework at this position should be enhanced to prevent breakage . Different from chiara's opinion9, in the present study, all 14 mandibular teeth were restored, and the tilt45-l was proved to be the best protocol . Because in the tilt45-l, the terminal implants were moved posteriorly by one tooth, tilted distally by 45 and extended to 14 mm; thus, the cantilever was correspondingly reduced to 7 mm, which was similar to the cantilever length that chiara used for the restoration of 12 teeth . It was theoretically reasonable to restore the complete set of 14 teeth without adding cantilever length by increasing the tilt angle and extending the implant length . Therefore, we believe that it was possible to restore all 14 teeth using the all - on - four protocol by optimizing the design . Notably, we only conducted the simulation analysis with 3d finite element models, and a number of experimental conditions were simplified during the experiment . Additional, in - depth studies followed by clinical validation should be required for tilt45-l protocol to be applied in clinic . With four - implant - supported fixed restorations for edentulous mandibles, configurations with the terminal implants tilted distally and extended in varying degrees resulted in various reductions in the stresses on implants and surrounding bone . Matching of the tilt angle and the length of terminal implants might maximize the reduction . Under the present experimental conditions, the tilt45-l group was the preferred configuration . By optimizing the design
Cerebro - cardiovascular diseases (cvd) such as stroke and ischemic heart disease are the second leading cause of death in korea . In 2008, death rates from stroke and ischemic heart disease among koreans were 56.5 and 25.7 per 100,000 people, respectively.1) the incidence rate of stroke in korea is twice as high as that in western countries.2) the rising incidence of cvd might be related to an increase in the aging population . The older population 65 years of age is 9.9% in korea, which may increase to 14.3% over the next 10 years.1) for people above the age of 65, stroke and ischemic heart disease have been identified as the 3rd and 4th major reasons for hospitalization, respectively.3) approximately half of the patients enrolled in the korean acute myocardial infarction registry and korean stroke registry were over the age of 65.4)5) old patients with acute myocardial infarction (ami) and stroke had a markedly high incidence of in - hospital mortality and an increased length of hospital stay, resulting in higher medical expenses.5 - 7) the proportion of older people at risk of developing chronic diseases living in rural areas has significantly increased, consequently increasing the financial burden of health care expenditure on families and communities in korea.11) the current survey was conducted in jeonnam, a province with the largest proportion of elderly people in korea: 14.9% of the total population is above the age of 65, with a high proportion of elderly women who live alone.1) disparities in seeking health services due to education and income levels are leading to emerging social and health issues in korea, especially for the elderly population in rural areas.8) elderly people living in rural areas are amongst the most vulnerable populations for developing cvd . Early recognition of symptoms and prompt medical care are essential for providing appropriate treatment to prevent adverse clinical outcomes in patients with stroke or ami.9) however, the korean registry studies showed that only 20.5% of patients with acute ischemic stroke were admitted within 3 hours after symptom onset5) and 40.1% of ami patients were admitted to hospital 6 hours after the acute event.4) in particular, older adults with stroke or heart attack (ha) symptoms were admitted significantly later to the hospital than younger patients.9) this might be associated with poor recognition of symptoms and risk factors of stroke and ha as well as of required actions . In addition, ischemic stroke and ha have the same lifestyle risk factors, and public awareness of these risk factors is essential . Therefore it is important to identify the level of awareness about stroke and ha symptoms and risk factors in rural elderly people, in an effort to help them prevent and detect cvd . This study was a descriptive survey to identify the level of knowledge of stroke and ha symptoms, as well as cvd risk factors among rural elderly people . Elderly people or patients who visited 3 public health care centers and 2 medical centers located in jeonnam province were included as subjects of this study . People above the age of 60 who had at least one cvd risk factor and were able to communicate verbally were selected . Written permissions from the directors of the community health care and medical centers were obtained for the study . After agreement of a survey method at the meeting, data were collected by 5 trained research assistants, who were senior nursing students . Four hundred and fifty - one individuals participated in the survey, of which 444 provided complete datasets and were selected for further analysis . The participants were interviewed individually and the questionnaires were assisted if required . The questionnaire consisted of items pertaining to warning symptoms of stroke and ha as well as cvd risk factors identified by the american heart association.10) the structured questionnaire contained 18 items, including 9 stroke and 9 ha warning symptoms, as well as 11 items for cvd risk factors . The questionnaire included terms that have been disclosed as public information by korean medical and nursing textbooks, and were validated by two nursing professors . One point was given for each correct answer and a zero was given for each incorrect answer; the individual scores were added to obtain a total score . Dichotomous data in the kuder - richardson 20 (kr-20) formula were used to calculate the internal consistency of stroke and ha symptoms, and cvd risk factors, and the alphas were 0.80 and 0.85, respectively . In addition, the subjects were also asked what they would do if they thought someone was having a stroke or ha, and their ability to perceive the possible occurrence of a stroke or a ha was assessed . Data were analyzed using statistical package for the social sciences version 14.0 (chicago, il, usa). Demographic characteristics of the subjects and response rates to symptoms and risk factors were calculated . Chi - square, t - test, and analysis of variance were used to examine the relationship between the demographic variables and knowledge scores of symptoms and cvd risk factors . The total knowledge scores of symptoms regarding stroke and ha, and the knowledge score of cvd risk factors were normally distributed, respectively . Stepwise multiple regression analyses were used to determine the predicting factors for low levels of knowledge of symptoms and cvd risk factors . This study was a descriptive survey to identify the level of knowledge of stroke and ha symptoms, as well as cvd risk factors among rural elderly people . Elderly people or patients who visited 3 public health care centers and 2 medical centers located in jeonnam province were included as subjects of this study . People above the age of 60 who had at least one cvd risk factor and were able to communicate verbally were selected . Written permissions from the directors of the community health care and medical centers were obtained for the study . After agreement of a survey method at the meeting, data were collected by 5 trained research assistants, who were senior nursing students . Four hundred and fifty - one individuals participated in the survey, of which 444 provided complete datasets and were selected for further analysis . The participants were interviewed individually and the questionnaires were assisted if required . The questionnaire consisted of items pertaining to warning symptoms of stroke and ha as well as cvd risk factors identified by the american heart association.10) the structured questionnaire contained 18 items, including 9 stroke and 9 ha warning symptoms, as well as 11 items for cvd risk factors . The questionnaire included terms that have been disclosed as public information by korean medical and nursing textbooks, and were validated by two nursing professors . One point was given for each correct answer and a zero was given for each incorrect answer; the individual scores were added to obtain a total score . Dichotomous data in the kuder - richardson 20 (kr-20) formula were used to calculate the internal consistency of stroke and ha symptoms, and cvd risk factors, and the alphas were 0.80 and 0.85, respectively . In addition, the subjects were also asked what they would do if they thought someone was having a stroke or ha, and their ability to perceive the possible occurrence of a stroke or a ha was assessed . Data were analyzed using statistical package for the social sciences version 14.0 (chicago, il, usa). Demographic characteristics of the subjects and response rates to symptoms and risk factors were calculated . Chi - square, t - test, and analysis of variance were used to examine the relationship between the demographic variables and knowledge scores of symptoms and cvd risk factors . The total knowledge scores of symptoms regarding stroke and ha, and the knowledge score of cvd risk factors were normally distributed, respectively . Stepwise multiple regression analyses were used to determine the predicting factors for low levels of knowledge of symptoms and cvd risk factors . A total of 38.3% of the participants lived alone and 25.5% never received regular health checkups . A total of 72.5% of participants had hypertension and 28.4% had diabetes for average lengths of 7.25.9 and 9.17.8 years, respectively . Among those who had hypertension or diabetes, 18.7% reported that they no longer visited the doctor nor took any medication . A total of 18% of the participants had a family history of stroke or ischemic heart disease and 39.6% had 2 or more risk factors for cvd . In response to the question on the possibility of stroke and/or ha occurrence, 32.4% reported a possibility of stroke and 31.1% reported a possibility of a ha . In response to the question on the actions that they would take if stroke or ha symptoms occurred, 54.1% reported that they would call emergency services, and 24.3% responded that they would go to the doctor's office and call their family members (table 1 and 2). In response to structured questionnaire items on the symptoms of stroke and ha, the participants, on average, accurately identified 5.8 out of the 9 stroke symptoms and 4.3 out of the 9 ha symptoms . The average knowledge score of participants about cvd risk factors was 7.3 out of 11; 78.4% of the participants accurately identified hypertension as a risk factor, and more than 60% of the participants recognized overall cvd risk factors . However, only 47.1% recognized diabetes as a risk factor for cvd . Less than 60% of the participants recognized sudden loss of vision in one or both eyes and nausea or vomiting, as stroke and ha symptoms . Chest pain or discomfort (75.2%) and shortness of breath (70.3%) were the most widely recognized symptoms of a ha . However, less than 50% of the participants were accurately able to identify other ha symptoms (table 3 and 4). There were significant differences between gender, age, education level, monthly income, and knowledge of stroke / ha symptoms and cvd risk factors . The level of knowledge about cvd risk factors was significantly lower in the participants who had no perceptions on the possibility of having a stroke or ha than those who had not (p<0.01). However, there were no significant differences in the levels of knowledge on symptoms and cvd risk factors according to clinical characteristics such as the presence of hypertension or diabetes, the lengths of period of those risk factors, and the number of cvd risk factors . Multiple stepwise regression analyses were conducted to determine the variables that can predict the lower level of knowledge of stroke/ ha symptoms and cvd risk factors . Lower education, older age, and lower monthly income were found to be independent predictors of the low knowledge of stroke and ha symptoms . In addition, a low perceived risk of stroke or ha was a predictor of the knowledge of cvd risk factors (table 6). A total of 38.3% of the participants lived alone and 25.5% never received regular health checkups . A total of 72.5% of participants had hypertension and 28.4% had diabetes for average lengths of 7.25.9 and 9.17.8 years, respectively . Among those who had hypertension or diabetes, 18.7% reported that they no longer visited the doctor nor took any medication . A total of 18% of the participants had a family history of stroke or ischemic heart disease and 39.6% had 2 or more risk factors for cvd . In response to the question on the possibility of stroke and/or ha occurrence, 32.4% reported a possibility of stroke and 31.1% reported a possibility of a ha . In response to the question on the actions that they would take if stroke or ha symptoms occurred, 54.1% reported that they would call emergency services, and 24.3% responded that they would go to the doctor's office and call their family members (table 1 and 2). In response to structured questionnaire items on the symptoms of stroke and ha, the participants, on average, accurately identified 5.8 out of the 9 stroke symptoms and 4.3 out of the 9 ha symptoms . The average knowledge score of participants about cvd risk factors was 7.3 out of 11; 78.4% of the participants accurately identified hypertension as a risk factor, and more than 60% of the participants recognized overall cvd risk factors . However, only 47.1% recognized diabetes as a risk factor for cvd . Less than 60% of the participants recognized sudden loss of vision in one or both eyes and nausea or vomiting, as stroke and ha symptoms . Chest pain or discomfort (75.2%) and shortness of breath (70.3%) were the most widely recognized symptoms of a ha . However, less than 50% of the participants were accurately able to identify other ha symptoms (table 3 and 4). There were significant differences between gender, age, education level, monthly income, and knowledge of stroke / ha symptoms and cvd risk factors . The level of knowledge about cvd risk factors was significantly lower in the participants who had no perceptions on the possibility of having a stroke or ha than those who had not (p<0.01). However, there were no significant differences in the levels of knowledge on symptoms and cvd risk factors according to clinical characteristics such as the presence of hypertension or diabetes, the lengths of period of those risk factors, and the number of cvd risk factors . Multiple stepwise regression analyses were conducted to determine the variables that can predict the lower level of knowledge of stroke/ ha symptoms and cvd risk factors . Lower education, older age, and lower monthly income were found to be independent predictors of the low knowledge of stroke and ha symptoms . In addition, a low perceived risk of stroke or ha was a predictor of the knowledge of cvd risk factors (table 6). The participants identified 7.3 of the 11 cvd risk factors: hypertension was the most frequently identified risk factor (78.4%), followed by stress (73.0%), whereas diabetes was the least frequently identified risk factor (47.1%). These findings are consistent with earlier reports in korea which showed that subjects lacked awareness of diabetes as a cvd risk factor.11)12) people who are aware of the risk of stroke and ha may be more likely to engage in preventive practices . Therefore, community - based public education, with particular emphasis on the management of diabetes is needed, in order to prevent stroke or ha among elderly people suffering from diabetes . More than 70% of the participants identified sudden dizziness, pain on one side of the body, and trouble speaking, as stroke symptoms . This finding was also consistent with a previous report.13) the least identified stroke symptoms were sudden loss of vision in one or both eyes and nausea followed by vomiting . This finding was in agreement with previous studies that showed vision impairment to be the least identified warning symptom.14) more than 60% of the participants of this study did not identify back pain, neck / jaw pain, cold sweat, and nausea as ha symptoms . These findings were also consistent with a previous study of korean immigrants,15) as well as an additional study of korean subjects.16) considering average accurate response rates, the participants of this study reported a limited knowledge about most ha symptoms (4.3/9) except chest pain and shortness of breath, as compared to stroke symptoms (5.8/9). Previous studies have reported that atypical symptoms such as gastrointestinal or respiratory symptoms without chest pain, were the most significant factors for predicting a lower use of thrombolytic therapy, and were associated with in - hospital complications.17)18) furthermore, patients with an atypical presentation were more likely to be older, female, and have hypertension and/or diabetes.17 - 19) thus, public campaigns targeting elderly people and those with diabetes are necessary to improve their awareness of all possible symptoms including the atypical symptoms . Multiple regression analyses showed that lower levels of education and monthly income, and older age significantly predicted the lower levels of knowledge of stroke and ha symptoms and cvd risk factors . These findings are supported by previous studies.11)20) similar findings have also been reported in a review of fifteen international studies which showed that elderly people and those with lower levels of education tended to have less knowledge of risk factors and warning signs of stroke as compared to younger age groups and those with higher levels of education.21) a low level of education and old age were also shown to affect ha symptom recognition.22) in this study, 78.6% of the participants attended only elementary school and 81.3% had a monthly income of under one million won . The low levels of education and socioeconomic status were the defining demographics of our elderly population . The knowledge disparity among elderly people needs to be addressed in programs aimed at helping them recognize and take prompt treatment - seeking action through simple guidelines, when early warning symptoms appear . Participants who had never considered the possibility of having a ha had significantly lower levels of knowledge of cvd risk factors, and this finding was consistent with a previous study.23) approximately 69% of participants with one or multiple cvd risk factors did not perceive themselves to be at risk of a stroke or ha in this study . The decision to seek treatment is made based on whether the patients believe their physical symptoms to be serious and life threatening.9) public education can be an effective way to improve awareness of the risk factors for stroke and ha . A previous study reported that a 4 month educational intervention program for elderly adults with a mean age of 75 years was effective in improving awareness about stroke and ha symptoms.24) therefore, public health education for the elderly at risk of cvd should focus on improving awareness of having a stroke or ha to increase the knowledge of symptoms and risk factors of cvd, and to facilitate preventive actions . The limitation of this study was that the study subjects were recruited from only one rural province with a high proportion of elderly women and from the clients of community health care centers . Hence, these findings cannot be generalized for all elderly people living in rural areas in other provinces or for home residents . Knowledge of ha symptoms among rural elderly people in korea was lower than that of stroke symptoms . Lower education level, older age, and a lower monthly income were predicting factors for a lower knowledge of stroke / ha symptoms and cvd risk factors . In addition, a low perceived risk of ha independently predicted a low knowledge of cvd risk factors . Educational interventions are needed to increase the recognition of early warning symptoms and risk factors for cvd among rural elderly clients visiting community health care or medical centers, particularly those with low levels of education and income . Knowledge of ha symptoms among rural elderly people in korea was lower than that of stroke symptoms . Lower education level, older age, and a lower monthly income were predicting factors for a lower knowledge of stroke / ha symptoms and cvd risk factors . In addition, a low perceived risk of ha independently predicted a low knowledge of cvd risk factors . Educational interventions are needed to increase the recognition of early warning symptoms and risk factors for cvd among rural elderly clients visiting community health care or medical centers, particularly those with low levels of education and income.
Intestinal capillariasis, caused by capillaria philippinensis, was first recognized in an autopsy case occurred in ilocos norte, luzon island, the philippines . Since the first case report in the philippines, this nematode infections have been subsequently reported from other parts of asia (thailand, japan, iran, taiwan, egypt, indonesia, united arab emirates, korea, india, and lao pdr) [3 - 12], and even from non - endemic areas (south america). In the republic of korea, the first case, with severe emaciation and malnutrition due to long - lasting diarrhea, was found in 1991 by detecting worm sections from the biopsied intestine, and also by identifying the eggs in fecal samples . After the first case report, 1 indeginous (namwon - si, jeollabuk - do) and 3 imported cases (from bali in indonesia and saipan island) were added [14 - 16]. In the present study, we describe clinical and parasitological findings of an intestinal capillariasis case with protein - losing enteropathy occurred in an inland area of korea . A 37-year old man, residing in sacheon - si, gyeongsangnam - do, visited the gyeongsang national university hospital (gnuh) for his long - lasting diarrhea and general edema on july 2010 . He had been treated at a private clinic before visiting gnuh, but symptoms aggravated gradually . At that time, the total serum protein and albumin levels were 5.0 and 2.4 g / dl, respectively, urine protein was negative, and thyroid function test was normal . In routine stool examinations, clonorchis sinensis eggs were detected; therefore, we prescribed him praziquantel . Ten months later, he admitted to our hospital because of persistent diarrhea, abdominal pain, general edema, and 15 kg weight loss for a year . He said that he frequently ate raw fish, especially common blackish goby (acanthogobius flavimanus) and has never been abroad . We observed tenderness around periumbilical area and general edema on physical examination . In the initial laboratory findings, the total serum protein and albumin levels were 3.7 and 1.3 mg / dl, respectively . The results of thyroid function test were 90.81 ng / dl of t3, 0.91 ng / dl of free t4, and 13.35 miu / l of tsh, but all of autoantibodies such as anti - thyroglobulin, anti - tpo, tsh - receptor - ab were negative . Synthyroid 0.05 mg was given to him for 2 weeks, but serum albumin was still low as 1.2 g / dl, and diarrhea and general edema were not improved . In the abdominal ct, there was proximal small bowel wall thickening and transient intussusception . Diffuse effacement of small bowel folds from the mid - jejunum to terminal ileum was seen at a small bowel double contrast study . There was diffuse mucosal edema and large amount of whitish exudates at the jejunum and terminal ileum . However, the lesion was not ulcerative and without mass in the whole small bowel mucosa by small bowel capsule endoscopy . Esophagogastroduodenoscopic and colonoscopic findings were non - specific except for mild mucosal edema at the terminal ileum . Blind random biopsy was performed at 10 cm and 30 cm proximal from the ileocecal valve . The intestinal villi were atrophic and there was an intense infiltration of plasma cells in the lamina propria . Sectioned nematode worms were seen in the epithelial layer . Some cross - sectioned worms (n=9) were 25 - 30 (av . 27) m and 33 - 38 (35) m in diameter, and revealed the characteristic features of c. philippinensis (stichocytes and male and female genital organs) (fig . 1). We could make the diagnosis as intestinal capillariasis and prescribed albendazole 400 mg daily for 3 days . A month later, all symptoms disappeared and the total serum protein and albumin levels were normalized to 6.4 and 3.8 mg / dl, respectively . Adults and eggs of c. philippinensis were recovered in the diarrheic stools collected before and after treatment . Male worms (n=10) were 1,770 - 2,150 (1,872 in average) m long and 27.5 - 30.0 (28.0 in average) m wide, and had a characteristically long esophagus (950 - 1,150 m long) with a chain of stichocytes and a long spicule within a sheath (fig . 2a). Female worms (n=10) were 2,290 - 2,970 (2,556 in average) m long and 30 - 38 (35 in average) m wide, and had a long esophagus (1,150 - 1,450 m long) with a chain of stichocytes and immature eggs (35 - 4418 - 23 m size) in the uterus . Their posterior ends were more blunt than anterior ends, and vulva openings were located posteriorly at 38 - 45 m from the esophago - intestinal junction (fig . Mature eggs (n=5) detected in the diarrheic stools were 42.5 - 45.0 (av . 43.3) m long and 20.0 - 21.3 (20.4) m wide, and had characteristic mucoid plugs at both ends (fig . 2c). The present study is the 6th korean case of intestinal capillariasis and the 3rd case indigenously occurred in korea . The outbreak place of the present case, sacheon - si, gyeongsangnam - do, is not so far from namwon - si, jeollabuk - do, in which 2 indigenous cases previously occurred . These indigenous cases suggest that the life cycle of c. philippinensis is maintained in the southern regions of the korean peninsula, such as namwon - si and sacheon - si . Accordingly, studies on epidemiologic characteristics of c. philippinensis, especially on the source of human infections, transmission modes, fish intermediate hosts, and natural reservoir (definitive) hosts, should be performed in the near future . On the other hand, the remaining 3 cases were imported from bali in indonesia and saipan island [14 - 16]. Recently, in some asian countries (india, egypt, lao pdr, thailand, taiwan, and the philippines), intestinal capillariasis is regarded as the emerging and/or reemerging parasitic disease, and it's outbreaks have been frequently reported [12,17 - 21]. Protein - losing enteropathy is a complication frequently manifested in the intestinal capillariasis [16 - 18]. It is a result of direct mucosal invasion by the adult worms, which is sometimes exacerbated by the host inflammatory responses . Although the intestinal tract frequently has grossly normal appearance, histopathologic examinations of the biopsied intestines reveal villus atropy, crypt hyperplasia, and occasional infiltration of inflammatory cells . In the present study, although mild mucosal edema was observed at the terminal ileum, ulcerative or mass lesion was not found in the whole small bowel mucosa . However, in the biopsied specimens, villus atrophy, an intense infiltration of plasma cells, and sectioned nematode worms were seen in the epithelial layer . Numerous worms embedded in the intestinal mucosa may be associated with ulcerative and degenerative changes, and the protein - losing enteropathy may be provoked as a result . Regarding the diagnosis of intestinal capillariasis, characteristic clinical symptoms, including long - lasting diarrhea, abdominal pain, severe weight loss, and low serum albumin level, may be considerable factors in the first step . A definite diagnosis can be made by detection of worms and eggs in the diarrheic stools . However, histopathologic findings of biopsied intestines, including villus atrophy, crypt hyperplasia, intense infiltration of inflammatory cells, and worm sections, may be useful as the second - step diagnostic procedure . However, the exact sampling of intestines is not easy because endoscopic examinations are not applicable to the jejunum of humans, the normal habitat of c. philippinensis . In the present study, biopsy was performed at the terminal ileum through colonoscopy, and diagnostic clues were found in the biopsy specimens before the recovery of worms and eggs in the stool samples . Diagnosed their case i by detection of worms and eggs in the biopsy specimens of the terminal ileum, like in our case, whereas kwon et al . Found worm sections in the biopsied jejunum . All korean cases were definitely diagnosed by the detection of eggs and/or worms in the diarrheic stools of patients [10,14 - 16]. Cases indigenously occurred in namwon - si experienced consuming raw freshwater fish, i.e., several species of cyprinoid fish and rainbow trout . The present case recalled that he frequently ate raw goby (acanthogobius flavimanus), which could be easily captured in the seashore near his residential place . However, he was mixed - infected with c. sinensis, which is an evidence of eating raw freshwater fish also . Accordingly, some fish species, including the common blackish goby, must be the source of human infections, and this should be clarified in the near future in korea.
Coffee is one of the most commercialized food products and may be prepared by different techniques depending on the consumers' preference . From a chemical point of view, the two main coffee species (arabica and robusta) may be a rich source of biologically active compounds and their potential human health effects depend on consumers' physiology and the amount of coffee consumed per day . Among several compounds present in coffee, chlorogenic acids (cgas) are one of the most important groups . Although with considerable variation, total cgas may account for 7.014.4% of dry matter basis in green robusta and 4.08.4% in green arabica beans . Caffeic, ferulic, and p - coumaric acids are the main phenolic compounds in coffee which derive from trans - cinnamic acid . Naturally, they may present as mono- or diesters with quinic acid, forming chlorogenic acids, which are known to be the most active antioxidant compounds . Cgas are water soluble compounds [4, 5] and they can be divided into caffeoylquinic acids (cqas) with 3 isomers (3-, 4-, and 5-cqa), feruloylquinic acids (fqa) with 3 isomers (3-, 4-, and 5-fqa), dicaffeoylquinic acids (dicqas) with 3 isomers (3,4-dicqa; 3,5-dicqa; 4,5-dicqa), and, to a lesser extent, p - coumaroylquinic acids (pcoqas) with 3 isomers (3-, 4-, and 5-pcoqa). Among them, cqas are found to be the most abundant compounds in coffee [2, 6]. Since cgas contribute to acidity, astringency, and bitterness of the brewed coffee, they are relevant to sensorial properties of the beverage . Besides that, the antioxidant properties of cgas are well documented in the literatures [7, 8]. Literature survey revealed that numerous production steps involved in coffee production may influence the cgas content in the final product; however, the roasting process is described as the most important step which has a profound effect on chemical composition of the products [1012]. The cgas content in brewed coffee may differ according to other parameters, like coffee species [11, 12], origin of beans, and subsequent brewing methods [14, 15]. Although the cqas content in coffee beans and the effect of processing conditions, especially roasting, on cgas have been widely reported [11, 12, 15], data related to the new brewing procedures are limited [16, 17]. Coffee can be brewed in many ways depending on consumers' preference but recently consumer choices for a particular type of coffee beverage have been affected by various parameters . Although data indicates that coffee brews are capable of delivering different levels of cqas (26.1295.6 mg/100 ml), there is limited information regarding the influence of brewing conditions on the level of cqas, especially through the new brewing techniques like capsules, pods, or easy drinking beverages such as iced coffee . Considering the significant consumption of coffee beverages among european countries and due to the contribution of cqas as the most important class of cgas to human health, a comprehensive study was performed to evaluate the effect of wide range of brewing techniques on cqas content (3-cqa, 5-cqa, and 4-cqa), prepared by various technologies . This would allow us to estimate the role of brewing techniques and the composition of coffee blends in cqas content of coffee brews and subsequently in equilibrating the acidity of brews for consumers who suffer from acid reflux symptoms . However, some care should be taken into account because some differences regarding nomenclature of 3-cqa and 5-cqa seem to appear in several publications . Besides that, few research papers reported the validation of the analytical methods with regard to cqas in the new brewing processes . Therefore, the aim of this work was to evaluate the cqas content and profile in different brewing processes, including homemade brews (boiled, filter, french, and mocha coffee prepared using economically important coffee species, arabica and robusta) and commercial brewed coffee . Indeed, in order to understand the potential variation in the amount of cqas consumed by coffee drinkers and to go deeper into the influence of brewing techniques on concentration of phenolic compounds, various commercial coffee brews (capsule, pod, instant, iced coffee, and iced cappuccino) were assayed for their cqas content . Referenced standard of 5-caffeoylquinic acid (cas: 906 - 33 - 2; purity of 95%) was purchased from cymit (barcelona, spain). Individual standards of 4-caffeoylquinic acid (cas: 905 - 99 - 7; purity of 98%) and of 3-caffeoylquinic acid (cas: 327 - 97 - 9; purity of 95%) were acquired from sigma - aldrich (mo, usa). The chemical structures of the main cqas analyzed in the present study are shown in figure 1 . Solvents were acetonitrile and methanol (hplc gradient grade) and were obtained from vwr (belgium). Citric acid and glacial acetic acid with purity of 99% were supplied from merck (germany). Zinc acetate dihydrate (purity of 99%) and potassium hexacyanoferrate ii trihydrate (purity of 98%) were acquired from vwr (belgium). Twenty - four coffee brews were tested for their cqas content as follows: eight classical brews (boiled, french, filter, and mocha) prepared using arabica and robusta coffee as well as sixteen different commercial samples . A description with regard to the coffees used for brew preparation was exhibited in table 1 . Roasted arabica (100% coffea arabica, 2.34% water content) and robusta (100% coffea robusta, 3.11% water content) coffee, packed in nitrogen - based protective atmosphere, were kindly supplied by a local company of porto, portugal . Roasted beans were ground by means of a home grinder (braun ksm 2 model 4041, mexico). In order to determine the particle size, 50 g of ground coffee was sieved by means of three laboratory test sieves (retsch, germany) with different mesh size (212, 300, and 500 m). Then the particles of each sieve were weighted and presented as percentage from the total mass . Ground arabica (particle size: 51%> 500 m; 24%> 300 m and <500 m; 13%> 212 m and <300 m; 11% <212 m) and robusta coffee (particle size: 48%> 500 m; 27%> 300 m and <500 m; 17%> 212 m and <300 m; 6% <212 m) have almost the same particles size distribution . Therefore, the influence of particle size on the extraction of cqas in both species is almost in the same manner . These ground coffees were used to prepare classical coffee brews (boiled, french, filter, and mocha). Arabica coffee beans were ground before brewing by means of la cimbali, grinder - doser 6/sa . In order to prepare a high quality espresso coffee, a range of particle size from course to very fine ground is advised (particle size: 2%> 500 m; 72%> 300 m and <500 m; 22%> 150 m and <300 m; 2% <150 m and> 63 m). Various brands of different types of coffee were also purchased randomly from local commerce in porto, portugal . The purpose of the sampling scheme was to comprehensively evaluate the cqas concentration in a wide range of coffee brews commonly consumed . A total of twenty - four coffee brews were prepared according to the manufacturers' instructions; however, in some cases, information about the coffee origins and species, or roasting conditions used to prepare the blends, was not available . Coffee brews (three replicates for each sample) were stored at 22c in polypropylene containers until analysis, made in duplicate . Nine different coffee samples were obtained using pure arabica and robusta coffee with coffee / water ratio of 7.5 g/100 ml to uniformize the comparison of brewing techniques in terms of cqas content . An exception was homemade espresso coffee which was brewed using arabica coffee with coffee / water ratio of 7.5 g/40 ml . This was prepared by boiling 11.25 g ground coffee with 150 ml of distilled water for 10 min followed by 2 min of settling time followed by decanting the liquid . This was brewed by pouring 150 ml of boiling water onto 11.25 g of ground coffee in glass french press pot followed by stirring . After 2.5 min, the coffee brew was separated from ground coffee by pressing the plunger . 22.5 g of roasted and ground coffee was put in a paper filter bag (n 2) and extracted with 300 ml of boiled distilled water by means of conventional percolation coffee machine krups aroma caf 5 (germany). This was prepared using 7.5 g of finely roasted and ground arabica coffee using a semiautomatic espresso machine (la cimbali m31 classic) with hot water (90 2c, temperature of water at the exit of the heating unit) under pressure (9.0 0.2 bar) during 21 3 s until the volume in the cup met 40 ml . Fifteen commercial coffee brews were prepared accordingly to the manufacturers' instructions as follows . Extraction of each capsule was performed using an automatic coffee maker (krups, xn2100, germany) at a pressure of 19 bar by hot water (93 2c). A - type 1 (6.01 0.01 g), a - type 2 (5.01 0.06 g), a - type 3 (5.01 0.03 g), a - type 4 (5.14 0.02 g), a - type 5 (6.13 0.11 g), b (5.19 0.11 g), and c (5.71 0.02 g). The size of a single serving was 40 ml derived from the brewing of a 7.08 0.15 g roasted and ground coffee . Instant coffee . For this purpose, 2 g of commercial instant coffee powder was extracted with 150 ml of boiled distilled water . Regarding instant espresso, one pack containing 1.8 g of soluble coffee this was prepared based on preparation instruction where 2 tablespoons of iced coffee powder (8 g) were put in a glass and 240 ml of cold distilled water was added and stirred well . This was prepared based on preparation instruction as one pack containing 18 g cappuccino powder was put in the glass and 100 ml of cold distilled water was added and stirred well . This was obtained from necta coffee vending machine (necta astro double brew) to draw a cup of coffee (30 ml). Carrez solutions i (21.9 g of zinc acetate and 3 ml of glacial acetic acid diluted to 100 ml distilled water) and ii (10.6 g of potassium hexacyanoferrate ii dissolved in 100 ml of distilled water) were used for precipitation of proteins and other interfering compounds as well as the elimination of turbidity and for breaking of the emulsion . Prior to extraction, three cups of each type of brew were defrosted, mixed, and heated to reach a homogeneous mixture at 4045c . Extraction of cqas was performed in duplicate according to the method of fujioka and shibamoto with minor modifications . For this purpose, 3.0 ml of coffee was transferred to a polyethylene test tube and treated with 0.1 ml of each carrez solution (i and ii) and 0.8 ml of methanol and the volume was made up with distilled water to 8.0 ml . The mixture was vortexed for 1 min and left to stand for 10 min . After centrifugation (rotofix 32a, germany) at 4000 rpm for 10 min, the upper phase was filtered through the 0.2 m ptfe filter membrane (vwr, usa) just before analysis with hplc - dad at 325 nm . After the precipitation of the interfering compounds, the average volume of final solution was considered 7.5 ml; therefore, the concentration of cqas was calculated after applying the dilution factor of 2.5 . The instrumental analysis of cqas was performed using hplc - dad, merck hitachi elite la chromatograph (tokyo, japan) equipped with a quaternary system of pumping (l-2130) and l-2455 uv / vis spectrophotometry diode array detector . Separation was achieved using lichrocart rp-18 endcapped (250 4 mm, 5 m) column, attached to a guard column (4 4 mm, 5 m) of the same kind . Quantitative analysis of chlorogenic acids was performed based on the method described previously by tfouni et al . With slight modifications . The mobile phase was constituted eluent a: 10 mm citric acid aqueous solution (ph of 2.4) and eluent b: acetonitrile . The gradient was programmed as follows: from 0 to 30 min 8% of b, 30 to 35 min increase to 80% of b, 35 to 40 min 80% of b, 40 to 45 min decrease to 8% of b, and 45 to 50 min 8% of b. injected volume was 10 l and the flow rate of analysis was 1 ml / min . Identification of the target compounds was confirmed by retention time and spectrum comparison with standard solutions . To evaluate differences in variation between coffee samples in each class of brewing and also to study the differences among arabica and robusta coffee brews, data are reported as mean standard deviations of two extractions followed by two injections . Under the experimental conditions referred to above, separation of cqas could be achieved during the first 30 min with isocratic elution of water (ph: 2.4)/acetonitrile . However, gradient elution was applied to clean the column and remove other interfering compounds for starting the next run . A stock solution containing all cqas calibration curves were prepared by plotting the peak area against the corresponding concentrations by duplicate injection of 10 l of standard solutions at nine different concentration levels for 3-cqa (2400 regarding the detector response, the regression lines were linear over the studied concentration range and the corresponding coefficients of correlation (r) of 0.999 were obtained for all analyzed compounds . The sensitivity of the method, expressed as the slope of the calibration curve, was maximum for 4-cqa . The limit of detection (lod) and limit of quantification (loq) were calculated at signal to noise ratio of three (s / n = 3) and ten (s / n = 10), respectively . The lods were 0.37, 0.39, and 0.18 mg / for loqs, 1.24 mg / l was obtained for 3-cqa and 1.29 mg / l and 0.58 mg / l were achieved for 4-cqa and 5-cqa, respectively . Repeatability of the method (intraday precision) was estimated when the cqas standards at three concentration levels (c1, c2, and c3, see the concentrations in table 2) were analyzed on the same day for six injections . Reproducibility (interday precision) was the result of the analysis of standards at three concentration levels (c1, c2, and c3; see the concentrations in table 2) during the three sequential days by injecting three times and the average% cv was reported in table 2 . Intraday precision and recovery of cqas in some coffee brews, spiked at two different concentration levels, were exhibited in table 3 . The recovery test was performed by spiking various types of coffee brews with known quantity of the cqas reference standards before the extraction procedure . The average recovery (%) was reported as the mean ratio between the obtained and the expected concentration of cqas in fortified samples . Different coffee brews were selected based on their initial cqas concentration (filter, instant, and capsule coffee) so after spiking, the concentration of cqas in spiked samples was within the linearity range . The mean recoveries ranged between 91.46% and 103.39% (table 3). In the present study, firstly, the effects of brewing procedures as well as the effect of coffee species (arabica and robusta) on cqas content of classical brewing techniques were evaluated and afterwards commercial coffee brews including capsule, pod, instant, iced coffee, and iced cappuccino were compared with regard to their cqas concentration . Chlorogenic acids content in brews prepared using roasted and ground arabica and robusta coffee including boiled, french, mocha, and filter coffee are shown in table 4 . As it can be clearly seen in table 4, the major isomer in these classes of samples was 3-cqa, accounting for about 50% of the total cqas, followed by 5-cqa and 4-cqa, accounting for about 25 - 26% for each one, both for arabica and robusta coffee . During the extraction, most of the water extractable components are extracted at the beginning of the extraction process but lower concentration of 5-cqa than 3-cqa could be explained by the fact that 5-cqa is less water - soluble than 3-cqa, yielding lower concentration in the brews . Although some bibliographic references reveal 5-cqa as the main isomer among cqas [10, 14, 18] our results were comparable with the ones obtained by gloess et al ., who found 3-cqa at higher concentration in various types of coffee brews . Crozier et al . Proved that during roasting, 3-cqa and 4-cqa are destroyed more slowly than 5-cqa . In another study, farah et al . Found the reduction of 5-cqa from green beans to light roasted beans while 3-cqa and 4-cqa content increased in light roasted beans and then gradually decreased at higher roasted degree . Therefore, due to the different sensitivity of cqas to various roasting conditions [11, 12], the higher concentration of 3-cqa than 4- or 5-cqa might be explained by the origin of the beans and their roasting degree, which is however unknown for us . In both species, when considering the brewing procedure, the mocha extraction was the most efficient brewing method followed by boiled, french, and filter coffee . Since these samples were prepared with coffee / water ratio of 7.5 g/100 ml, the effect of this parameter on cqas content could be eliminated . Besides that, ground arabica and robusta coffee have almost the similar particle size distribution so the degree of grinding seems to have similar effect on cqas content . The most influencing parameters seem to be extraction temperature and pressure because mocha extraction was performed under pressure (0.5 relative atmospheres, corresponding to 110c). The decreasing order of total cqas of samples prepared with robusta coffee was mocha (872.93 mg / l)> boiled (771.29 mg / l)> french (666.67 mg / l)> filter coffee (624.03 mg the decreasing order was similar to robusta, although the total concentrations of cqas in mocha (744.04 mg / l) and boiled coffee (744.70 mg / l) were almost the same (p> 0.05) followed by french (645.56 mg / l) and filter coffee (638.58 mg filtered brews are the ones that least contribute to cqas intake and provide the lowest content of cqas . Indeed, despite the other studied brewing techniques, during the filter coffee brew preparation, ground coffee was only washed out with hot water at ambient pressure without any flotation, therefore yielding lower cqas contents than other brewed coffees . Tfouni et al . Also found the higher content of cqas in boiled coffee (26295 mg/100 ml) than filter coffee (24219 mg/100 ml). This could be due to the higher contact time between ground coffee and hot water during the boiled extraction procedure . In previous work, prez - martnez et al . Observed that mocha coffee was the richest source of cgas, followed by the filter and plunger coffee makers . Concerning the cqas content of french press, results were opposite to gloess et al . Who indicated higher extraction efficiency of 3-cqa and 5-cqa in french press than in mocha or even espresso coffee . This difference could be explained by different coffee / water ratio and extraction time that they used for mocha and french press brew preparations . Considering the influence of the raw material, in general, the cqas content of different coffee brews was significantly (p <0.05) affected by the coffee species, as robusta samples yielded greater cqas content than the arabica ones . The obtained results were in accordance with tfouni et al . Where robusta coffee brews contain higher cqas than the arabica ones . The biggest difference was found among mocha coffees with concentration of 872.93 mg cqas / l for robusta and 744.04 mg exception was filter coffee, where there was no remarkable difference between the values of total cqas for arabica (95.79 mg / l) and for robusta (93.60 regarding the sum of 3-, 4-, and 5-cqas in arabica filter coffee (81.0 mg/100 ml) which was higher than robusta filter coffee (56.2 mg/100 ml). Similar behaviour of arabica and robusta coffee at different roasting degree was also reported previously . Some authors attribute the lower concentration of cgas in arabica than in robusta to the coffee production step (wet or dry method). Generally, wet method is used for arabica coffee and requires substantial amounts of water . It could be a reason for loss of cgas in arabica coffee in comparison to the robusta coffee that is commonly processed by the dry method . According to leloup et al . And clifford, although green robusta beans have a higher cgas content, the sensitivity of cgas in robusta coffee matrix seems to be more than that in arabica coffee matrix which could explain the same behaviour of arabica and robusta coffee brews in some cases . Although, based on the concentration basis (mg / l), mocha produced a high concentrated brew than others in terms of cqas, that finding is different when content per cup size is considered . As it can be seen in figure 2, boiled coffee has the greatest amount of cqas per cup (115.69 and 111.71 mg/150 ml in robusta and arabica, resp . ), and mocha has the less content both in robusta (52.38 mg/60 ml) and arabica coffee (44.64 mg/60 ml). It means that consumption of a cup of boiled coffee contributes to higher intake of cqas by consumers followed by french, filter, and mocha . It should be mentioned that espresso lab - made prepared with roasted and ground arabica coffee was compared with commercial brews prepared under pressure . In order to understand the variation in the amount of cqas consumed by coffee drinkers and to go deeper into the influence of brewing techniques on the concentration of phenolic compounds, indeed, analysis of commercial coffee brews is of interest because they are representative of real samples which are delivered outside the laboratory conditions . As previously mentioned, comparison of commercial coffee brews was more complicated due to the lack of information regarding ratio of each species in the blend, roasting conditions, grinding degree, and the origin of the beans used for brewing . The results of cqas concentration in different commercial coffee brews expressed as mg / l are displayed in table 4 . Analysis of the coffee samples indicates the presence of 3-, 4-, and 5-cqa in all samples . The most abundant cqas in all considered samples (except instant natural a) was 3-cqa accounting for 3450% of the total cqas followed by 2336% for 5-cqa and 25 to 28% for 4-cqa . Generally speaking, the results of the processes studied varied according to the brewing mechanisms and total cqas ranged from 45.79 mg the results were in the range of 748.40 to 1656.82 mg cqas / l, much higher than those reported for classical brew preparation . Capsule a - type 1 was found to produce the higher concentrated brew in terms of total cqas (1656.82 mg since all capsules were brewed with the same machine and conditions, the effect of water temperature and pressure on cqas contents would be similar . Information of the label of the product revealed that capsule a - type 1 is a blend of arabica and robusta from different origins which were ground finely; however, the ratios between species are unlikely to be identical and both are known to influence the cqas content . Frequently, among capsules a, the lowest cqas content was reported in capsule a - type 5 that has the highest roasting intensity, which may possess more degradation of cqas during the roasting . Although the coffee quantity of capsule a - type 5 (6.13 0.11 mg / capsule) was almost similar to a - type 1 (6.01 0.01 g / capsule), their country of origins and coffee variety may be different which could explain the diversity of cqas among these two types of capsules from the same brand . Among all analyzed capsule coffee, capsule b had less quantity of coffee powder (5.19 0.11 g / capsule) which may explain its less concentration of cqas (748.40 mg there are limited studies regarding cqas content in capsule coffee [16, 17]. Observed 3-cqa in concentration of 15 mg/30 ml and found 5-cqa in lower concentration (6 mg/30 ml) in nespresso coffee variety of arpeggio . According to the results of hplc analysis, pod espresso revealed the greatest content of cqas (1662.01 mg / l), corresponding to 823.45, 436.30, and 402.30 mg / l for 3-cqa, 4-cqa, and 5-cqa, respectively . These high concentrations could be attributed to the high quantity of coffee per pod (7.08 0.15 g / pod). Grinding degree, ratio of arabica to robusta in the blend of coffee pod, could also influence the extraction of cqas, together with other technological factors like water pressure, which was unknown . Another concentrated brew with regard to cqas seems to be vending coffee (1521.05 mg / l) and espresso lab - made (1220.35 mg lab - made, the variety of coffee (100% arabica) may play an important role in cqas content . The results obtained in the present study confirmed the presence of high concentration of cqas in various espresso coffee (capsules, pod, or normal espresso) ranging from 748.40 to 1662.01 mg / l . However, caprioli et al . Reported the cqas content up to 2223.4 mg the presence of cqas in various types of espresso coffee was also affirmed by niseteo et al . Who obtained 495.56985.73 mg cqas / l, which is in compliance with the cqas content determined in the present study (748.401662.01 mg although espresso coffees (capsules, pod, or normal espresso) contain high concentration of cqas, their average content per cup was found to be less than classical techniques such as boiled, french, and filter coffee (figure 2). The total cqas content per cup ranging between 29.94 and 66.27 mg/40 ml was found in brewed coffee using capsule b and pod espresso, respectively . Presented 3-cqa and 5-cqa in the levels of 18 and 8 mg/30 ml of espresso coffee prepared with semiautomatic machine, respectively . In the case of instant brewing technique, despite the other brews, the main isomer in instant natural a was 5-cqa (36%) followed by 3-cqa (34%) and 4-cqa (28%). Accounting for the total cqas, the greatest amount was obtained for instant espresso (991.85 mg / l) followed by instant natural b (227.57 mg / l), natural a (179.16 mg / l), and instant decaffeinated (171.86 mg these values are in accordance with some authors which developed a study for comparison of normal coffee over decaffeinated coffee [4, 14, 27] and loss of cgas in decaffeinated coffee was reported . However, it must be taken into account that soluble coffee suffers an additional thermal extraction treatment at high temperature after roasting which decreased their antioxidant capacity . These additional processes may affect the cqas content due to the interaction of cgas with maillard reaction intermediates . These data demonstrate that when comparing commercial soluble coffee as mg / cup, they could be accounted as the potential source for delivery of moderate level of cqas as instant espresso delivered around 50 mg cqas per cup of 50 ml . Reported the cgas ranging from 37.04 to 121.25 mg/200 ml in various soluble coffees . The higher content than our study is probably due to the higher consumed cup size (200 ml). Despite our results, niseteo et al . Found the instant coffee as one of the richest sources of cqas with concentration ranging from 2300.77 to 4034.41 mg the lowest concentration of cqas was found in iced cappuccino (45.79 mg / l, corresponding to 4.58 mg/100 ml) and iced coffee (104.19 mg / l, corresponding to 25.00 mg/240 ml). The presence of cqas in cappuccino prepared with hot water was previously reported by niseteo et al . In the range of 15.89104.65 it must be taken into account that for iced coffee and iced cappuccino, there are an additional process including adding other ingredients like milk and sugar which will influence the presence of cqas in final product . According to narita and inouye presence of 5-cqa is ph dependent where at lower ph it is more stable and by incubation at 37c in high ph (7.4, 8.0, 8.5, and 9.0), 3-cqa and 4-cqa were produced from isomerization of 5-cqa . Besides that, total cqas were decreased gradually at ph of 5.09.0 . The effect of milk on antioxidant capacity can be attributed to the precipitation of polyphenols due to the binding with milk proteins such as casein [27, 30]. It is worth noting that although iced coffee and in particular iced cappuccino contain ingredient such as milk which result in high ph beverages and subsequently degradation of cqas, the less ratio of coffee in these products than other brews which are prepared only from pure coffee powder may also affect the amount of cqas in final products . This investigation clearly demonstrated that coffee brews commonly consumed are capable of delivering high amounts of cqas as the major isomer in analyzed samples was 3-cqa, followed by 5-cqa and 4-cqa . Besides that it was confirmed that brewing mechanisms have a profound effect on the amount of cqas delivered per cup . Since chlorogenic acids play an important role in human health, this study allowed us to elucidate the role of brewing techniques and type of coffee on cqas content of brewed coffee and subsequently allowing us to equilibrate the acidity of brews for consumers . This equilibration lets consumers to avoid consequences of high cgas consumption and at the same time they intake sufficient amount for medicinal purposes.
Clinical research is responsible for current scientific progress in the medical field, and the participation of human patients is vital for the confirmation of new therapeutic strategies . Despite the efforts to ensure ethical rigor in research over the past 60 years, ethical concerns have been an area of concern to both researchers and government agencies, as studies may involve the participation of potentially vulnerable populations, especially in developing countries . Ethical issues are evoked in such studies, as the study investigators or sponsors are from developed countries, and the clinical trials are often conducted in developing nations . Study participants are vulnerable due to their poor education level, unfamiliarity with scientific jargon, poverty, and general lack of access to health care services (1). Individuals with reduced autonomy, that is, those who are vulnerable, must be protected so that they are not used only as objects with which to conduct research and to aid in scientific development (2). Although participant autonomy is facilitated by the use of an informed consent form in accordance with the helsinki declaration, and, in brazil, with resolution 196/96 (3), many studies have questioned the effectiveness of informed consent documents as the common clarification method used in clinical trials (4). Such concern has arisen because many patients are incapable of understanding the details in the document presented to them (5) and often consent as a result of the socioeconomic conditions under which they live and the role that clinical trials play in their search for solutions to health problems (6). Under these circumstances, participants may become frustrated due to a lack of understanding of the actual role of a trial in which they have consented to participate . Studies have shown that the decision to participate in a clinical trial is mainly motivated by altruism, that is, the possibility of helping other people and science (7 - 9), as well as by the desire for personal benefits, such as access to better care or treatment (10). Nevertheless, little is known regarding the factors that motivate participation in cardiology clinical trials focused on heart failure (11,12). This study was conducted to evaluate the factors that motivate participation in cardiology clinical trials, as well as those leading to participant frustration . The present study was a descriptive, exploratory, and quantitative study conducted in a public hospital specializing in cardiology . The study protocol was approved by the universidade de so paulo hospital das clnicas ethics committee (reference number 1223/2005) on january 26, 2006, in accordance with the national ethics committee of the brazilian ministry of health (3). The following were established as inclusion criteria: age between 20 and 80 years; participation in phase ii, iii, or iv clinical trials for drug testing that were conducted in outpatient units from 2002 to 2006; placebo usage in the washout phase or that was compared to the tested drug; randomization into the clinical trial and consent to participate . Individuals who did not have the ability to remember their participation in the selected clinical trials and/or did not agree to participate in this study were excluded . Two groups were then formed: group i, comprising patients who had participated in placebo - controlled trials after randomization, and group ii, comprising patients who had participated in clinical trials in which the tested drug was compared to another drug after randomization and in which the placebo was only administered during the washout period . The participants in both groups had the same clinical profiles, had chronic coronary arterial disease, and had been followed as outpatients at the institution . Some of these trials were published with all patients here enrolled (13,14) and other were part of the casuistic of the final trial (15). Data were obtained by consulting patient ambulatory records and by conducting an interview with patients via a questionnaire containing 29 items that had been previously designed and developed after considering all of the items on the consent form necessitated by resolution 196/96 . The first part aimed to collect identifying and demographic information of the participants, such as name, hospital registration number, age, gender, marital status, occupation, time of follow - up at the institution, study in which he / she participated and whether he / she completed the study . In addition to general participant characteristics, this part of the questionnaire evaluated education according to the following levels: 1) elementary school, 2) secondary school and 3) college . The second part of the questionnaire consisted of open and multiple - choice questions concerning participation in the clinical trial . It began with questions related to the invitation and motivation for participation (why did you choose to accept the invitation to participate in the study? ; five answer choices: a) for the sake of science, b) for your own benefit, c) because the doctor assisting you asked you to do so, d) because the doctor said it would be good for you and e) for fear of not being given care or missing a treatment opportunity due to the fact that the study took place at a public hospital, or due to other reasons). In the questionnaire, there were yes / no questions concerning information that must be included in a consent form, such as the importance of the study in which one was invited to participate, risks and inconveniences, the existence of other therapies, information confidentiality, expense refunds, compensation in the case of occasional damage or problems, the need to read the consent form before signing it, discussion about the consent document with the researcher, confirmation of comprehension of the information in the document and confirmation of comprehension of the word placebo . Next, the questionnaire asked whether the participants had considered withdrawing from the study, with yes or no as possible answers . If the answer was affirmative, they were asked for their reason and to state why the researcher had not been informed, as all of the participants did complete the study . Finally, we asked the participants, in an open - answer format, what it meant for them to participate in a clinical trial . All questionnaires were answered by the patients themselves without any influence but with the help, when necessary, of one investigator . Casuistic: of the 106 participants selected for the interview, two had died prior to the interview, eight were excluded, two refused to participate, five did not come on the scheduled interview date, and nine were unable to be reached . Overall, 80 participants comprised the study sample of 60 males (75.0%) and 20 females (25.0%). Of those participants, 47 were in group i (mean age 58.3 years), and 33 were in group ii (mean age 59.4 years). The classificatory variables are presented in tables with absolute (n) and relative (%) frequencies . To evaluate whether there was an association between the questions of interest, the student's t test, the chi - square test the content analysis methodological framework was used to analyze the descriptive data obtained by means of the open questions (16). Initially, pre - analysis, which consisted of the content organization phase and was aimed at systematizing ideas, was conducted . The classificatory variables are presented in tables with absolute (n) and relative (%) frequencies . To evaluate whether there was an association between the questions of interest, the student's t test, the chi - square test the content analysis methodological framework was used to analyze the descriptive data obtained by means of the open questions (16). Initially, pre - analysis, which consisted of the content organization phase and was aimed at systematizing ideas, was conducted . In both groups, most of the participants were illiterate or had an incomplete elementary school education (63.8%), thus characterizing the overall sample as having a poor education level . Sixty - four (80.0%) participants had a partner and were either married or had a common - law relationship . Regarding employment, half of the participants (51.3%) did not have a formal job, with 37.5% retired and 13.8% performing household activities . When the participants were asked about their reason for participating in a clinical trial, 53 subjects (66.2%) in the combined groups cited the personal benefit expected as the primary reason, followed by the sake of science 34 subjects (42.5%) (table 2). It was also found that 25.0% of the participants participated in the clinical trials due to their doctor's request or to recommendations to participate in the clinical trial, indicating the influence of professionals in the decision - making process . Other individuals participated in the trial because it was conducted at a public hospital (10.0%) and because they feared no longer being given care at the institution (3.8%) if they refused . Other cited reasons, which represented 6.2% of the responses, were related to the possibility of clinical tests that would not be available during routine hospital care and to the influence of their participation on the care received . Nine patients (11.2%) reported the desire to withdraw from the clinical trial (table 3), but they did not inform the investigator and completed the trial . The main reason that led individuals from group i (6.4%) to consider withdrawing was the risk involved in certain tests during the trial . In group ii, the main cited reason for considering withdrawal was the frequency of medical consultations required (6.0%). The main reason reported by the participants for not having interrupted their participation in the clinical trial was fear of being prevented from receiving care at the institution and discontinuation of their treatment (5%). Additionally, it was found that the attempt to find a treatment to improve their heart condition led the participants to not withdraw from the study despite their dissatisfaction (2.5%). Clinical research with pharmaceutical industry fund in brazil is still a relatively new area of concern, and it was only in 1996 that guidelines and regulating standards for studies involving humans were approved (3). Consequently, the number of clinical studies sponsored by pharmaceutical companies increased (particularly in cardiology), which was mainly due to the prevalence of diseases and large population found in developing countries, easy recruitment and good clinical research centers (17). This increasing number of clinical research has been a source of concern because the participants in clinical trials in these countries may be more vulnerable during the consent process due to their socio - economic conditions, poor education level and limited access to health care services (18). All of these issues were observed in the present study . Regarding the factors associated with the decision to participate in these studies, the expectation of personal benefit and altruism were the main motivating factors . (19) of 150 patients who participated in a randomized and double - blinded clinical trial of a drug developed for acute myocardial infarction . The study revealed that 43% of the patients participated in the study to receive better treatment, 12% expressed a desire for a good follow - up, 35% reported a desire to help promote medical research, 14% did not report a definite reason, and 8% reported being afraid to refuse to participate . The expectation of personal benefit from participation in the clinical trial is considered to be the major determinant of successful patient recruitment (20). However, when a participant erroneously believes that a study can offer substantial clinical benefit, a therapeutic misconception arises (21). In this situation, a patient who understands that his / her doctor's main goal is to find the best treatment for his / her disease may overestimate the personal therapeutic benefit from participating in the study . Nevertheless, these trials can produce a large amount of useful scientific knowledge and provide important benefits to future patients even if they do not provide a direct benefit to the current participants (22). Additionally, as an investigator, a doctor makes treatment decisions based on the specific design of each study (23). However, many patients, as exemplified by this study, also participate in clinical trials because they are motivated by altruism, which corroborates the findings from other studies, particularly oncological and prevention studies (11,24). In ethical terms, such an attitude is ideal, as the participant's individual benefit cannot be guaranteed, nor is that the objective of a clinical trial (24). (25) interviewed 250 patients who participated in cardiovascular clinical trials and observed that the main cited reasons for participation were the possibility of access to medical care and a desire to contribute to the advancement of science . Another point of interest in our study is that 25% of the participants participated in the clinical trial because of a doctor's request or recommendation . This information reflects the influence that a doctor's recommendations can have on a patient's life trajectory and on his / her decision - making process (26). However, the fact that people tend to change their behavior when they are the targets of interest and attention, regardless of the specific nature of an intervention, must also be considered . This phenomenon is known as the hawthorne effect (27), and in such situations, patients become eager to please their doctors and make them feel successful . Additionally, patients wish to participate so that good results can be achieved in the study . Although the doctors who conducted the clinical trials and enrolled the patients in this study were not always the same as those who had cared for the patients in the institution's outpatient clinic, it is necessary to consider the potential conflicts of interest that exist in studies sponsored by pharmaceutical companies nevertheless, secondary interests are not considered to be negative per se, and the problem does not lie in whether these interests can be defined as good or bad; rather, the problem is that damage occurs when secondary interests overlap and improperly influence, distort, or corrupt the professional's judgment with regard to the patient's health, the clinical trial, or education (28). Another point of interest in our study was the dissatisfaction expressed by nine individuals (11.2%) with regard to continuing participation in the clinical trial that was not expressed to the researcher during the course of the study . It is possible that these omissions occurred due to an asymmetry in the relationship between the researchers and subjects, which is largely analogous to a doctor - patient relationship . The doctor figure usually reflects an image of power and knowledge; thus, questioning his / her judgement may not facilitate the researcher - subject relationship (29). The results of the present study also demonstrate that even after patients read and discuss the consent form, which is written in lay language, with the investigator, it may be important to recheck during the follow - up which participants may have had the intention to abandon the trial but feared to spontaneously express such an opinion, or even to identify those individuals who did not understand the consent form or the trial . We were not able to analyze the consent forms from the trials we elected to evaluate, but all of them were written according to the guideline approved by our institutional ethics committee . The main reasons leading the participants to consider withdrawing from the clinical trials were the perception of risk in undergoing the tests required by the trial (group i) and the frequency of required medical appointments (group ii). These findings indicate that many patients signed the consent form and agreed to participate in the study without actually understanding what was being asked of them . In these circumstances, studies evoke ethical issues, lead to great researcher responsibility and may cause personal risks to research subjects (30), as we have already seen (31). Additionally, it was found that most of the participants did not withdraw from the study due to fear of discontinuation of their treatment at the hospital . This finding is in stark contrast to the principle of autonomy and leads us to reflect on how free the participants in clinical trials in developing countries really are to decide about remaining as a participant in such trials when they depend on the care provided by a public institution . Our sample size may have been too small to achieve accurate information regarding our question of interest . In addition, this investigation, as a retrospective study, is influenced by recall bias, even though recall ability was one of the criteria used for participant exclusion . The use of other interviewing techniques, and qualitative techniques in particular, could certainly contribute to the exploration of all of these concerns . Our sample size may have been too small to achieve accurate information regarding our question of interest . Nevertheless, we think the study provides important information . In addition, this investigation, as a retrospective study, is influenced by recall bias, even though recall ability was one of the criteria used for participant exclusion . The use of other interviewing techniques, and qualitative techniques in particular, could certainly contribute to the exploration of all of these concerns . The results of the present study indicated that the main motivation for participating in a cardiology clinical trial was the personal benefit expected by the participants in both groups . Although many of the patients confirmed their wish to withdraw from the trial, their dependence on the care provided by this medical center forced them to remain as subjects in the study . Additionally, the relative lack of understanding of the study by some of the participants was evident, indicating that the use of an informed consent form in developing countries is a complex process that needs to be reviewed by institutions participating in multi - center clinical trials.
Catel manzke syndrome (mim: 616145) is a rare genetic disorder characterized by pierre robin sequence with hyperphalangy and clinodactyly of the index finger, . Affected individuals typically present with the classic features of pierre robin sequence including micrognathia, airway obstruction secondary to displacement of the tongue base, and often, cleft palate . The orthopedic abnormalities are the result of an accessory bone between the second metacarpal and proximal phalanx that causes radial deviation of the second digit (manzke dysostosis). Cardiac abnormalities, facial dysmorphisms, and additional skeletal abnormalities have also been described in a subset of patients with catel manzke syndrome,, . Until recently, the genetic basis of catel manzke syndrome was unknown, although inheritance appeared to be autosomal recessive, . Recently implicated homozygous or compound heterozygous pathogenic variants in tgds (dtdp - d - glucose 4,6-dehydratase) as the causative factor in a series of seven unrelated patients with features of catel manzke syndrome . Of the seven unrelated patients reported, c.298 g> t (p.ala100ser) was the most common pathogenic variant and was hypothesized to be a founder mutation . Tgds encodes a member of the short - chain dehydrogenase / reductase (sdr) family; however the specific function of the protein in humans is unknown . In the present report, we describe an additional patient with molecularly confirmed catel manzke syndrome who has pierre robin sequence (without cleft palate) and manzke dysostosis, and we compare his phenotype with the phenotype of the seven previously described patients with pathogenic variants in tgds . He required tracheostomy for severe airway hypotonia with pharyngomalacia and laryngomalacia, and gastrostomy tube placement for failure to thrive likely secondary to his pharyngeal dysphagia and airway obstruction . The proband was born at 38 4/7 weeks of gestation via spontaneous vaginal delivery without complications to a 29 year - old female . The mother had a history of three prior first - trimester spontaneous abortions and one healthy son with the same partner . No prior genetic workup had been performed for the spontaneous abortions, and the cause for these spontaneous abortions was unknown . His postnatal course was complicated by significant failure to thrive noted as early as three weeks of age . His failure to thrive persisted despite increased caloric density and feeding therapy, and at two months of age, a modified barium swallow showed pharyngeal dysphagia, and he was noted to have significant fatigue with feeding . He was also diagnosed with gastroesophageal reflux . During an admission for failure to thrive, stridor and desaturations to 85% he was diagnosed with severe pharyngomalacia and laryngomalacia by otolaryngology, and a supraglottoplasty was performed . By six months of age, he was diagnosed with mixed sleep apnea and respiratory insufficiency due to airway hypotonia and oropharyngeal weakness . These respiratory issues were suspected to contribute to his feeding difficulties and failure to thrive . Tracheostomy was ultimately placed at seven months of age, and he required nocturnal ventilator use . He also underwent myringotomy and tympanostomy for recurrent ear infections . By eight months of age, a repeat modified barium swallow still showed pharyngeal dysphagia, but his fatigue with oral feedings appeared to be improved . In addition, by this time, his weight gain slowly began to improve . At his most recent exam at 12 months of age, his weight was 8.42 kg (z score = 1.22, who growth chart), his length was 72.4 cm (z score = 1.36, who growth chart), and his head circumference was 45.9 cm (z score = 0.13, who growth chart). He was using the gastrostomy tube for the majority of his caloric intake but was tolerating small amounts of oral feeds . He was noted to have prominent overriding sutures, a tubular - appearing nose with high nasal bridge and pinched nares, retrognathia, high and narrow arched palate with small groove of the posterior soft palate, and ankyloglossia (fig . His fingers and toes appeared long, and his index fingers were deviated and overlapping . A mild pectus deformity was noted . On hand radiographs, the second proximal phalanges appeared hypoplastic with an accessory bone located at the base of the second digit, and there was medial deviation of the distal second digits (fig . Lateral view of the spine demonstrated anterior beaking of the l2 and l3 vertebral bodies . Echocardiogram at three months of age showed a small patent foramen ovale and a small patent ductus arteriosus, both with left to right flow and both of which resolved by six months of age . Routine chromosome analysis, comparative genomic hybridization microarray (affymetrix cytoscan hd microarray system, scott and white healthcare molecular genetics laboratories), very long chain fatty acids (peroxisomal diseases laboratory, kennedy krieger institute), and lysosomal storage panels (lysosomal diseases testing laboratory, thomas jefferson university) were also normal . The proband and both parents were enrolled in our skeletal dysplasia study according to the baylor college of medicine's institutional review board . Informed consent was obtained specifically for molecular studies and for the publication of clinical information and photographs . Dna was extracted from peripheral blood monocytes and precipitated for polymerase chain reaction and sanger sequencing . Polymerase chain reaction was performed using primers designed to cover the entire coding region of tgds (supplemental table 1) with an annealing temperature of 55 c . Products were verified by agarose gel electrophoresis and then sequenced by chain - termination (sanger) sequencing using the same primers at beckman coulter genomics (danvers, ma). The proband and both parents were enrolled in our skeletal dysplasia study according to the baylor college of medicine's institutional review board . Informed consent was obtained specifically for molecular studies and for the publication of clinical information and photographs . Dna was extracted from peripheral blood monocytes and precipitated for polymerase chain reaction and sanger sequencing . Polymerase chain reaction was performed using primers designed to cover the entire coding region of tgds (supplemental table 1) with an annealing temperature of 55 c . Products were verified by agarose gel electrophoresis and then sequenced by chain - termination (sanger) sequencing using the same primers at beckman coulter genomics (danvers, ma). Sanger sequencing revealed that the proband was apparently homozygous for the c.298 g> t (p.ala100ser) pathogenic variant, the most common pathogenic variant identified by ehmke et al . . Sanger sequencing of dna samples from the parents revealed that both parents are heterozygous for this variant and thus, confirmed that the patient is homozygous (fig . To date, this report is only the second publication describing molecularly confirmed catel manzke syndrome, and our patient is only the eighth reported patient . We compared our patient's features with the features of the seven previously reported patients in the original manuscript describing pathogenic variants in tgds as the cause for catel manzke syndrome (table 1),,, . Our patient has pierre robin sequence and manzke dysostosis, the two classic features of catel manzke syndrome . In addition, he had a minor congenital cardiac defect that resolved and facial features (narrow nose, full cheeks) that have been described in a subset of the previously reported patients, . Notably, he did not have a cleft palate, a feature seen in six of the seven other molecularly confirmed patients . Our patient's respiratory complications place him on the more severe end of the spectrum of reported patients . In addition, he had significant failure to thrive requiring hospital admission, supplemental calories and gastrostomy tube placement . Although postnatal growth failure was noted in two of the seven previously reported patients, only one of these two patients was reported to require tube feedings, . Although it is unclear if early postnatal growth issues and failure to thrive are primary features of catel manzke syndrome, we suspect that our patient's failure to thrive was secondary to his severe complications from pierre robin sequence because weight gain improved with both gastrostomy and tracheostomy in place . Significant feeding difficulties and failure to thrive are common complications in patients with pierre robin sequence . Tgds encodes dtdp - d - glucose 4,6-dehydratase / growth - inhibiting protein 21, a protein whose function remains unknown although it has been hypothesized to play a role in proteoglycan metabolism . The finding of the c.298 g> t (p.ala100ser, rs140430952) pathogenic variant in our patient supports the hypothesis of ehmke et al . That this is the most common pathogenic variant associated with catel manzke syndrome . Taken together, the eight reported patients with pathogenic variants in tgds have the c.298 g> t variant at an allele frequency of 68.75% . Prior work has suggested that this variant is harbored on a 50 kb haplotype and thus suggests a common founder mutation, found primarily in the european population . Our patient is of mixed european ancestry and previously described patients with this mutation were of german, french, dutch and british / south american ancestry . A search of the exac database reveals that this variant has been observed 37 times in 121,256 alleles (allele frequency of 0.0003) in the heterozygous state . In addition to individuals reporting european ethnicity, the variant has also been observed in two individuals with african ethnicity, and one individual with ethnicity described as the amino acid substitution is present in the predicted nad binding domain, and it is suggested that the mechanism is loss of function . The absence of individuals with this variant in the homozygous state in the exac database provides further evidence of the pathogenicity . Our patient is on the severe end of the spectrum of previously reported patients with respiratory complications and failure to thrive requiring gastrostomy tube placement, supplemental calories, and tracheostomy but without cleft palate . Our finding of a homozygous p.ala100ser variant in tgds in our patient supports that this is a common mutation in catel manzke syndrome, and his phenotypic presentation underlies the severe respiratory and growth complications that can occur in association with pierre robin sequence in this disorder . The following is the supplementary data related to this article.supplemental table 1primers used for sequencing the coding region of tgds.
The number of complications associated with therapeutic colonoscopy has increased with the increasing number of these procedures . Of these complications, the incidence of perforation is even higher during endoscopic submucosal dissection (esd) in the colorectum [1 - 3]. The risk factors for colorectal esd - associated perforation include inexperience on the part of the endoscopist (i.e., has performed <50 esd procedures), a large tumor size, and submucosal fibrosis . Colonic perforation during colonoscopic polypectomy or colorectal esd occurs as a result of unintended endoscopic resection / dissection and/or thermal injury . In the former case, immediate perforation predominantly occurs; that is, colonic wall defects are seen during the procedure . However, in the case of thermal injury, the postprocedural ulcer base may demonstrate whitish coagulation damage, and delayed perforation mainly occurs . We here report a case in which delayed perforation occurred after performing esd on a laterally spreading tumor (lst) in the colon . This perforation was completely unexpected because there were no esd - associated risk factors for this complication in the patient, and no severe coagulation damage was apparent at the post - esd ulcer base . A 43-year - old man was referred to our center for the endoscopic resection of a nongranular lst (lst - ng) in the transverse colon . The colon was well prepared, and a flat, elevated lst - ng measuring 2022 mm was observed in the transverse colon . The lesion was type iv with a kudo pit pattern, and clinically suggestive of noninvasive neoplasia . A submucosal cushion was created with a saline injection, and no nonlifting sign or submucosal fibrosis was noted . Submucosal dissection was performed with a fixed flexsnare knife (kachu medico co., seoul, korea). After the dissection of the lesion (except for the central portion), the residual attached portion was snared and removed en bloc . The duration of electrocauterization (i.e., the duration of submucosal dissection and snaring) was 23 minutes, and the total procedure time was 43 minutes . No barotraumas, deep thermal injuries, or other complications such as immediate bleeding or perforation were noted during the procedure . The post - esd ulcer base was clean without bleeding, deep coagulation injury, or perforation (fig . 1). The patient had no abdominal pain and his vital signs were stable after colonoscopic resection . Sips of water were allowed, and he complained of severe abdominal pain several hours later . The same symptoms returned when sips of water were allowed a few hours later . Direct tenderness of the whole abdomen was noted on physical examination . His vital signs were stable; however, free air under the right diaphragm was noted on simple abdominal images (fig . His white blood cell count was 2800/l, and the c - reactive protein concentration was 5.85 mg / dl . Abdominopelvic computed tomography was performed 30 hours after the procedure, which revealed a pneumoperitoneum and wall irregularities at the endoscopic resection site (fig . 2). Empirical antibiotics were administered, and an emergency laparoscopic operation was performed because the symptoms rapidly aggravated and the whole abdomen demonstrated rigidity with rebound tenderness, suggesting severe, aggravating peritonitis . Primary repair was performed after massive irrigation of the soiled feces and removal of necrotic colon tissue . The final pathologic diagnosis of the endoscopic resection specimen was mixed traditional serrated adenoma and tubular adenoma with clear resection margins . 3). The surgically resected colon specimen demonstrated wall necrosis, neutrophil infiltration, and a large hematoma in the adjacent proper muscle layer (fig . Therapeutic colonoscopy demonstrates a higher perforation rate than does diagnostic colonoscopy (0.15% to 3%), and the mortality rate can be as high as 13% [1 - 3,5]. Our current case demonstrated delayed perforation, which is not uncommon, accounting for 6% to 40% of colon perforations, mainly after therapeutic colonoscopies [5 - 7]. Most notably, esd, which was originally developed for the en bloc resection of large or ulcerative gastric epithelial neoplasms, demonstrates a high perforation rate ranging between 1.7% and 20.4% [4,8 - 11]. In our current case, esd with snaring is a simplified alternative to esd alone for the en bloc resection of large colorectal lesions . Although the procedure duration of esd with snaring is usually shorter than that of esd without snaring, the perforation risk associated with esd with snaring was not significantly lower than that of esd without snaring (3% vs. 7%, p=0.24) in our previous study . Perforation can occur after colonoscopy and it is not a negligible complication, and therefore determining if the patient or the procedure has any risk factors for colon perforation would help promote safe clinical practice . The risk factors for colonoscopic perforation include female sex, comorbidities, older age (> 75 years), diverticulosis, bowel obstruction, and a history of intra - abdominal surgery . In the case of colorectal esd, a large tumor size (i.e., 50 mm), an inexperienced endoscopist, and submucosal fibrosis are well - known risk factors for perforation . However, none of these factors were present in our current case . In addition, during the procedure, there was neither a deep dissection that injured the proper muscle layer nor excessive thermal injury that resulted in severe coagulation damage to the submucosa and proper muscle . Furthermore, the endoscopic resection specimen did not reach the proper muscle layer or indicate severe tissue injury . We suggest several possible explanations for the unexpected perforation in our case, on the basis of the histopathology of the surgical specimen . First, when considering a large hematoma in the proper muscle layer adjacent to the perforation site, a high - tension colonic wall might lead to perforation . However, perforation did not occur at the center of hematoma where the wall tension might be highest . A second possibility is that acute infection followed by massive inflammation and necrosis in the colonic wall may have occurred because there was severe neutrophil infiltration . Therefore, it is difficult to know the causal relation between neutrophil infiltration and necrosis . Third, delayed perforation may occur because of the excessive electrical coagulation of vessels in the submucosa and proper muscle layer . A long duration of electrocauterization may predispose vessels to coagulation injury, thereby resulting in delayed perforation . In our case, this cautery duration is longer than the average cautery time of conventional polypectomy, which is usually <1 minute . Thus, the perforation mechanism in our current case remains unclear, and we believe several mechanisms together may have contributed to this adverse event . The treatments for perforation include surgical and nonsurgical management . In the past, surgery was the mainstay of treatment and medical management was only performed on select patients . However, as endoscopic clipping techniques have progressed, the need for and the frequency of surgery have decreased . Our group has reported the clinical courses of 38 perforations after both diagnostic (n=13) and therapeutic (n=25) colonoscopies . Of these cases, only nine patients (23%) required surgery . However, patients who receive a nonsurgical intervention should receive close follow - up examinations because immediate surgery needs to be performed if clinical deterioration occurs . Surgery is indicated for patients with large perforations, generalized peritonitis, ongoing sepsis, and deterioration despite being managed conservatively . In our current case, rapid progressive abdominal pain with tenderness and rebound tenderness developed in the whole abdomen, indicating severe, generalized peritonitis . Severe peritoneal soiling with large necrotic tissue at the endoscopic resection site was noted in the surgical field . We believe that endoscopic clipping may have resulted in the adverse outcome in our case because we could not clean the soiled content, and successful clipping may have been prevented by the necrotic wall . Thus, we believe the decision to perform emergency surgery was appropriate . Therapeutic colonoscopy, which has dramatically evolved in recent years, is an important modality for the treatment of colonic neoplasm . However, as shown in our current case, unexpected critical complications such as perforation can develop . Endoscopists should always keep in mind that perforation can develop even after clean procedures, and should closely evaluate any complaints about unexplained abdominal pain after therapeutic colonoscopy procedures, including polypectomy and esd.
Autoimmune overactivation, vascular malfunction, and collagen overproduction are the primary causes of ssc; a multisystem fibrotic disease causing vasculopathy the main complications of which affect the skin, muscles, joints, and internal organs . Musculoskeletal involvement is common among patients with ssc and it is also a main cause of disability . The hands are the most commonly affected part of the body including: hand deformities, loss of flexion of the metacarpophalangeal joints, loss of extension of the proximal and distal interphalangeal joints, loss of thumb abduction, flexion, and opposition, and loss of wrist motion in all planes resulting in the classic claw - hand deformity . Ssc generally results in disfigurement to visible and socially relevant parts of the body (particularly the hands, mouth, and face), resulting in body image dissatisfaction not unlike patients with severe burn injuries . Perhaps because deteriorating psychological function and psychological distress erode a person's self - esteem . Women with ssc have a lower self - esteem than burn patients with observable deformities, as thickening skin is both disabling and cosmetically disconcerting . The typical hand deformations result in functional disability as related to activities in daily living requiring object manipulation, grasping, and pinching . Approximately 90% of patients with ssc confirm the loss of both hand movement and dexterity (gross and fine motor control). Hand deformity, expanded fingers, and decreased wrist extension are the main risk factors leading to hand disability . Hand function is one of the greatest concerns among patients with ssc including: muscle strength, grasp patterns, precision and accuracy, anatomical integrity, sensation, motivation, coordination and dexterity, unilateral and bilateral tasks, and daily living tasks . Pharmacological treatments used include: (a) vasodilators and antiplatelet aggregation drugs to improve peripheral blood circulation, (b) immunosuppressant drugs to prevent the synthesis and release of harmful cytokines, and (c) agents that reduce collagen synthesis to inhibit or reduce fibrosis; while nonpharmacological treatments include manual therapies . Crucially, a hand rehabilitation program is needed for prevention of hand deformity and maintaining quality of life . Recent studies by vannajak and colleagues reported that traditional thai massage (ttm) on the upper extremities in patients with ssc can increase hand mobility in the immediate and short - term and increase hand skin temperature for half an hour . Hand exercises including finger stretching, upper extremity stretching, occupational therapy, and mcmennell joint manipulation have been reported to improve hand function through active exercise (active range of motion) combined with connective tissue massage . Both prior studies measured hand mobility in scleroderma (hamis) and the test results showed that the use of heating modalities in conjunction with physical therapy improved extensibility of collagen tissue along with tendon and joint capsules; underscoring the importance of superficial heating modalities or thermal glove insulation for keeping the hands warm and increasing extensibility . Secondary raynaud's phenomenon (srp) generally occurs and is serious in ssc when the patient is exposed to cold or to emotional stressors . In a cold environment, and especially in winter, even a small decrease in temperature can aggravate the vasculitis in the hands . In order to maintain good hand function for adls, when patients with ssc are exposed to cold the hands need to be protected against heat loss from heat convection and/or direct contact with cold . Wearing gloves is a simple, safe, and easy way to prevent hand heat loss and direct contact with cold . The objective of this study was to evaluate the short - term effect on hand mobility of thermal insulation gloves in combination with a daily home program (including ttm, stretching, and superficial heating) among patients with ssc . This was a randomized control trial conducted with 28 scleroderma (ssc) patients (both males and females) from the rheumatology department, srinagarind hospital, khon kaen university, at the faculty of associated medical sciences, khon kaen university, thailand . The study was conducted in patients who were already on vasodilator drugs (depending on rheumatologist, i.e. Nifedipine), which was administered by rheumatologist, specialist in scleroderma . The study was approved by the khon kaen university ethics committee for human research based on the declaration of helsinki and the international conference on harmonization ich good clinical practice guidelines (order: 4.2.01: 38/2011, number: he541337). Scleroderma patients (diffuse subtype) were recruited to the study, which was conducted in the thai cool season (december 2012-january 2013). Exclusion criteria were: (a) a history of hand surgery within 6 months, (b) an open wound or ulceration on the hand, (c) loss of tactile and proprioceptive sensation in the hand, (d) diabetes mellitus, (e) smoking, or (f) a psychiatric condition . Participants were asked to sign a consent form after which they were randomly allocated into one of two groups (experimental treatment or control group). Then each was given a hamis hand function test by a physical therapist . The experimental group performed the daily home program wearing gloves, while the control group did the program without gloves . All of the participants had superficial heat (i.e. Warm water ~ 40c) applied to the hands for 20 min . Then a relative gave ttm to the upper extremities, from the neck to the finger tips, for 30 min (15 min on each side). Seven stretching positions were done following the ttm: (a) forearm supination / pronation, (b) wrist flexion / extension, (c) radial / ulnar deviation, (d) finger abduction / flexion / extension, (e) thumb abduction / adduction, (f) flexion / extension, and (g) thumb opposition [figure 1]. Patients began by gradually stretching to the point of resistance, then holding for 30 seconds . They repeated this four times on each side, once a day, for 2 weeks . (patients did the stretching independently after instruction and supervision by the physical therapist .) Next, the patients in the glove group put on the gloves for 6 h per day . Stretching exercise in daily home program, (a) forearm supination, (b) forearm pronation, (c) wrist flexion, (d) wrist and finger extension, (e) wrist radial deviation, (f) wrist ulnar deviation, (g) finger abduction, (h) finger flexion, (i) thumb abduction, (j) thumb adduction, (k) thumb flexion, and (l) thumb extension data for hamis was collected and expressed as a median: (q1q3). Any significant difference between the daily home program with gloves and without after 2 weeks of treatment differences between the groups would be considered significant at a p <0.05 [figures 2 and 3]. Change in left and right hand mobility after 2 weeks of daily home program flow chart of the study the study had 28 participants: the glove - wearing group (nine females; five males) and daily home program group (four females; 10 males). The diagnosis was the diffuse, cutaneous systemic sclerosis (dcssc) subtype in the indurative phase . The modified rodnan skin thickness score (mrss) was 1 - 2 at the dorsum of the forearm, hand and between 3 and 6 at the proximal phalange of the 3 finger (0 = normal skin thickness, 1 = mild, 2 = moderate, and 3 = severe). Hamis is a performance index comprising nine items normally constituting in an ordinary range of motion (a score between 9 and 18 reflects a high degree of deformity). Baseline characteristics of participants are presented in table 1, while changes in hand mobility after the 2 week study is shown in table 2 and figure 1 . Baseline characteristic of patients with ssc hand mobility in scleroderma patients change after gloving in combination with ttm prevention of hand deformity is a core goal and is one reason for the need for physical therapy . The aim of the current study was to compare the therapeutic effect between a daily home program with and without thermal insulation (wearing gloves) as measured by hand mobility (hamis). Accordingly, wearing gloves as part of the daily home program represented a benefit viz - - viz hand mobility . Vannajak and colleagues reported that ttm also aids with hand mobility among patients with ssc . Although their study did not include any stretching exercise because they were focused on isolating the effect of ttm on hand mobility, other studies used ttm augmented by daily home programs with heat and/or stretches appropriate to the individual's condition, disease stage, and expectation for improvement of hand mobility in the indurative phase . Wearing gloves in combination with ttm and a daily home program may be particularly useful in winter . The combination of ttm + heat + stretching produced a synergistic therapeutic effect . In the current study, the gloves were made of nylon fabric, selected because of the results of a previous in vitro study about the characteristics of material and insulation properties . After the 2 weeks of routine treatment there was no difference between the two groups in mobility, on the basis of ttm and stretching alone, but wearing gloves plus the other treatments clearly improved finger flexion, finger extension, thumb abduction, and pincer grip aspects . Even though the median hamis score was equal between groups, wearing gloves in combination with ttm and the daily home program was a better method . In winter, gloves provide protection from exposure to cold which aggravates severe srp in ssc . If an srp attack is not prevented, vascular permeability and vasospasm will result in sclerosis of vascular, severe pain, and disturbed daily living . The daily home program in our study indicated an increase in hand mobility among patients with ssc, which agrees with a previous study on active range of motion and another on individual rehabilitation programs . Vannajak and colleagues demonstrated that ttm can increase hand skin temperature and hand mobility in both the immediate and short - term . The results of the current study underscore that primary self - care by hand exercise is beneficial in increasing hand mobility which leads to improved quality of life among patients with ssc, and is especially effective when combined with manual therapy and wearing gloves to prevent cold exposure leading to srp . A limitation of this study was its short duration; notwithstanding, the results indicate a measurable benefit for the group wearing gloves with the daily home program . Further study will be conducted on microvascular structure alteration with a longer duration follow - up . After 2 weeks, the daily home program helped to improve hand mobility among patients with ssc . Wearing gloves in combination with the daily home exercises was effective, safe, and led to increased self - esteem . The quality of life score among patients with ssc also improved as a result of increased hand mobility . The suggestion was larger sample size and longer duration of study is desired to further confirm these observations . Finally, wearing gloves in combination with ttm, heat, and stretching may help to improve hand mobility among patients with scleroderma.
Aortocaval fistula (acf) mostly results from ruptured abdominal aortic aneurysm (raaa), with a reported overall prevalence of 2% to 6% in patients with raaa . These patients are always under emergency situations, which have a high mortality, but their clinical presentations may be quite variable . Because this disease has variable clinical manifestations, the diagnosis is usually delayed, which increases the difficulties in treatment . Both conventional open repair (or) and endovascular aneurysm repair (evar) have been used to treat this condition . However, no patients with abdominal aortic dissecting aneurysm rupturing into inferior vena cava (ivc) have been reported, and there is also no standard therapy for this condition . Because morphologic and hemodynamic characteristics of dissection are different from abdominal aortic aneurysm (aaa), evar may be a more valid alternative over or in these patients . We present a patient with acf from rupture of abdominal aortic dissecting aneurysm, which had a chronic course of disease . Evar was successfully performed for this patient, without endoleak and fistula at the follow - up . Prior to referral to our institution, the 70-year - old man had recurrent cough, anhelation, and chest distress . About half a year before he came to our hospital, he had temporary abdominal pain, which disappeared spontaneously and suddenly . He was treated as pneumonia at the local hospital . However, these symptoms aggravated gradually, so he was transferred to our hospital for further treatment . He had a history of tobacco abuse and chronic obstructive pulmonary disease, but no history of hypertension or heart disease . Physical examination revealed blood pressure of 126/63 mm hg, heart rate of 85 beats / min, and slight pitting edema of both lower extremities below the knee . Moist crackles could be documented on both lungs, and respiratory sounds were weakened at bilateral lower lobe of lung . No abdominal tenderness or rebound tenderness was found, but a pulsating mass with continuous vascular bruit was recognized at the lower abdomen, without abdominal thrill . Laboratory results showed no hepatic or kidney dysfunction, hgb of 124 g / l, wbc of 12.29 10/l, bnp of 2625 pg / ml, troponin - t of 40.6 ng / l . Chest computed tomography scans before admission showed severe pulmonary edema and inflammation (figure 1a). A later computed tomography angiography (cta) revealed an abdominal aortic dissecting aneurysm with an associated aortocaval fistula, no bleeding or hematoma in retroperitoneal space (figure 1b). Echocardiography demonstrated that left ventricular ejection fraction was 45%, without abnormality of heart structure . A, chest ct scans before admission revealed bilateral diffuse interstitial infiltrations and patchy shadow in both lungs, partial pulmonary atelectasis of both inferior lobes, together with a little pleural effusion . B, preoperative reconstructed ct scan showed dissecting aneurysm and early opacification of inferior vena cava (ivc), which indicated dissecting aneurysm had ruptured to form an aortocaval fistula (arrow). This elderly patient had a severe pulmonary disease and chronic heart failure (chf), thus traditional or was extremely risky for him . However, because of the ruptured abdominal aortic dissecting aneurysm and the persistent venous hypertension, this patient had a critical need for operation . Minimally invasive endovascular intervention was performed immediately after the diagnosis was confirmed . Under general anesthesia, bilateral common femoral arteries this patient's anatomy was suitable for evar according to cta and intraoperative angiography (figures 2 and 3a). Because the angle of neck was about 65 and this patient's rarely complex condition, we implanted medtronic endurant suprarenal aortic devices . A bifurcated stent graft (enbf2516c170ee), 2 contralateral iliac limbs (enlw1613c120ee, enlw1620c120ee), and an ipsilateral extension (enlw1616c95ee) were placed . After these stent grafts were placed, proximal type i endoleak and fistula could be seen from angiography . To close the endoleak and fistula, finally, the endoleak and aortocaval fistula disappeared, which suggested evar was successful (figure 3). A, computed tomographic angiography (cta) revealed false lumen of aorta (ao) directly rupturing into inferior vena cava (ivc) through a 6.9 mm fistula (arrow a), and the maximal diameters of this dissecting aneurysm was 89 mm . B, cta showed the aorta divides into true and false lumen through a 27.4 mm tear (arrow b), which formed the abdominal aortic dissecting aneurysm . C, cta showed the diameter of neck was 21.3 mm (arrow c). D, reconstructed ct scan revealed the length of neck is about 20 mm (arrow d), and the angle of the neck is about 65. ct = computed tomography . A, angiography showed fistula (arrow a) between false lumen of dissecting aneurysm and early opacification of inferior vena cava (ivc). B, after endografts were placed, angiography revealed proximal type i endoleak (arrow b) and early opacification of ivc (arrow c). C, after balloon dilatation was performed, angiography showed the endoleak and aortocaval fistula had disappeared . He experienced a lengthy postoperative course including 20 days in intensive care unit due to pulmonary edema and pneumonia . He was initially maintained on venous thrombus embolism (vte) prophylaxis with heparin during operation and then switched to low molecular weight heparin after operation . When he discharged, edema of both lower limbs had resolved, and not only the pulmonary edema but also pneumonia had significantly relieved (figure 4a). On the follow - up of 9th month, cta showed successful endovascular exclusion without endoleak or recurrent fistula . A, chest ct scan before discharge showed pulmonary edema and inflammation had significantly relieved . From (b) and (c), cta at the follow - up of 9th month after operation revealed no endoleak or fistula . It is reported that the overall prevalence of acf is quite low, about 2% to 6% in patients with raaa . The most common cause of acf is aneurysm erosion, especially in patients with raaa . On account of increased venous hypertension however, acf usually results from raaa, so its most frequent symptoms should be abdominal pain and hemorrhagic shock . In our case, without bleeding in the retroperitoneal space, abdominal aortic dissecting aneurysm ruptured directly into ivc to form a fistula, which led to venous hypertension . Therefore, the hemodynamics and clinical presentations of this patient were different from cases before . In addition, fistula between false lumen of dissecting aneurysm and ivc is small, and the blood flow of false lumen in this case is much lower than that of true lumen . Therefore, the low blood flow of this fistula leads to chf and chronic pulmonary congestion, and the chronic pulmonary congestion may be the reason of repeated pneumonia for this patient . The dramatic clinical presentation in this case is rare, which leads to delay of diagnosis . Another potential reason of delayed diagnosis may lie in the local doctor's lack of associated knowledge . According to some reports, early diagnosis and intervention can double survival rates from 25% to 50% . The diagnosis was not made until enhanced computed tomography was performed half a year after initial presentation, which was unfortunate for the patient . The venous hypertension had leaded to a series of severe complications, such as pneumonia and heart failure . In addition, patients with ruptured aortic aneurysm need emergency operation according to the guideline . It is unknown whether endovascular intervention improves survival compared with traditional or for this disease . With the risk related to chf, massive intraoperative blood loss, and pulmonary infection, the surgical mortality of open repair ranges from 16% to 66% . Evar is valid in certain anatomical configurations and may be the optimal selection in terms of stent graft available . And evar has potential benefits of less blood loss and no need for aortic cross - clamping during operation . Several cases of evar for raaa with acf have been described, with about 30% mortality, which had partially due to delays in diagnosis . Accompanied with chf and chronic pulmonary congestion, the general condition of the elderly patient in our case was poor, he was not suitable for or . Meanwhile, the anatomical configurations of our patient were suitable for evar . Furthermore, the morphologic and hemodynamic characteristics of dissecting aneurysm in our case are different from aaa or dissection before . Therefore, evar was the best choice for this patient . In our case, evar alone appears to have successfully isolated abdominal aortic dissecting aneurysm and closed the acf without the need of a stent - graft in the ivc . Because of aortocaval fistulas and type ii endoleak, persistent communication between the aneurysm and the ivc may exist . In some literatures, early placement of stent graft in the ivc . However, no evidence demonstrated stent graft of ivc should be placed at the time of initial evar . Because the chance of persistent aortocaval fistula after evar is rather small, endoleak and fistula if persistent aortocaval fistula occurs, stent graft in ivc can be performed in the future . In our case, no fistula or endoleak were found at the follow - up of the 9th month . In addition, because aneurysm sac debris may dislodge, prophylaxis of pulmonary embolism should be addressed . In our case, however, the chance of clinically significant pulmonary embolism is minute, and ivc filter placement may complicate the possible further ivc stent - graft placement . Diagnosis of this patient was delayed, but he fortunately had a successful operation and recovered finally . Despite his poor general status and the high mortality associated with this condition, his complete recovery presents the potential for recovery with appropriate treatment . Further educational programs should be developed, which may give rise to earlier diagnosis and treatment with better outcomes.
The term macrolide encompasses a diverse family of unrelated compounds with large macrolactam rings . Erythromycin a, the prototype macrolide antibiotic was isolated from a philippine soil sample in the 1940s and was first marketed in 1952 as an alternative therapy to beta lactam agents for the treatment of infections with gram - positive cocci . During the 1990s clarithromycin, roxithromycin, and azithromycin were introduced . Macrolide antibiotics inhibit rna - dependent protein synthesis by reversibly binding to the 50s ribosomal subunit of a susceptible microorganism . Macrolides are widely used to treat infections of soft tissues and of the respiratory tract due to their efficacy against gram - negative and gram - positive bacteria, including intracellular germs such as chlamydia and legionella [24]. Their main side effects are nausea, vomiting, diarrhea, and abdominal pain, which become more evident when erythromycin is used in place of the other macrolides . Mounting evidence suggests that macrolide antibiotics have both anti - inflammatory and immune - modulatory properties and are thus beneficial to chronic pulmonary diseases such as diffuse panbronchiolitis, cystic fibrosis, asthma, and bronchiectasis . These properties were suspected upon the realization that erythromycin decreased the need for corticosteroids in asthma treatment . It must be pointed out that immune modulation is the suppression of inflammation and immune hyperactivation without causing immune depression (immunosuppression). Effects on the generation and release of various cytokines involved in the inflammatory process have been studied both in vivo and in vitro . Interest in the immunomodulatory effects of macrolides began in the 1960s with the observation that the 14-member antibiotic, troleandomycin, was an effective steroid - sparing it has been more than 20 years since the immunomodulatory effects of macrolides were accepted as a standard of care for the treatment of diffuse panbronchiolitis (dpb) in japan . Erythromycin and clarithromycin are also widely used in japan for the therapy of sinusitis and chronic obstructive pulmonary disease (copd). In more recent years, azithromycin has been widely adopted as immunomodulatory agents for the treatment of cystic fibrosis (cf) and bronchiectasis . The anti - inflammatory effects of macrolides are significant . The historical change in the natural course of diffuse panbronchiolitis (dpb), a fatal disorder of the airways, following the introduction of erythromycin in its treatment has focused attention of researchers on the anti - inflammatory properties of macrolides . The clinical impact on diffuse panbronchiolitis (dpb) the immunomodulatory activity of macrolides has been a source of mechanistic research as well as clinical research in non - dpb inflammatory airway disease . Suppression of neutrophilic inflammation of the airways has been demonstrated as the most robust immunomodulatory response from 14- and 15-membered ring macrolides . Macrolide antibiotics are known for their efficacy in treating acute airway infections, but just as importantly, they are also effective anti - inflammatory agents . Their anti - inflammatory properties have been studied most thoroughly in chronic inflammatory airway diseases, particularly diffuse panbronchiolitis (dpb). Erythromycin, azithromycin, clarithromycin, and roxithromycin inhibit chemotaxis and infiltration of neutrophils into the airway and, subsequently, decrease mucus secretion . Mucus formation, a significant cause of morbidity and mortality in patients with chronic airway inflammation, is directly inhibited by macrolides and suppressed by decreased inflammation in the airway . The mechanisms of action for the anti - inflammatory properties of the macrolides are clearly multifactorial . Macrolides inhibit the production of many proinflammatory cytokines, such as interleukin (il)-1, il-6, il-8, and tumor necrosis factor - alpha, perhaps by suppressing the transcription factor nuclear factor - kappa b or activator protein-1 . Inhibition of cytokine production has been seen in vitro and also in bronchoalveolar lavage fluid, which contains less il-8 and fewer neutrophils after treatment with macrolides . Macrolides also inhibit formation of leukotriene b4, which attracts neutrophils, and inhibit the release of superoxide anion by neutrophils that may be present in the airway . Together, these anti - inflammatory effects result in improved pulmonary functions and fewer airway infections . In patients with dpb, . These effects might be pharmacological functions of the macrolide itself, independent of antibiotic effects . Apart from antibacterial effects, macrolides have effects on neutrophil function (decreased oxidant production, apoptosis) and on the production of cytokines involved in the inflammation cascade (decreased production of il-1, il-6, il-8, and tnf and increased production of il-10 and, possibly, il-4). With regard to t lymphocytes, erythromycin (em) and its derivatives inhibit t - lymphocyte proliferation and induce t - lymphocyte apoptosis [14, 15]. In this paper, we present a collection of clinical studies and case reports dealing with the potential benefits of macrolides antibiotics in the treatment of selected dermatoses which have primarily been classified as noninfectious . Studies that dealt with the effects of macrolides as anti - inflammatory and immune - modulator in skin and hair disorders were included . (a) macrolides and intractable rosacearosacea is a common cutaneous disorder which occurs most frequently in light - skinned, middle - aged women . There are variable cutaneous signs of rosacea such as flushing, erythema, telangiectasia, edema, papules, and pustules .conventional treatment of rosacea is based on a combination of systemic and topical antibiotics . Since the 1950s, the therapeutic activity of commonly used antimicrobials including tetracycline, doxycycline has been mainly attributed to their anti - inflammatory activities . However, long - term treatment with antibiotics is not well tolerated due to requiring frequent administration, poor compliances and side effects including gastrointestinal intolerance, photosensitivity, and candidiasis .azithromycin is effective in treating rosacea . Facial skin biopsies were taken from 17 subjects with papulopustular rosacea and 25 healthy controls . Rosacea patients had greater skin reactive oxygen species levels than healthy controls (p <0.001). Rosacea subjects then received oral azithromycin 500 mg on three days each week for 4 weeks . A statistically significant decrease in chemiluminescence, a measurement of the generation of reactive oxygen species, was demonstrated after treatment with azithromycin .the utility of oral azithromycin was confirmed by several other clinical studies . Fernandez - obregon reported that all of ten patients who were not tolerated or controlled by conventional treatment of rosacea demonstrated a significant improvement with the oral use of azithromycin . In addition, modi et al . Treated a 67-year - old man who had photosensitivity to the doxycycline and hyperpigmented dyschromia to the minocycline with an oral use of azithromycin in a dose of 250 mg 3 times weekly . Reported that treatment with oral azithromycin led to 75% decreases in the total number of lesions and an 89% decrease in inflammatory lesions compared with basal status . Another open - label study showed that azithromycin is as effective as standard dose of doxycycline and has a positive impact on the quality of life of patients compared with conventional treatment regimens .kim et al . Treated a 52-year - old woman who had intractable rosacea not responding to various conventional treatments including topical benzoyl peroxide and metronidazole as well as oral metronidazole, isotretinoin, and doxycycline, by using oral azithromycin 500 mg per day for 2 weeks . The authors reported that the lesions had mostly disappeared, and no specific side effects related to the azithromycin were noted . Rosacea is a common cutaneous disorder which occurs most frequently in light - skinned, middle - aged women . There are variable cutaneous signs of rosacea such as flushing, erythema, telangiectasia, edema, papules, and pustules . Conventional treatment of rosacea is based on a combination of systemic and topical antibiotics . Since the 1950s, tetracycline and erythromycin are the most commonly used oral antibiotics . The therapeutic activity of commonly used antimicrobials including tetracycline, doxycycline has been mainly attributed to their anti - inflammatory activities . However, long - term treatment with antibiotics is not well tolerated due to requiring frequent administration, poor compliances and side effects including gastrointestinal intolerance, photosensitivity, and candidiasis . Facial skin biopsies were taken from 17 subjects with papulopustular rosacea and 25 healthy controls . Rosacea patients had greater skin reactive oxygen species levels than healthy controls (p <0.001). Rosacea subjects then received oral azithromycin 500 mg on three days each week for 4 weeks . A statistically significant decrease in chemiluminescence, a measurement of the generation of reactive oxygen species, fernandez - obregon reported that all of ten patients who were not tolerated or controlled by conventional treatment of rosacea demonstrated a significant improvement with the oral use of azithromycin . In addition, modi et al . Treated a 67-year - old man who had photosensitivity to the doxycycline and hyperpigmented dyschromia to the minocycline with an oral use of azithromycin in a dose of 250 mg 3 times weekly . Reported that treatment with oral azithromycin led to 75% decreases in the total number of lesions and an 89% decrease in inflammatory lesions compared with basal status . Another open - label study showed that azithromycin is as effective as standard dose of doxycycline and has a positive impact on the quality of life of patients compared with conventional treatment regimens . Kim et al . Treated a 52-year - old woman who had intractable rosacea not responding to various conventional treatments including topical benzoyl peroxide and metronidazole as well as oral metronidazole, isotretinoin, and doxycycline, by using oral azithromycin 500 mg per day for 2 weeks . The authors reported that the lesions had mostly disappeared, and no specific side effects related to the azithromycin were noted . (b) macrolides and adult - onset still's disease (aosd)adult - onset still's disease (aosd), an autoinflammatory syndrome of unknown etiology, typically manifests with spiking fevers, polyarthritis, and characteristic evanescent rash . Thanou - stavraki et al . Described a young woman with aosd complicated by calf fasciitis that serendipitously responded to clarithromycin administered for another indication . Although aosd pathogenesis remains unclear, a role for dysregulation of innate immunity is suggested . Based on this possible innate immune mechanism, the investigators suspected that macrolides may have induced a therapeutic response in this patient with aosd.saviola et al . Treated six cases of aosd with clarithromycin (cm) in combination with low - mild dose of glucocorticoids (gc), and methotrexate (mtx). Four of them were not responsive to high - dose gc added to disease - modifying antirheumatic drugs (dmards), while two of them were treated with low - mild dose of gc added to cm from the beginning . Cm, 500 mg b.i.d ., was added to a mild - low dose of gc and to mtx . The dose of the drugs was reduced (and stopped where possible) following clinical and laboratory parameters . 5 patients reached acr 70% and could stop any therapy in 618 months; 1 continued chronic therapy with low - dose gc added to cm and mtx to maintain acr 50% . The authors reported that cm can be a useful drug for the treatment of aosd, even in patients not responsive to high - dose gc and dmards . Adult - onset still's disease (aosd), an autoinflammatory syndrome of unknown etiology, typically manifests with spiking fevers, polyarthritis, and characteristic evanescent rash . Thanou - stavraki et al . Described a young woman with aosd complicated by calf fasciitis that serendipitously responded to clarithromycin administered for another indication . Although aosd pathogenesis remains unclear, a role for dysregulation of innate immunity is suggested . Based on this possible innate immune mechanism, the investigators suspected that macrolides may have induced a therapeutic response in this patient with aosd . Treated six cases of aosd with clarithromycin (cm) in combination with low - mild dose of glucocorticoids (gc), and methotrexate (mtx). Four of them were not responsive to high - dose gc added to disease - modifying antirheumatic drugs (dmards), while two of them were treated with low - mild dose of gc added to cm from the beginning . Cm, 500 mg b.i.d ., was added to a mild - low dose of gc and to mtx . The dose of the drugs was reduced (and stopped where possible) following clinical and laboratory parameters ., 5 patients reached acr 70% and could stop any therapy in 618 months; 1 continued chronic therapy with low - dose gc added to cm and mtx to maintain acr 50% . The authors reported that cm can be a useful drug for the treatment of aosd, even in patients not responsive to high - dose gc and dmards . (c) macrolides and sapho syndromein 1987, synovitis, acne, pustulosis, hyperostosis, and osteitis (sapho) syndrome was proposed as an umbrella term for a group of diseases with similar musculoskeletal manifestations, in particular hyperostosis of anterior chest wall, synovitis, and multifocal aseptic osteomyelitis, observed in association with dermatologic conditions such as palmo - plantar pustulosis, severe acne, and hidradenitis suppurativa . Despite recent advances in the understanding of the epidemiologic, pathophysiologic, and immunogenetic mechanisms involved in sapho syndrome, etiopathogenesis remains poorly understood . Propionibacterium acnes, the microorganism associated with acne, has been recovered on bone biopsy in some patients, but the possible pathogenetic role of an infectious agent in a genetically predisposed individual, resulting in exaggerated inflammatory response as reactive osteitis, is a largely unproven hypothesis .schaeverbeke and colleagues reported one case of successful treatment of a sapho patient with azithromycin . Kirchhoff and colleagues presented data for seven patients being treated successfully with azithromycin over 5 months . . Reported five patients with sapho syndrome (3 women; 2 men), ages 27 to 44 years, showed remarkable response to treatment with macrolide antibiotic (clindamycin) and nonsteroid anti - inflammatory drugs (lornoxicam). All patients did well and remained symptom - free for up to four years, after a 38-month course of treatment . The authors concluded that appropriate therapy with antibiotics and nsaids can produce rapid symptom resolution, while avoiding unnecessary procedures and long - term antibiotic therapy . In 1987, synovitis, acne, pustulosis, hyperostosis, and osteitis (sapho) syndrome was proposed as an umbrella term for a group of diseases with similar musculoskeletal manifestations, in particular hyperostosis of anterior chest wall, synovitis, and multifocal aseptic osteomyelitis, observed in association with dermatologic conditions such as palmo - plantar pustulosis, severe acne, and hidradenitis suppurativa . Despite recent advances in the understanding of the epidemiologic, pathophysiologic, and immunogenetic mechanisms involved in sapho syndrome, propionibacterium acnes, the microorganism associated with acne, has been recovered on bone biopsy in some patients, but the possible pathogenetic role of an infectious agent in a genetically predisposed individual, resulting in exaggerated inflammatory response as reactive osteitis, is a largely unproven hypothesis . Schaeverbeke and colleagues reported one case of successful treatment of a sapho patient with azithromycin . Kirchhoff and colleagues presented data for seven patients being treated successfully with azithromycin over 5 months . . Reported five patients with sapho syndrome (3 women; 2 men), ages 27 to 44 years, showed remarkable response to treatment with macrolide antibiotic (clindamycin) and nonsteroid anti - inflammatory drugs (lornoxicam). All patients did well and remained symptom - free for up to four years, after a 38-month course of treatment . The authors concluded that appropriate therapy with antibiotics and nsaids can produce rapid symptom resolution, while avoiding unnecessary procedures and long - term antibiotic therapy . (d) macrolides and psoriasispsoriasis is a well - known clinical description of an inflammatory skin disorder with other manifestations of what, until now, has been considered as a single disease entity . The characteristic skin lesion is persistent, erythematous, indurated and scaly, reflecting infiltration of inflammatory cells and increased proliferation and turnover of keratinocytes . The infiltrates in the dermis and the deeper layer of the epidermis mostly comprise of macrophages and t cells . Stimulation of dendritic cells and macrophages, which are called antigen - presenting cells, results in the activation of t - helper (th) cells . These differentiate into ifn - gamma, producing th 1 cells, and il-17, producing th 17 cells . Interaction of these cells with macrophages, mast cells, and neutrophils results in cytokine release and inflammation, leading to keratinocyte proliferation .psoriasis is characterized by the presence of neutrophil overactivation and overproduction of interleukin (il)-6 and il-8 from keratinocytes . It is now clear that macrolide antibiotics inhibit the production of many proinflammatory cytokines, such as il-6, il-8, and tumor necrosis factor (tnf)-, perhaps by suppressing the transcription factors nuclear factor (nf)-b or activator protein-1, and reduce neutrophil activity . Although in some studies it has been reported that intervention by antibiotics is not beneficial [35, 36], other studies have shown efficacy of macrolides in psoriasis .a high incidence of streptococcal throat infection as the main trigger for psoriasis exacerbations favors streptococcal antigens as a causative agent, which may induce cross - reactive t - cell responses against skin components [37, 38]. Deserves special attention, where ten patients with chronic plaque psoriasis were enrolled and advised to take 150 mg roxithromycin (a macrolide) orally twice daily for 1 to 7 weeks . Six out of the ten patients exhibited a decrease in psoriasis area and severity index (pasi) score . The mechanism by which macrolides downregulates the host inflammatory response was unclear but certainly multifactorial.macrolides, as a class, and azithromycin in particular, have a characteristic immunomodulatory and anti - inflammatory potential, in addition to their main antibacterial action against streptococci . This macrolide probably also suppresses immunological events in interferon gamma - treated keratinocytes, including expression of mhc class ii, secretion of il-1 alpha, and superantigen presenting ability [40, 41].saxena and dogra tried oral azithromycin in a single blind randomized case - control trial . 50 patients with moderate - to - severe chronic plaque psoriasis were enrolled . Of these, 30 randomly selected patients received azithromycin for 48 weeks as a single oral 500 mg daily dose for 4 days with a gap of 10 days (total 24 such courses). The remaining 20 patients received a vitamin c tablet (nonchewable) in the same dosage schedule . A significant improvement in pasi score was noted from 12 weeks in the majority of patients in the azithromycin group . At the end of 48 weeks, 18 patients (60%) showed excellent improvement, while 6 patients (20%) showed good improvement, and 4 patients (13.33%) showed mild improvement . A significant improvement in the skin lesions was noted at 12 weeks of azithromycin therapy . Based on this study, the authors reported that the results substantiated the hypothesis that chronic ongoing stimulus by the streptococci or its superantigen was indispensable in maintaining the disease.17 subjects participated in an open trial of macrolides for treatment of psoriasis . Mean pasi scores dropped significantly, and itch was reduced in 11 subjects after therapy . This study showed that macrolide antibiotics may be effective for treatment of psoriatic skin lesion, and that they may have antipruritic effects the reason for the antipruritic effect is not known; however, it is suggested possibly that macrolide antibiotics inhibit production of cytokines or neuropeptides that cause pruritus .polat the patients were divided into two treatment groups: one to receive erythromycin and topical steroids and the other only topical steroids: the first group were treated with erythromycin 1000 mg / day and topical corticosteroids for 4 weeks, while the control group were treated only with topical corticosteroids . There was no significant difference between the baseline mean psoriasis area and severity index (pasi) of the two groups . They reported that the treatment used for the study group was more effective against pruritus than that used for the control group . Six patients with severe pruritus and six patients with moderate pruritus in the study group found that itch disappeared completely after the treatment . In the control group, none of the patients with severe or moderate pruritus found that itch disappeared completely . Psoriasis is a well - known clinical description of an inflammatory skin disorder with other manifestations of what, until now, has been considered as a single disease entity . The characteristic skin lesion is persistent, erythematous, indurated and scaly, reflecting infiltration of inflammatory cells and increased proliferation and turnover of keratinocytes . The infiltrates in the dermis and the deeper layer of the epidermis mostly comprise of macrophages and t cells . Stimulation of dendritic cells and macrophages, which are called antigen - presenting cells, results in the activation of t - helper (th) cells . These differentiate into ifn - gamma, producing th 1 cells, and il-17, producing th 17 cells . Interaction of these cells with macrophages, mast cells, and neutrophils results in cytokine release and inflammation, leading to keratinocyte proliferation . Psoriasis is characterized by the presence of neutrophil overactivation and overproduction of interleukin (il)-6 and il-8 from keratinocytes . It is now clear that macrolide antibiotics inhibit the production of many proinflammatory cytokines, such as il-6, il-8, and tumor necrosis factor (tnf)-, perhaps by suppressing the transcription factors nuclear factor (nf)-b or activator protein-1, and reduce neutrophil activity . Although in some studies it has been reported that intervention by antibiotics is not beneficial [35, 36], other studies have shown efficacy of macrolides in psoriasis . A high incidence of streptococcal throat infection as the main trigger for psoriasis exacerbations favors streptococcal antigens as a causative agent, which may induce cross - reactive t - cell responses against skin components [37, 38]. Ohshima et al . Deserves special attention, where ten patients with chronic plaque psoriasis were enrolled and advised to take 150 mg roxithromycin (a macrolide) orally twice daily for 1 to 7 weeks . Six out of the ten patients exhibited a decrease in psoriasis area and severity index (pasi) score . The mechanism by which macrolides downregulates the host inflammatory response was unclear but certainly multifactorial . Macrolides, as a class, and azithromycin in particular, have a characteristic immunomodulatory and anti - inflammatory potential, in addition to their main antibacterial action against streptococci . This macrolide probably also suppresses immunological events in interferon gamma - treated keratinocytes, including expression of mhc class ii, secretion of il-1 alpha, and superantigen presenting ability [40, 41]. Saxena and dogra tried oral azithromycin in a single blind randomized case - control trial . 50 patients with moderate - to - severe chronic plaque psoriasis were enrolled . Of these, 30 randomly selected patients received azithromycin for 48 weeks as a single oral 500 mg daily dose for 4 days with a gap of 10 days (total 24 such courses). The remaining 20 patients received a vitamin c tablet (nonchewable) in the same dosage schedule . A significant improvement in pasi score was noted from 12 weeks in the majority of patients in the azithromycin group . At the end of 48 weeks, 18 patients (60%) showed excellent improvement, while 6 patients (20%) showed good improvement, and 4 patients (13.33%) showed mild improvement . A significant improvement in the skin lesions was noted at 12 weeks of azithromycin therapy . Based on this study, the authors reported that the results substantiated the hypothesis that chronic ongoing stimulus by the streptococci or its superantigen was indispensable in maintaining the disease . Mean pasi scores dropped significantly, and itch was reduced in 11 subjects after therapy . This study showed that macrolide antibiotics may be effective for treatment of psoriatic skin lesion, and that they may have antipruritic effects . The reason for the antipruritic effect is not known; however, it is suggested possibly that macrolide antibiotics inhibit production of cytokines or neuropeptides that cause pruritus . The patients were divided into two treatment groups: one to receive erythromycin and topical steroids and the other only topical steroids: the first group were treated with erythromycin 1000 mg / day and topical corticosteroids for 4 weeks, while the control group were treated only with topical corticosteroids . There was no significant difference between the baseline mean psoriasis area and severity index (pasi) of the two groups . They reported that the treatment used for the study group was more effective against pruritus than that used for the control group . Six patients with severe pruritus and six patients with moderate pruritus in the study group found that itch disappeared completely after the treatment . In the control group, none of the patients with severe or moderate pruritus found that itch disappeared completely . (e) macrolides and alopecia areata, associated with h. pylori infectioncampuzano - maya described a case of a 43-year - old man with patchy alopecia areata and h. pylori infection; the patient had hair regrowth after bacterial eradication . The patient was prescribed first - line h. pylori eradication with proton pump inhibitor (omeprazole) 20 mg twice daily, amoxicillin 1000 mg twice daily, and clarithromycin 500 mg twice daily for 14 days and was followed photographically every 2 wks . He was instructed not to take or apply any medications for alopecia areata . The patient's condition started to improve within 4 wks of completing h. pylori eradication . By week 16, the patient had completely reversed the hair loss, and by week 44, he remained h. pylori - negative and completely cured of alopecia areata . The author reported that this is the first documented case of reversed hair loss after h. pylori eradication and, if such an association is confirmed by epidemiological studies designed for this purpose, new therapeutic options could be available for these patients, especially in areas where infection with h. pylori is highly prevalent . Campuzano - maya described a case of a 43-year - old man with patchy alopecia areata and h. pylori infection; the patient had hair regrowth after bacterial eradication . The patient was prescribed first - line h. pylori eradication with proton pump inhibitor (omeprazole) 20 mg twice daily, amoxicillin 1000 mg twice daily, and clarithromycin 500 mg twice daily for 14 days and was followed photographically every 2 wks . He was instructed not to take or apply any medications for alopecia areata . The patient's condition started to improve within 4 wks of completing h. pylori eradication . By week 16, the patient had completely reversed the hair loss, and by week 44, he remained h. pylori - negative and completely cured of alopecia areata . The author reported that this is the first documented case of reversed hair loss after h. pylori eradication and, if such an association is confirmed by epidemiological studies designed for this purpose, new therapeutic options could be available for these patients, especially in areas where infection with h. pylori is highly prevalent . (f) macrolides and chronic urticaria, associated with h. pylori infectionchronic urticaria is one of the most frequent skin diseases in medical practice . Urticaria is defined as acute if the whealing persists for less than six weeks and as chronic if it persists for longer . Chronic urticaria that lasts from several years to decades significantly impairs the quality of life . There is evidence that helicobacter pylori has a critical role in different extragastric diseases such as chronic urticaria . Ben mahmoud et al . Presented a case of chronic urticaria in an adult patient with h. pylori infection and disease regression after triple anti - h . Pylori therapy . In contrast to the autoimmune mechanisms involved in chronic urticaria against which no specific treatment strategy has been developed, infections with h. pylori could be treated with triple therapy . The authors suggested that laboratory tests for the detection of this pathogen should be performed in patients with chronic urticaria . Urticaria is defined as acute if the whealing persists for less than six weeks and as chronic if it persists for longer . Chronic urticaria that lasts from several years to decades significantly impairs the quality of life . There is evidence that helicobacter pylori has a critical role in different extragastric diseases such as chronic urticaria . Ben mahmoud et al . Presented a case of chronic urticaria in an adult patient with h. pylori infection and disease regression after triple anti - h . Pylori therapy . In contrast to the autoimmune mechanisms involved in chronic urticaria against which no specific treatment strategy has been developed, infections with h. pylori could be treated with triple therapy . The authors suggested that laboratory tests for the detection of this pathogen should be performed in patients with chronic urticaria . (g) macrolides and pityriasis rosea sharma et al . Performed a clinical study to evaluate the efficacy of erythromycin in patients with pityriasis rosea (pr). Ninety patients over a period of 2 years were alternatively assigned to treatment group or placebo group . Complete response was observed in 33 patients (73.33%) in the treatment group and none in the placebo group . The authors concluded that oral erythromycin was effective in treating patients with pityriasis rosea, and that the effect of erythromycin may be related to its anti - inflammatory properties.rasi et al . Conducted a placebo - controlled study on 184 patients with pityriasis rosea attending the outpatient dermatology department clinic . Adult patients were treated with 200 mg of erythromycin 4 times daily, and children were treated with 20 to 40 mg / kg daily in 4 divided doses . Controls were given a placebo (an emollient cream) that was not identical in appearance . Subjects were seen at follow - up visits 2, 4, 6, and 8 weeks after starting treatment . Both groups were comparable with regard to sex, age, and mean duration of disease at the time of attending the clinic . They found no significant difference between the 2 treatment groups at weeks 4, 6, and 8 after beginning of treatment.other authors believe that the use of macrolides is best considered experimental and should not be adopted into routine clinical practice until further studies are conducted and results are published . Even if macrolides are finally proven to be effective in modifying the course of pr, this does not substantiate that pr is caused by a bacterial rather than a viral infection . Macrolides have anti - inflammatory and immunomodulating effects that might affect the course of pr or other cutaneous eruptions independent of their antibacterial properties . Sharma et al . Performed a clinical study to evaluate the efficacy of erythromycin in patients with pityriasis rosea (pr). Ninety patients over a period of 2 years were alternatively assigned to treatment group or placebo group . Complete response was observed in 33 patients (73.33%) in the treatment group and none in the placebo group . The authors concluded that oral erythromycin was effective in treating patients with pityriasis rosea, and that the effect of erythromycin may be related to its anti - inflammatory properties . Conducted a placebo - controlled study on 184 patients with pityriasis rosea attending the outpatient dermatology department clinic . Adult patients were treated with 200 mg of erythromycin 4 times daily, and children were treated with 20 to 40 mg / kg daily in 4 divided doses . Controls were given a placebo (an emollient cream) that was not identical in appearance . Subjects were seen at follow - up visits 2, 4, 6, and 8 weeks after starting treatment . Both groups were comparable with regard to sex, age, and mean duration of disease at the time of attending the clinic . They found no significant difference between the 2 treatment groups at weeks 4, 6, and 8 after beginning of treatment . Other authors believe that the use of macrolides is best considered experimental and should not be adopted into routine clinical practice until further studies are conducted and results are published . Even if macrolides are finally proven to be effective in modifying the course of pr, this does not substantiate that pr is caused by a bacterial rather than a viral infection . Macrolides have anti - inflammatory and immunomodulating effects that might affect the course of pr or other cutaneous eruptions independent of their antibacterial properties . (h) macrolides and pityriasis lichenoidespityriasis lichenoides is an uncommon reactive papulosquamous eruption of unknown origin . Seven of the children who experienced a remission were off erythromycin and free of lesions after 2 to 5 months of therapy . The authors concluded that a trial of erythromycin should be considered in children with pityriasis lichenoides before other, possibly more toxic, measures are instituted.skinner and levy reported two cases of persistent pityriasis lichenoides et varioliformis acuta (pleva) unresponsive to tetracycline and erythromycin that rapidly resolved with bimonthly treatment with azithromycin for 5 days . The first case was a 51-year - old female started on azithromycin 500 mg on day 1 and 250 mg on days 2 through 5, to be taken on the first and third weeks of the month . One week after starting the first course, she reported that no new lesions had formed, and that the current lesions were resolving . After 3 weeks and two courses of azithromycin, the patient was clear of all lesions . The second case was a 5-year - old boy in whom erythromycin taken for 3 months did not improve the rash . He was continued on azithromycin for one more course and was clear of all lesions on 1-month followup and again 2 months later . Seven of the children who experienced a remission were off erythromycin and free of lesions after 2 to 5 months of therapy . The authors concluded that a trial of erythromycin should be considered in children with pityriasis lichenoides before other, possibly more toxic, measures are instituted . Skinner and levy reported two cases of persistent pityriasis lichenoides et varioliformis acuta (pleva) unresponsive to tetracycline and erythromycin that rapidly resolved with bimonthly treatment with azithromycin for 5 days . The first case was a 51-year - old female started on azithromycin 500 mg on day 1 and 250 mg on days 2 through 5, to be taken on the first and third weeks of the month . One week after starting the first course, she reported that no new lesions had formed, and that the current lesions were resolving . After 3 weeks and two courses of azithromycin, the patient was clear of all lesions . The second case was a 5-year - old boy in whom erythromycin taken for 3 months did not improve the rash . He was continued on azithromycin for one more course and was clear of all lesions on 1-month followup and again 2 months later . (i) macrolides and bullous pemphigoidbullous pemphigoid is the most common autoimmune - mediated bullous disease in men . Mensing and krausse tested erythromycin combined with a low - dose methylprednisolone in eleven patients in a prospective study . A historical collective of the last 33 patients treated before this study was started served as the control group . The duration of hospitalization as an expression of therapeutic response, but also of lowered side effects dropped down from 43 to 33 days in the erythromycin treated group . Reported that the macrolide antibiotic erythromycin has been effective in bullous pemphigoid in their studied patients . Fox et al . Reported two patients with bullous pemphigoid treated with erythromycin demonstrated improvement . Mensing and krausse tested erythromycin combined with a low - dose methylprednisolone in eleven patients in a prospective study . A historical collective of the last 33 patients treated before this study was started served as the control group . The duration of hospitalization as an expression of therapeutic response, but also of lowered side effects dropped down from 43 to 33 days in the erythromycin treated group . Reported that the macrolide antibiotic erythromycin has been effective in bullous pemphigoid in their studied patients . Fox et al . Reported two patients with bullous pemphigoid treated with erythromycin demonstrated improvement . (j) successful treatment of idiopathic thrombocytopenic purpura with macrolidesohe and hashino reported 3 cases of primary immune thrombocytopenia (itp) patients who were successfully treated with macrolides, irrespective of helicobacter pylori (h. pylori) infection status . Case 1, an 88-year - old woman who was an h. pylori - positive itp patient, was treated with clarithromycin (cam). As an alternative to cam, she was successfully treated with erythromycin (em) for more than 7 months . Case 2, a 61-year - old man who was an h. pylori - negative itp patient, was unsuccessfully treated with cam but successfully treated with em . Case 3, a 75-year - old woman who was a h. pylori - negative itp patient, was treated with cam . After approximately 6 months, she was treated with em for a common cold, and her platelet count increased rapidly . The authors concluded, based on these findings, that macrolide treatment was effective for itp . The effectiveness of macrolides might suggest immunomodulatory effects as well as antibacterial effects for h. pylori.in a previous work, the authors have already reported 3 cases of idiopathic thrombocytopenic purpura (itp), also known as primary immune thrombocytopenia, which show increased platelet counts following clarithromycin treatment, irrespective of h. pylori infection status .the authors attributed this therapeutic success of macrolides in treating cases of itp to the immunomodulatory effects of macrolides . Immunomodulatory effects from macrolide antibiotics might be obtained by the eradication of bacteria or by modulation of the immune system involving the mucosa on which commensal bacteria reside . Ohe and hashino reported 3 cases of primary immune thrombocytopenia (itp) patients who were successfully treated with macrolides, irrespective of helicobacter pylori (h. pylori) infection status . Case 1, an 88-year - old woman who was an h. pylori - positive itp patient, was treated with clarithromycin (cam). As an alternative to cam, she was successfully treated with erythromycin (em) for more than 7 months . Case 2, a 61-year - old man who was an h. pylori - negative itp patient, was unsuccessfully treated with cam but successfully treated with em . Case 3, a 75-year - old woman who was a h. pylori - negative itp patient, was treated with cam . After approximately 6 months, she was treated with em for a common cold, and her platelet count increased rapidly . The authors concluded, based on these findings, that macrolide treatment was effective for itp . The effectiveness of macrolides might suggest immunomodulatory effects as well as antibacterial effects for h. pylori . In a previous work, the authors have already reported 3 cases of idiopathic thrombocytopenic purpura (itp), also known as primary immune thrombocytopenia, which show increased platelet counts following clarithromycin treatment, irrespective of h. pylori infection status . The authors attributed this therapeutic success of macrolides in treating cases of itp to the immunomodulatory effects of macrolides . Immunomodulatory effects from macrolide antibiotics might be obtained by the eradication of bacteria or by modulation of the immune system involving the mucosa on which commensal bacteria reside . Despite the small number of studies shedding light on the anti - inflammatory and immunomodulatory mechanisms of the macrolides, there is strong evidence providing support to the benefit of using this type of drug for the long term and in low doses to treat some chronic inflammatory skin disorders . Although additional studies are needed, macrolide therapy in some of chronic dermatoses has the potential of modifying the morbidity and possibly ameliorating the severity of some, but not all, of these conditions.
During march 2010october 2011, we conducted a nationwide survey of persons from all 3 zones in the country who had pulmonary sputum smear positive tb . The survey was designed according to the guidelines of the world health organization (who) (5) by using cluster sampling of 39 diagnostic centers . Cases were classified as newly diagnosed or previously treated according to who definitions (6). We collected the following variables through a questionnaire administered during sputum collection: patient sex, age, country of birth, and treatment history (new or previously treated). A laboratory in south africa that is accredited for molecular testing by the national accreditation system tested sputum samples for resistance to rifampin and isoniazid by using genotype mtbdrplus assay (hain lifescience gmbh, nehren, germany) (7,8). The sensitivity of this assay to detect mutations known to confer resistance is higher for rifampin than for isoniazid (98.4% vs. 88.7%, respectively). Statistical analyses were performed in stata (version 12; stata corp ., college station, tx, usa). Prevalence estimates were adjusted for fluxes in tb notifications from 2007, the year on which sampling calculations were based, through 2010, the year in which the survey started (table footnote; technical appendix figure). * prevalence estimates were obtained by using logistic regression (stata s svy: logit command, stata corp ., college station, tx, usa) on the binary treatment history variable; each new / retreatment case with a drug - susceptibility test result was weighted by the number of new / retreatment cases notified in its cluster in 2010 (the year in which the survey started), divided by the total number of new / retreated cases with a drug - susceptibility test result in its cluster . The estimation of odds ratios reported elsewhere in the text also included the expansion of categorical sex, age group, and zone variables (xi: logit), with clustering and cis of variance . The findings were robust to multiple imputation of missing data (adding another 78 new and 18 retreatment cases to the sample), use of sampling weights based on 2007 notifications (the year in which cluster samples were calculated), and no use of sampling weights at all; prevalence estimates were equivalent to or slightly higher than those reported here . Ninety - six enrollees were subsequently excluded because of sample contamination (52 patients), insufficient material to perform the genotype mtbdrplus assay (41 patients), and isolation of mycobacteria spp . The overall total drug susceptibility recovery rate was 89.9%, in line with the country expectations and who recommendations (5). Of the patients retained in the study (754 persons with new cases and 96 persons with previously treated cases), the male - to - female ratio was 2.4:1.0, and median age was 30 years (range 486 years, interquartile range 2344 years). Mdr tb was detected in 5.2% (95% ci 2.87.5) of persons with newly diagnosed tb and 40.8% (95% ci 24.757.0) of persons with previously treated tb . Levels of resistance to isoniazid and rifampin and frequencies of any resistance and monoresistance are shown in the table . History of previous anti - tb treatment was the strongest independent factor for mdr tb (odds ratio [or] 23.0, 95% ci 9.456.1, p<0.001), and living in the south - central region or in puntland was associated with a significantly higher risk for mdr tb than was living in somaliland (or 3.6, 95% ci 1.96.9 p<0.001 for living in puntland and or 4.3, 95% ci 1.711.3, p = 0.003 for living in the south - central region). Associations between mdr tb and sex, age, and country of birth were not significant . Compared with a study conducted in neighboring ethiopia in 2005, where mdr tb was found in only 1.6% of new and 11.8% of previously treated tb cases (9,10), and with surveys conducted in countries of the eastern mediterranean region, where the average proportion of mdr tb in new and previously treated tb cases was of 3.4% and 29.9%, respectively (4), the proportions of mdr tb detected in somalia are high . At this level of resistance, one would expect that among the 9,760 pulmonary tb cases notified in somalia in 2011 (4), 750 were mdr tb and therefore required treatment with second - line drugs; this number does not include other (non - mdr) cases of rifampin - resistant tb that would probably require an mdr tb regimen . This finding presents a real emergency for the national tuberculosis control program considering the duration of second - line treatment (> 2 years) (11,12), the current availability of such treatment in somalia for a only few patients, and the country s lack of laboratory capacity to diagnose drug resistance . A systematic review of the cost effectiveness of mdr tb care (13) suggests that it would cost us$3,5005,900 to treat mdr tb on an outpatient basis in somalia . At <us$400 per disability - adjusted life - year efforts are being made to treat mdr tb patients detected in the survey . Because the cost of treating all 750 mdr tb patients is 32%54% of the us$8.2 million (all of it from the global fund to fight aids, tuberculosis and malaria) that was available to somalia in 2012 for its entire tb control program (4), additional funding will be needed . In a drug quality survey conducted in somalia in 2010, 60% of 10 products containing first - line anti - tb drugs that can be easily purchased from pharmacies and informal health care providers met international quality standards (i. sindani, pers . The extensive use of drugs of suboptimal quality, the widespread practice of using wrong medical prescriptions, and incomplete adherence of patients to treatment are the most likely reasons for the high levels of mdr tb in somalia . These levels appear to be highest in the south - central region, where the security situation is most volatile and disruption of care more frequent . This region also is most affected by recent food shortages (14) and has the most internally displaced persons (15), factors that are expected to exacerbate disease progression and transmission of m. tuberculosis . This study shows that nationwide surveys to monitor drug - resistant tb are possible even when social conditions are unstable . Two middle eastern countries, afghanistan and yemen, also were able to conclude nationwide drug resistance surveys in 2011 despite social unrest (4). Sample collection, transportation, and monitoring of survey operations in our study have been challenging . The analysis had to account for population movements and changes in the availability of and access to health services . This study, conducted under difficult circumstances in a country with unstable social conditions, showed that mdr tb is a serious underdetected and widespread public health problem in somalia . The documented levels of mdr tb are among the highest reported in africa and the middle east and suggest that 750 patients in the country had mdr tb in 2011 . Urgent measures should be introduced to improve access to diagnosis of drug resistance and availability of second - line medication for all patients who need them . Percentage change in total tuberculosis notifications by diagnostic center participating in the survey, somalia, 20072010.
Integration of functional magnetic resonance imaging (fmri) with electroencephalography (eeg) has offered the possibility of understanding new insights into neuroscientific studies because of the higher temporal and spatial measurements of brain activity when compared with the use of each technique separately . Rather than only an additional tool, coregistered eeg - fmri has been shown to be a promising and powerful technique for the mapping of brain activity and has drawn the attention of several researchers and clinicians in recent years [17]. Meanwhile, consolidation of simultaneous eeg - fmri and enlargement of its range of applications still depend on enhancing the quality of the eeg signal acquired simultaneously with the fmri data . The mr scanner constitutes a quite hostile environment for eeg because of the voltages induced by the magnetic fields used for acquisition of fmri data . Such voltages correspond to three different types of artefact and may corrupt and distort the eeg signal, measured by the scalp electrodes . The first type of artefact is the movement artefact associated with motion of the subject head, electrodes, and wires into the static magnetic field (b0) of the mr scanner, which introduces temporary voltage fluctuations in the measured scalp potential [8, 9]. A second type of artefact is the pulse or ballistocardiogram artefact, provoked by the pulsatile movement of the blood in scalp arteries within b0 [1013]. Finally, the gradient or imaging acquisition artefact is the voltage induced in the measured scalp potential by the application of rapidly varying magnetic field gradients for spatial encoding of the mr signal and radiofrequency pulses (rf) for spin excitation [1416]. The occurrence of the movement artefact within a scenario of abrupt head motions as well as the pulse artefact is out of the scope of this work, and further details about their characteristics and methods to suppress them can be found in the abovementioned references . Regarding the gradient artefact, its amplitudes can be several orders (up to 10 v) higher than the neuronal eeg signal . The gradient fields and rf pulses used in the mr pulse sequences induce a characteristic and repetitive artefact waveform in the electrical potential picked up by the scalp eeg electrodes (scalp potential), which is approximately the differential waveform of the corresponding gradient pulse . The onset of the artefact waveform corresponds to the occurrence of a rf pulse in the mr sequence, such that the time in - between consecutive rf pulses (termed repetition time, tr) matches the period of the artefact waveform . The stack of repetitive individual mr slices within a single tr occurs in the recorded scalp potential as signal peaks, and the time corresponding to the acquisition of one slice or slice - time (tr - slice) lies in the range of 50150 ms . In the frequency - domain, the repetitive feature of the gradient artefact can be observed as discrete harmonic artefact frequency intervals or the fundamental of each respective frequency bin corresponds to multiples of the inverse of the slice repetition time (1/tr - slice). For periodic or interleaved fmri acquisition, in which delays are left between mr volumes, harmonics in the frequency range of 1/tr appear convolved with the frequency bins associated with the slice repetition frequency, 1/tr - slice [1519]. In the literature, a number of solutions have been proposed to attenuate the effects of the gradient artefact at the source . For instance, it is possible to reduce its magnitude by laying out and immobilising the eeg leads, twisting the leads or modifying the lead paths, using a bipolar electrode configuration, and using a head vacuum cushion [16, 20]. The use of interleaved fmri acquisition approaches has been shown to be suitable for certain forms of brain activity, such as slowly varying rhythms and evoked responses . However, they are generally less flexible and experimentally efficient than continuous measurements [9, 21]. According to mullinger et al ., the amplitudes of the gradient artefact can also be attenuated by adjusting the subject position within the fmri scanner . Chowdhury et al . Have proposed the use of an eeg cap that incorporates electrodes embedded in an external layer and can record the gradient artefact separately from the eeg signal . Thus, subtraction between the signals recorded by internal and corresponding external electrodes allows the attenuation of the artefact . Although these solutions permit achieving a considerable attenuation of the gradient artefact, its effective suppression and satisfactory eeg correction must be performed by using dedicated postprocessing signal approaches . The average artefact subtraction (aas) methodology is the most established postprocessing technique for gradient artefact suppression . Such an approach makes use of the assumption of periodic and stationary nature of the artefact to calculate an average template from occurrences of the artefact waveform, which is then subtracted from the scalp potential . It also assumes the artefact and the eeg signal are not correlated, so that the subtraction of the averaged template permits an estimation of the corrected eeg . The performance of the aas method highly depends on the reproducibility of the artefact waveform from epoch to epoch, which can be facilitated by utilising a setup that yields more accurate sampling of the gradient artefact waveform over time . Have demonstrated that the use of synchronisation between the fmri clock and the eeg sampling frequency allows more precise sampling of the artefact and construction of a more accurate artefact template, in consequence . Thus, a cleaner eeg can be obtained after application of aas in the recorded scalp potential . Head motions of the subject provoke alterations in the morphology of the artefact waveform over the artefact period, in such a way that the average artefact template cannot characterise individual occurrences of the artefact waveform . To address this problem, allen et al . And becker et al . Proposed the use of a sliding average window implementation whereby the artefact template may be individually calculated for a particular occurrence of the artefact waveform . However, the correct choice of the number of averaging epochs poses difficulties to implementation of this approach, since few windows can result in removal of the neuronal eeg, whereas the use of many windows can lead to remaining residual artefacts after aas . Hence, to effectively suppress the gradient artefact with a satisfactory preservation of the neuronal eeg, additional approaches like low - pass filtering with a cut - off frequency around 5080 hz and adaptive noise cancelling must be employed to attenuate residual artefacts [14, 15, 20, 21, 2629]. Some variants of aas have been devised in attempt to improve the accuracy of template calculation by using principal component analysis, independent component analysis, and spatial filtering . To correct the jitter between eeg sampling frequency and fmri clock, more precise computing of the timing error has been addressed by negishi et al ., and huang et al . . Nevertheless, estimation of an optimal artefact template is still the object of study . In addition, the study of ultra - high - frequency neuronal activity as currently performed requires the use of interleaved approaches as well as customised fmri sequences that are generally not available to all investigators . Thus, further improvements of aas and development of novel correction methods are still required and highly desirable to enhance the quality of the eeg signal, mainly regarding eeg signals with low amplitude and with frequency activity in the gamma band (30100 hz) and high - frequencies oscillations between 100 and 500 hz [20, 34]. Because of the risk of simultaneous removal of neuronal eeg activity during application of the gradient artefact correction approach, assessment of the preservation of the eeg signal should be carried out together with the efficacy of the artefact suppression . This, however, has seldom been made systematically or in a consistent way [3437]. In many eeg - fmri studies, a single algorithm is chosen without proper justification, and often the quality of gradient artefact correction and eeg preservation is assessed by visual inspection only . The classical (gold standard) way of analyzing eeg signals relies on visual judgement and recognition of sometimes very subtle or short duration phenomena such as spike - wave patterns in epilepsy studies or k - complexes in sleep research . Nonetheless, those patterns may easily be distorted or obscured after application of the artefact correction approach . A difficulty that arises with regard to the analysis of spontaneous eeg excerpts is the stochastic and nonstationary nature of the neuronal eeg . On the other hand, identification of single events in the corrected eeg is not suitable for a scenario in which the signal of interest is the spontaneous eeg and, thus, larger eeg excerpts over time should be analyzed . In addition, the lack of knowledge of the true eeg signal makes it difficult to compare the power spectra of artefact - corrected eeg excerpts with the spectra of the eeg recorded inside or outside the scanner . Thereby, a more systematic approach to assess and compare the performance of the gradient artefact correction methods is advised in some applications, rather than only relying on the analysis of single events or the quantification of eeg power in certain spectral bands . Moreover, to date, generalisation of the correction results for different types of eeg data has been poorly made as well [16, 3537]. This paper presents a novel methodology for gradient artefact correction based upon optimised moving - averaging (oma) filtering . Oma filtering constitutes a modality of iterative filtering decomposition [39, 40] and has been exploited in a research project that our group has undertaken to investigate characteristics and features of the gradient artefact that might be used to attenuate, correct, and improve the quality of the corrected eeg signal [38, 4143]. Optimised moving - average makes use of forward - backward application of a moving - average (ma) filter as an integration procedure to suppress the artefact and estimate partial components of the corrected eeg at the same time . Recursive application of such a procedure allows estimation of the corrected eeg as a sum of the calculated partial components . Rather than estimation of an average template, as performed by the aas implementation, the artefact is calculated not for epochs, but sample - by - sample, as described in section 2 . To assess the degree of eeg preservation, we have devised a novel and simple evaluation approach that allows accounting for the stochastic nature of the neuronal eeg and was used to perform a comparative analysis of oma with aas . Comparison between the performances of oma and aas reveals that our method can provide an improved balance for the suppression of the artefact together with a satisfactory preservation of the neuronal eeg signal, as shown in section 4 . In parallel, the use of low - pass filtering or another correction approach to suppress residual artefacts after application of oma can be avoided, and thus our method potentially better preserves highly relevant high - frequency eeg features . Furthermore, the results indicate that our approach is capable of satisfactorily correcting the eeg data even within a scenario of misalignment between eeg sampling interval and the mr slice - time and without accurate information about mri triggers . Analysis of the application of the gradient artefact correction in eeg data sets recorded by using mr scanners from two different vendors is also provided in section 4 . Our devised methodology was tested in two types of eeg data simultaneously acquired with fmri data . The eeg data sets were kindly provided by brain products gmbh, gilching, germany, which gave consent for their publication . Data acquisition was conducted in accordance with the declaration of helsinki, approved by the responsible ethics committee, and the subjects gave their informed written consent before participating in the study . One of the eeg data sets was recorded within a philips scanner (hereafter referred to as philips data), whereas the other eeg data set was recorded within a ge scanner (hereafter referred to as ge data). The eeg data in both the philips and ge scanner were recorded in two volunteers by using an mri - compatible 64-channel eeg system (brainamp mr, brain products gmbh, gilching, germany). An mr - compatible eeg cap (braincap mr, easycap gmbh, herrsching, germany) containing sintered ag / agcl electrodes the eeg cap was arranged in accordance with the standard 10 - 5 electrodes' positioning, with the fcz position used as reference and the ground electrode located at the afz position . The impedance of all electrodes was set below 30 k, and one additional electrode was placed on the subject back to record the ecg signal . The eeg amplifiers were positioned inside the scanner bore near the middle axis and connected via fiber optic to a pc interface located outside the scanner room . Syncbox (brain products gmbh, gilching, germany) was used to synchronise the internal sampling clock of the eeg amplifier and the mri scanner 10 mhz master clock . The signal acquisition was performed using a sampling rate at 5000 hz and measurement resolution at 0.5 v . Hardware - filtering in the frequency band between 0.016 hz and 250 hz was applied before data digitalisation in order to prevent saturation and reduce the gradient artefact amplitude . During acquisition, the volunteers were instructed to perform a simple opening / closing eyes manoeuvre at regular time intervals . Regarding acquisition of fmri data, acquisition of the philips data set was carried out using a 3 t achieva scanner (philips, eindhoven, the netherlands). One volunteer was scanned using a functional echo - planar imaging (epi) sequence with 40 transversal slices and volume repetition time (tr) equal to 2000 ms . The fmri clock and the eeg sampling frequency have been synchronised, so that tr was set as a multiple of the eeg sampling interval . Fmri data acquisition was continuously performed, and tr was adjusted as a multiple of the slice - time (tr - slice). Hence, tr - slice was equal to 2000 ms/40 slices = 50 ms . Acquisition of the ge data set was carried out using a 3 t discovery mr750 scanner (ge, waukesha, usa). A second volunteer was scanned using an epi sequence with 28 transversal slices and volume repetition time (tr) equal to 2000 ms . Fmri data acquisition was continuously performed, and the fmri clock and the eeg sampling frequency have been synchronised for a period equal to 500 ms, corresponding to seven times of tr - slice . Thereby, although tr was approximately adjusted as a multiple of the slice - time (tr - slice), tr - slice was not aligned and did not match a multiple of the eeg sampling interval . Our proposed methodology for gradient artefact correction was implemented in two steps: (i) peak detection and tr - slice estimation and (ii) optimised moving - average filtering . The recorded scalp potential, sn, in one specific eeg channel was mathematically modelled as a linear superposition of the neuronal eeg, etrue, n, and the induced voltage associated with the gradient artefact interference, gartf, n:(1)sn = etrue, n+gartf, n, where n is the time sample . An initial detection of the peaks corresponding to the onset of the mr slices observed in the recorded scalp potential must be performed according to our proposed methodology . Such detection permits estimation of the slice - time (tr - slice) according to the time basis of the eeg sampling system, which is utilised during implementation of the optimised moving - average filtering . Within a scenario of alignment between the mr slice - time and the eeg system sampling interval, the estimated tr - slice precisely corresponds to a multiple of the eeg sampling interval [25, 28, 29]. However, when there is misalignment between the mr slice - time and the eeg sampling interval, the value estimated for tr - slice may not match a multiple of the sampling interval and, thereby, vary . This variation can be accounted for by application of optimised moving - average filtering, as described in section 2.4 . Figure 1 shows an excerpt of the scalp potential picked up from the philips data, in which tr is a multiple of tr - slice . The time measured between two consecutive peaks in the signal matches the slice - time or tr - slice . Assuming that the artefact waveform is stationary, any moving - average window m with length equal to tr - slice along the signal contains the artefact waveform period, but with a different onset of those samples localised in the signal peaks . Thereby, assuming that the gradient artefact waveform is stationary and has zero mean, integration of (1) over the period m results in cancellation of the artefact waveform . Also assuming that the terms of (1) are uncorrelated, the resulting value of the integral along the scalp potential, s, corresponds to a mean estimate of the neuronal eeg, e^n. This integral can be described as a moving - average filter with order m:(2)1mk=0m1snk1mk=0m1etrue, nk+gartf, nk(3)1mk=0m1etrue, nk+1mk=0m1gartf, nk = e^n.because of the phase distortion provoked by the moving - average filter [4446], the mean value e^n is not in phase with the neuronal eeg, etrue, nk . In order to make them in phase, the moving - average must be backward applied in (3):(4)1mk = m10e^n+k=1mk = m101mk=0m1snkn+k=1m2snm+1 + 2snm+2++m1sn1+msn+m1sn+1++2sn+m2+sn+m1(5)=1m2k=m+1m1mksn+k = ecomp,1,n.thereby, according to (4) and (5), forward - backward application of the moving - average filter in the recorded scalp potential results in the signal ecomp,1 that is in phase and constitutes a mean approximation of the neuronal eeg: (6)ecomp,1e^true.equation (5) acts as a smoothing filter, in such a way that the signal ecomp,1 contains low - frequency activity associated with e^true . In turn, the frequency activity associated with the gradient artefact is contained in the signal, ehigh,1, resulting from the subtraction of ecomp,1 from s:(7)ehigh,1=secomp,1.since high - frequency components associated with e^true remain in ehigh,1, it is possible to obtain an estimate of such components by the iterative application of (5) in ehigh,1 . The second component, ecomp,2,n, results from the application of (5) in ehigh,1:(8)ecomp,2,n=1m2k=m+1m1mkehigh,1,n+k, and the signal ehigh,2 can be obtained afterwards:(9)ehigh,2=ehigh,1ecomp,2.this procedure was repeated so forth, for a number j of iterations, allowing estimation of the component ecomp, j:(10)ecomp, j, n=1m2k=m+1m1mkehigh, j1,n+k.this procedure also constitutes a modality of iterative filtering decomposition (ifd) in which (5) is termed double average filter [39, 40]. The convergence of ifd has been demonstrated in lin et al . And is ensured by the coefficients (masks) of the double average filter having value between 0 and 1 . Finally, the estimate e^true of the corrected eeg can be calculated by the sum of j estimated components: (11)e^true=j=1jecomp, j.implementation of (11) can be visualised in the scheme of figure 2 . Application of the z - transform in the oma filter, homa, described in (10), permits finding its transfer function:(12)homaz=1m21zm1zm1z11z.making use of (12), the frequency response of homa can be calculated by setting z = e. figure 3 shows such a frequency response for some values of m. the magnitude response reveals the presence of spaced zeros at the frequency 2/m . For a hypothetical value m = 1, the oma filter represents an all - pass band filter, as expected for a moving - average filter . The phase response confirms the zero - phase characteristic of the oma filter . From (7), we can find the transfer function between ehigh,1 and s:(13)h111m21zm1zm1z11z=1homaz.hence, ehigh, j corresponds to(14)ehigh, jz=1homazjsz.therefore, (12), (13), and (14) allow removing j cascade components shown in figure 2 and establishing the transfer function between e^true and s:(15)e^truezsz=11homazj = hcz.in figure 4, the magnitude response of hc(z) in (15) is depicted, taking into account m = 16 and m = 250 and some values of j. it can be observed that increasing of j is followed by substantial droop reduction (increasing gain) in the filter pass - bands . On the other hand, increasing of j is also followed by reduction in the attenuation in the filter stop - bands . In order to improve the attenuation in the stop - bands, hc(z) might be applied within a cascade implementation as indicated in(16)hlz = hczl, where l is the number of cascades . Figure 5 depicts the magnitude response of hl(z), for l = 2 and l = 5, taking into account m = 250 and some values of j. as noticed, (16) can be used to provide reduced droop in the pass - bands together with higher attenuation in the stop - bands according to the values of j and l. in addition, (15) and (16) possess a zero - phase characteristic as well, similar to that observed in figure 3(b). Therefore, (15) and (16) can be used to estimate the artefact - corrected eeg in z - domain . On the other hand, in time - domain, the corrected eeg, e^true, can be calculated by using either (11) or (17):(17)e^true = sehigh, j.as indicated in (10) and (12), the estimate ecomp, j is calculated sample - by - sample, rather than calculation and subtraction of an average artefact template . Thus, the optimised moving - average filtering implementation depicted in figure 2 permits an individual calculation and subtraction of the artefact for each signal sample rather than epochs averaging, as performed by aas . Hence, the inherent uncertainty associated with averaged samples and the influence of alterations of the artefact waveform provoked by subject head motions might be minimised by using our proposed approach . To assess the performance of the proposed approach in a scenario of misalignment between the eeg sampling interval and the slice - length, oma has been applied by using (16), taking into consideration some values of l, as shown in section 4 . According to ritter et al ., two measures must be complimentarily used to evaluate the performance of the gradient artefact correction approach: (i) the effectiveness of gradient artefact attenuation and (ii) the degree of preservation of the neuronal eeg after artefact correction . To assess the gradient artefact attenuation, the rms and amplitude of the artefact voltage over time were calculated taking into account the subtraction between s and the estimation of the corrected eeg, e^true . Also we performed calculation of the spectral power attenuation around the fundamental of the frequency bins associated with the slice repetition frequency (1/tr - slice). To this end, we have estimated and took into consideration a bandwidth of 1 hz around the fundamental of each frequency bin . Calculation of the spectral power attenuation was carried out for the frequency bins below 500 hz . Although a band limiter set at 250 hz was employed during data acquisition, we would rather evaluate the attenuation in frequency bins up to 500 hz because of the artefactual energy that may remain above the band limiter edge frequency . Equation (18) was used to compute the spectral power attenuation in decibel:(18)attenuation=20logpapb db, where pb and pa correspond to the spectral power within the harmonic artefact bins, before and after application of the gradient artefact correction, respectively . The scheme depicted in figure 6 was used to perform the quantitative evaluation of the eeg preservation . As indicated in figure 6, a reference eeg signal, eref, n, has been linearly added to the measured scalp potential, sn, thus generating the modified signal smod, n . As eref, n, we used eeg excerpts recorded inside the mr scanner during nonscan periods . Thereby, the letters and t indicate that the reference eeg excerpt has been recorded at a different time than sn . The gradient artefact correction was then applied to smod, n and sn, resulting in the estimates e^true,1,n + e^ref, n and e^true,2,n, respectively . Thereby, the subtraction between these estimates allows obtaining an estimate of the reference signal, e^ref, n, which was finally compared with eref, n . Equations (19) and (20) were used to calculate the signal - to - noise ratio (snr) and the mean squared error (mse) as complimentary measures of temporal and frequency contents of e^ref, n in comparison with eref, n:(19)snr = coveref, e^referefe^ref,(20)mse=1nn=1neref, ne^ref, n2.the snr calculated by (19) corresponds to a measure of cross - correlation and allows an evaluation and comparison of frequency characteristics between eref, n and e^ref, n . Values of snr closer to unity mean higher similarity between eref, n and e^ref, n . In turn, smaller values of mse computed according to (20) indicate higher correspondence between eref, n and e^ref, n over time . In addition to being of simple implementation, the evaluation scheme of figure 6 has the advantage of allowing the assessment of longer eeg excerpts by accounting for the stochastic nature of the neuronal eeg signal and not single events only . The results obtained by application of oma were compared with those obtained by subtraction of a mean template, according to the average artefact subtraction (aas) methodology [14, 21]. Both oma and aas, we developed and applied our proposed methodology to the eeg recordings in matlab (the mathworks inc ., natick, usa) environment . In turn, the average artefact subtraction was carried out by utilising the software brain vision analyzer 2 (version 2.1.0.327; brain products gmbh, gilching, germany). As benchmark, a sliding moving - average window implementation of 21 epochs was used for construction of the average artefact template . The reason for using 21 epochs was based upon the default settings of the brain vision analyzer . The epoch length to be averaged and construct the artefact template was set at 50 ms (tr - slice) for the philips data and 500 ms for the ge data . Such values were chosen to match the minimum period in which the fmri data were synchronised as a multiple of the eeg sampling interval, so that the influence of head motions in longer sliding average windows might be prevented . After application of oma and aas, assessment of artefact attenuation and evaluation of the eeg preservation, as described above, were carried out using matlab . All 63 eeg channels were used to perform the analysis of the ge data . For the philips data, the channel tp8 was excluded from the analysis because the entire recordings were corrupted by artefacts, which made it impossible to pick up a signal excerpt representative of eref, n . Figure 7(a) depicts an exemplary scalp potential excerpt, picked up from the philips data, electrode position fz . In figure 7(b), the power spectrum of the signal of figure 7(a) is shown, and the harmonic activity associated with the gradient artefact can be visualised as spectral peaks at multiples of the fundamental frequency equal to 20 hz, corresponding to the frequency bins associated with 1/tr - slice . By performing the peak detection associated with the gradient artefact in the signal of figure 7(a), tr - slice was estimated in even lengths of 250 samples, thus confirming the alignment between tr - slice and the eeg sampling interval for the philips data . Hence, for application of the optimised moving - average (oma), we set m = 250 . Figure 8(a) depicts the artefact - corrected eeg by using (15), for j = 200, j = 2000, and j = 200000 . It can be seen in figure 8(b) that the harmonic activity associated with the gradient artefact has been attenuated for the used values of j. increasing the value of j is shown to provoke better preservation of the power activity along the spectrum of the corrected eeg signal, mainly in high - frequencies . Smaller attenuation in the frequency bins associated with 1/tr - slice is also noticed when the value of j is increased, which agrees with the comb - filtering response depicted in figure 4 as well . Therefore, the choice of the value of j should be made in such a way as to provide adequate attenuation of the artefact activity in the frequency bins and satisfactory preservation of the eeg signal . However, (15) might not provide enough attenuation of the artefact, and (16) can be used instead, as shown in the next example . An illustrative scalp potential excerpt picked up from the ge data, eeg electrode position fp1, is depicted in figure 9(a), and its corresponding power spectrum is shown in figure 9(b). For these data, the slice - length has not been aligned with the eeg sampling interval, in such a way that the value of tr - slice was estimated at 357 1 samples . To demonstrate the performance of the proposed method to suppress the gradient artefact in this scenario, we used (16), taking into account m = 357 and j = 200000, for l = 1 (see (15)), l = 30, and l = 100 . Figure 10(a) depicts the corresponding power spectra of the corrected eeg . For l = 1, oma was unable to satisfactorily attenuate the gradient artefact, so that residual spectral power artefact remained in most of artefact frequency bins (dark trace). Figure 10(b) shows a detail of the time - course corrected eeg signals around 408 s in which the presence of such residuals can also be noticed (thin dark traces). Nevertheless, higher values of l allow increased attenuation of the harmonic artefact activity (figures 10(a) and 10(b)). For l = 5 (pink trace), residual spectral power associated with the artefact was substantially attenuated in the bandwidth up to 300 hz . In turn, when l = 100 (green trace), such attenuation was even higher, and artefact harmonic activity in the frequency bins could be strongly reduced in the bandwidth below 500 hz . As observed in the spectrum details around 56 and 336 hz (figure 10(a)), by increasing l, it provokes larger attenuation around the frequency bins . Thereby, (16) can be used to account for the enlargement of the spectral artefact harmonic lines provoked by the alignment error between the eeg sampling interval and tr - slice, thus being able to correct the scalp potential within this scenario . Figure 10(c) depicts the power spectra of the corrected eeg after application of oma (blue trace, j = 200000 and l = 100) and the mean template subtraction by aas (red trace) in the signal of figure 9(a). As can be noticed for the signal corrected by the aas method, residual power associated with the artefact activity arose in higher - frequencies bins, above around 200 hz . In figure 10(d), a detail around 408 s of the time - course of the corrected eeg by oma and aas is also depicted . Some small amount of residual artefacts corresponding to the artefact residual power can be noticed in the time - course signal corrected by aas . Both oma and aas play a role of comb - filtering approaches [24, 38] whereby harmonic frequency components associated with the slice repetitive frequency (1/tr - slice) can be attenuated . On the one hand, aas implementation consists of a coherent detection - based comb - filtering process [24, 48] that is carried out by subtraction of the template with period tr - slice . As such, the aas method is highly dependent on precise sampling of the scalp potential as well as accurate alignment amongst the averaging epochs to construct the average template . Moreover, small drifts and subject head motions can provoke broadening of the high - frequency artefact spectral lines, in such a way that aas may fail to attenuate them, and residual artefacts arise in the corrected eeg as a consequence . This helps to explain why aas is not effective in eliminating the high - frequency artefact activity shown in figures 10(c) and 10(d), whose attenuation is more affected by imprecise sampling than low - frequency artefact activity as well [25, 29]. On the other hand, oma performs comb - filtering making use of the filtering implementation described in section 2.4 . By using proper values of m, j, and l in (16), thereby, it allows oma to effectively account for the attenuation of high - frequency artefact activity, as depicted in figure 10 . The results presented in tables 15 for the oma correction (16) refer to j = 200000 and l = 1 (philips data) and j = 200000 and l = 100 (ge data). As observed in table 1, the median rms and amplitude calculated for the artefact voltages estimated by both approaches are quite similar . However, when the power spectra of the corrected signals are compared, the attenuation of the artefact activity in the frequency bins provoked by the aas and oma is different (table 2). Although both approaches are shown to provoke attenuation approximately similar in some artefact frequency bins, oma provided more attenuation than aas in higher - frequency bins . To perform the quantitative evaluation of eeg preservation, we used the scheme depicted in figure 6 and calculated the median values of snr (19) and mse (20) between eref, n and e^ref, n . The results of such measures are shown in tables 3, 4, and 5 . In tables 4 and 5, we show the individual results for some exemplary channels, fp1, f3, oz, cpz, fz, fc5, and af3 . In these tables, we also included the values of snr and mse, considering application of low - pass (lp) filtering in eref, n and e^ref, n . The lp filter cut - off frequencies are indicated and were used to assess the eeg preservation in different eeg bandwidths for both oma and the aas method . Calculation of the median global values shown in table 3 also took into account the results considering lp filtering . It can be observed that the overall values of snr and mse for the oma method are better than those for aas . It is also noteworthy that low - pass filtering of eref, n and e^ref, n substantially increased the snr and decreased the mse considering the oma approach, unlike the aas method . Therefore, by using oma, the signals eref, n and e^ref, n have become more similar after lp filtering, attesting even better preservation of the neuronal eeg in low - frequencies . We also noticed that the mean subtraction by aas produced signals eref, n and e^ref, n less similar because of the higher influence of small drifts and subject head motions . This led to the small differences of the values of snr and the mse for the considered lp cut - off frequencies, as observed in tables 4 and 5 (aas). Figure 11 shows a comparison of the typical attenuation in the frequency bins provoked by oma (15) and aas, taking into account different values of j and different number of averaging epochs, as well as similar eeg preservation (same snr and mse). The results shown in figure 11 were obtained from an eeg excerpt picked up from the channel cpz of the philips data . As observed, even though oma and aas can provide similar eeg preservation according to the values of j and the number of averaging epochs, respectively, oma provokes larger attenuation than aas in the overall frequency bins, mainly in frequencies higher than 100 hz . In addition, the larger the values of j, the higher the difference of attenuation by oma in a certain frequency bin . In turn, the attenuation by aas is more uniform among different numbers of averaging epochs taking into consideration a certain frequency bin . The attenuation provided by aas is shown to be slightly higher and less uniform than oma only for the frequency bins 20 and 40 hz . Therefore, figure 11 demonstrates that our proposed comb - filtering approach can be more effective in attenuating the artefact activity and simultaneously in achieving eeg preservation similar to that provided by the aas method . As the repetitive gradient artefact waveform recorded in the scalp potential during continuous acquisition of eeg - fmri is approximately the differential waveform generated within the mr sequence, a proper integration over the artefact period might be used to cancel out the artefact, as indicated in figure 1 and (3). To implement such integration, we have investigated and proposed the forward - backward application of a moving - average filter in the scalp potential, described in (5). When this procedure is recursively carried out, as depicted in the scheme of figure 2, it permits suppressing the gradient artefact from the scalp potential and obtaining an estimation of the neuronal eeg . Such kind of moving - average procedure is referred to as iterative filtering decomposition, which has been investigated as an alternative implementation for empirical mode decomposition [39, 40]. It also constitutes a comb - filtering approach whereby harmonic signal components can be filtered out (figures 3, 4, and 5). As observed in figures 4 and 5, the oma comb - filtering approach described in (15) and (16) can provide increased gain in the filter pass - bands together with effective attenuation in the stop - bands, in addition to possessing a zero - phase characteristic (figure 3). Thus, it is able to effectively suppress the harmonic artefact activity associated with the repetitive artefact waveform that occurs in the slice repetition time (tr - slice) and satisfactorily preserve the neuronal eeg signal at the same time, as observed in figures 711 and tables 15 . When compared with subtraction of an average artefact template, according to the aas method implementation [14, 21], both oma and aas show a quite similar rms and amplitude attenuation associated with the gradient artefact over time (table 1). Meanwhile, oma is shown to provoke different attenuation in the artefact frequency bins in comparison with aas, as can be observed in table 2 and figure 11 . As a consequence, our proposed method can lead to different preservation of the eeg signal than aas, as indicated by the snr and mse (tables 3, 4, and 5). In addition, taking into account similar eeg preservation (same snr and mse), oma is shown to be more effective in attenuating the artefact in the overall frequency bins (figure 11), especially in higher - frequencies (above 100 hz). Thus, this characteristic can avoid the use of further processing methods such as adaptive noise cancelling (anc) and low - pass (lp) filtering that may contribute to suppressing high - frequency activity of the neuronal eeg . This is a substantial conceptual advantage compared to aas, in which postprocessing is essential to remove residual artefacts [14, 25, 26, 28]. Therefore, these results indicate that oma can lead to a better balance for the trade - off artefact attenuation and eeg preservation, thus outperforming the slice - average subtraction by aas . Better preservation of the corrected eeg in low - frequencies was also achieved by oma, as shown in tables 3, 4, and 5 . The snr closer to unity and the smaller values of the mse reflect smaller difference between the reference eeg, eref, n, and its estimate, e^ref, n, after application of oma than aas . Thus, it confirms an improved preservation and less distortion of the eeg signal by using oma . Yet, baseline correction has not been performed before application of oma, whereas it has been carried out before aas . This evidences that oma is more robust to the alterations of the artefact waveform caused by small drifts and movements of the subject head . The inherent uncertainty (standard deviation) associated with averaging epochs provoked by alterations in the artefact waveform because of small drifts as well as subject head motions may lead to an inaccurate estimation of the average artefact template . Contrary to aas, gradient artefact estimation by our method does not rely on calculation of an average template . Rather, implementation of oma is individually performed sample - by - sample according to application of the oma filter indicated in (10), whose uncertainty is influenced by artefact waveform alterations that occur in samples of the scalp potential raging from snm+1 to sn+m1 . In case of aas, it is affected by artefact waveform alterations that occur in those 2 m + 1 sliding average windows considered for average template construction, whose samples range from snmm to sn+mm (see appendix). Hence, it explains why oma is less affected by small drifts and subject head movements than aas . As reported by mandelkow et al ., synchronisation between the eeg sampling interval and the fmri acquisition clock leads to increasing of the usable bandwidth in the corrected eeg up to around 150 hz after average template subtraction . Since oma is capable of provoking larger attenuation of the artefact activity in higher - frequency bins, this shows that our proposed approach could be used to produce further broadening of the usable bandwidth of the corrected eeg . As shown in figures 10 and 11, by using oma, satisfactory attenuation in the artefact frequency bins the study of high - frequency neuronal activity between 100 and 500 hz has currently received increased attention and requires the use of customised fmri sequences that are generally not available to all investigators [17, 20, 34]. Furthermore, it makes use of an interleaved fmri protocol, which has been shown to be less effective and flexible than continuous fmri measurements . Therefore, the usage of oma could be useful in eeg - fmri studies that address those high - frequencies oscillations . As depicted in figure 10, implementation and application of oma also have no dependency on interpolation or slice - timing correction between the slice - length and the eeg sampling interval [14, 21, 27, 33]. This characteristic suggests that our approach can produce satisfactory results even when there are jitter errors between fmri clock and eeg sampling rate . Albeit reduction of jitter by using hardware synchronisation solutions has become increasingly available and oma is shown to provide better results within a scenario of alignment between tr - slice and the eeg sampling interval (philips data), (16) reveals representing a general analytical expression that allows satisfactory estimation and suppression of the gradient artefact in scenarios either with or without the occurrence of such timing errors . Moreover, our method is data - driven, not requiring accurate information about mri triggers and events to be implemented [14, 25, 26, 34]. The approach depicted in figure 6 proposed for evaluation of eeg preservation is shown to simplify the implementation of this measurement, so that there is no need to perform comparison of spectral power of excerpts of the reference eeg, eref, n, with corrected eeg excerpts, e^true, n, as is usually performed in the literature [14, 34, 35]. Since the power spectrum represents an average measure of the frequencies contained in the time - domain signal, spectral power associated with artefact residuals might be masked in the power spectrum of e^true, n, which can compromise this kind of analysis . Application of the artefact correction approach directly in the reference eeg [34, 35] also has the disadvantage of not accounting for the influence of the artefact, which might impair the accuracy of the assessment of eeg preservation by using such a procedure . Furthermore, the stochastic nature and the lack of knowledge of the neuronal eeg make it imprecise to compare the power spectrum of the artefact - corrected eeg with the spectrum of the reference eeg recorded inside or outside the scanner . Therefore, evaluation of eeg preservation by quantification of power in certain spectral bands should always be performed together with evaluation of eeg preservation in time - domain in order to obtain a more precise measurement . On the other hand, the assessment of single events is not suitable to account for the stochastic and nonperiodic nature of the neuronal eeg . All these characteristics can be effectively accounted for by using the time - domain scheme depicted in figure 6 . The selectivity of the oma filtering, that is, the amount of attenuation of the gradient artefact together with the degree of eeg preservation, is influenced by the slice - length (tr - slice or m) and the values of j and l. therefore, combination of proper values of those parameters should be taken into account to obtain an optimal balance between artefact attenuation and eeg preservation . In this respect, the assessment indicated in tables 25 and figure 11 could be used to choose the values of j and l according to the value estimated for m, as well as obtaining an optimal selectivity . Additionally, the eeg sampling frequency should be taken into account as well, since it can influence the value of m. some preliminary investigations suggested that oma might be used to satisfactorily produce eeg correction for lower eeg sampling frequencies (around 500 hz), provided that the gradient artefact waveforms are well reproduced in the scalp potential . Further, oma might be applied when empty gaps occur between volume acquisitions, by setting m as tr - slice followed by tr, without substantial degradation of the eeg quality . These characteristics should be better assessed in future work . As further suggestion for future work, oma should be applied and generalised for other types of eeg data recorded in other types of mr scanners as well as for correction of other types of periodic artefacts, such as the artefact that affects the scalp potential during electrical impedance tomography . Because of the computational efficiency of the oma filter (homa), (15) and (16) are not computationally demanding either, in addition to possessing quite low complexity of implementation . Thus, the time to compute our proposed approach is quite low and comparable with the processing time of the mean template subtraction performed by the brain vision analyzer software . Equations (15) and (16) also allow using noninteger values of m, according to the estimated value of tr - slice, which can be used to reduce the effect of jitter errors on the comb - filtering performance as well . Regarding the use of (11), some problems that arise are the substantial ringing effects in the signal ends provoked by the recursive application and subtraction of the components of the corrected eeg as well as the increased computational load . Thereby, (15) and (16) should preferably be used in order to minimise such problems . Such characteristics and the evaluation metrics utilised here constitute the innovative outcomes of this work in comparison with some previous results presented by our group [38, 41, 42]. Last, we noticed that the performance of oma might be compromised with the occurrence of eeg amplifier saturation, as also observed for the aas method . Thus, band limiters are needed to reduce the eeg system dynamic range and prevent saturation of the eeg amplifier during eeg data acquisition . In this work, we have shown the efficacy of using optimised moving - average filtering (oma) for suppression of the gradient artefact from the scalp potential recorded during continuous eeg - fmri . Oma constitutes a comb - filtering approach, whose application in the scalp potential allows suppression of the gradient artefact and simultaneous estimation of the neuronal eeg . When compared with the slice - average subtraction as performed according to the established average artefact subtraction (aas) methodology, oma is revealed to be capable of obtaining an improved balance compared to aas for the trade - off between effective suppression of the gradient artefact and preservation of the eeg signal . In this respect, oma can provide larger attenuation in higher - frequency artefact bins and be less affected by artefact waveform alterations owing to small drifts and subject head motions . In addition to being data - driven and not requiring accurate information about mri trigger and events, oma also is shown to satisfactorily correct the eeg signal in scenarios either with or without misalignment between the eeg sampling interval and the mr slice - time . Finally, besides effectively accounting for the stochastic and nonperiodic nature of the neuronal eeg, our proposed approach for evaluation of eeg signal preservation is shown to simplify the implementation of this measurement . Such characteristics indicate that our methodology can help to improve the quality of the eeg signal recorded during fmri as well as the performance evaluation of the gradient artefact correction approaches and thus contribute to the consolidation of coregistered eeg - fmri.
The ihs proposed diagnostic criteria for cervicogenic headache that include pain referred from the neck and involving the head and/or face, clinical or radiographic evidence that suggests a cervical spine lesion, elimination of the headache after diagnostic blockade, and pain resolution 3 months after treatment.3 for the diagnostic blockade, pain anesthesiologists target the c1c2 joint, the c2c3 joint, and c3c4 joint to provide possible relief and objective evidence of a cervical source of the headache.6 7 various mechanisms to explain cervicogenic headache have been proposed, including the role of spinal kinematics.3 although cervicogenic headache was previously thought to be associated with upper cervical spine pathology (c1c3), the treatment of lower surgical pathology results in headache relief.8 9 10 11 recent work by schrot et al has illustrated not that only was lower cervical disk disease an apparent headache generator, but also that differences in headache relief after upper- or lower - level subaxial surgery was not observed.9 cervical total disk replacement in comparison with fusion has equivalent or better outcomes.12 13 in addition, the recent long - term data demonstrates that cervical disk arthroplasty (cda) not only preserves motion at the index level but also reduces adjacent - segment disease compared with fusion procedures.7 14 15 16 although riina et al found that patients undergoing single - level arthroplasty had greater headache relief in comparison with patients undergoing arthrodesis, our group did not find a significant difference between the two groups using a similar data set for single - level surgeries.8 9 comparing the data from a two - level arthroplasty versus fusion may better resolve this difference if improved cervical spine biomechanics contributes to headache relief . A prospective, randomized, controlled multicenter study of two - level anterior cervical disk surgery conducted by davis et al reported improved neck disability index (ndi) and visual analog scale (vas) neck pain scores for arthroplasty compared with fusion at 2 years.17 if headache relief is facilitated by improved cervical kinematics after arthroplasty, patients undergoing two - level arthroplasty may experience even greater headache relief than patients undergoing two - level fusion surgery . In this study, we analyzed the 5-year follow - up data from a prospective randomized investigational device exemption (ide) clinical trial to evaluate the following: the incidence of cervicogenic headache in patients suffering from myelopathy or radiculopathy who undergo surgery, the temporal change in headache score after surgery, and the difference, if any, between one- and two - level cda and arthrodesis . In addition, demographic variables of interest (i.e., age, gender, and body mass index [bmi]) were analyzed to determine their effect on headache relief . No study to date has compared headache relief between one- and two - level anterior cervical surgery and between cda and arthrodesis . A detailed description of surgical technique and the ide study design has been previously reported.17 patients underwent surgery as part of a food and drug administration ide prospective, randomized clinical trial between april 2006 to march 2008 at 24 clinical sites in the united states . This study was approved by the university of california davis institutional review board (no . 217014). 1) were patient age 18 to 69 years with a diagnosis of degenerative disk disease with associated radiculopathy or myeloradiculopathy at a single level or at two contiguous levels from c3 to c7 . The study required patients to have radiographic findings that correlated with their symptomatology and to have failed nonoperative management for at least 6 weeks or to have demonstrated progressive worsening of symptoms, calling for urgent surgery . Abbreviations: ct, computed tomography; mri, magnetic resonance imaging; ndi, neck disability index . Patients were randomized in a 2:1 ratio (cda to anterior cervical diskectomy and fusion [acdf]) resulting in 164 patients receiving treatment with a mobi - c cervical artificial disc (ldr medical, troyes, france) at a single level, 225 patients receiving treatment with a mobi - c cervical artificial disc at two contiguous levels, 81 patients receiving corticocancellous allograft and anterior cervical plate using the standard acdf technique at a single level, and 105 receiving the same acdf treatment at two contiguous levels . An additional 24 patients (15 one - level, 9 two - level) were treated with the cda device as training cases . This study only includes data from the randomized patients; the training cases were excluded from the analysis . The patients were unblinded after surgery, because the postoperative radiographs viewed by the patient and clinician made the treatment readily apparent . Patients were evaluated preoperatively and postoperatively at 6 weeks and 3, 6, 12, 18, 24, 36, 48, and 60 months . Patients were asked to refrain from taking nonsteroidal anti - inflammatory drugs from a week before surgery to 3 months after surgery . An exception was made for patients having cda who were diagnosed with postsurgical heterotopic ossification . The baseline demographics collected included height, weight, bmi, age, gender, race, ethnicity, working status, smoking status, and driving status . Each question is scored on a scale of 0 to 5 . In this study, the response scoring scale was as follows: 0, i have no headaches at all; 1, wilcoxon signed rank tests were used to compare the change from baseline within the treatment groups . Two - tailed t tests were used to compare the mean improvement between the treatment groups at each postoperative time point . Analysis of variance was used to test the mean improvement across demographic groups . A p value a detailed description of surgical technique and the ide study design has been previously reported.17 patients underwent surgery as part of a food and drug administration ide prospective, randomized clinical trial between april 2006 to march 2008 at 24 clinical sites in the united states . This study was approved by the university of california davis institutional review board (no . 217014). 1) were patient age 18 to 69 years with a diagnosis of degenerative disk disease with associated radiculopathy or myeloradiculopathy at a single level or at two contiguous levels from c3 to c7 . The study required patients to have radiographic findings that correlated with their symptomatology and to have failed nonoperative management for at least 6 weeks or to have demonstrated progressive worsening of symptoms, calling for urgent surgery . Abbreviations: ct, computed tomography; mri, magnetic resonance imaging; ndi, neck disability index . Patients were randomized in a 2:1 ratio (cda to anterior cervical diskectomy and fusion [acdf]) resulting in 164 patients receiving treatment with a mobi - c cervical artificial disc (ldr medical, troyes, france) at a single level, 225 patients receiving treatment with a mobi - c cervical artificial disc at two contiguous levels, 81 patients receiving corticocancellous allograft and anterior cervical plate using the standard acdf technique at a single level, and 105 receiving the same acdf treatment at two contiguous levels . An additional 24 patients (15 one - level, 9 two - level) were treated with the cda device as training cases . This study only includes data from the randomized patients; the training cases were excluded from the analysis . The patients were unblinded after surgery, because the postoperative radiographs viewed by the patient and clinician made the treatment readily apparent . Patients were evaluated preoperatively and postoperatively at 6 weeks and 3, 6, 12, 18, 24, 36, 48, and 60 months . Patients were asked to refrain from taking nonsteroidal anti - inflammatory drugs from a week before surgery to 3 months after surgery . An exception was made for patients having cda who were diagnosed with postsurgical heterotopic ossification . The baseline demographics collected included height, weight, bmi, age, gender, race, ethnicity, working status, smoking status, and driving status . Each question is scored on a scale of 0 to 5 . In this study, the response scoring scale was as follows: 0, i have no headaches at all; 1, wilcoxon signed rank tests were used to compare the change from baseline within the treatment groups . Two - tailed t tests were used to compare the mean improvement between the treatment groups at each postoperative time point . At 60 months, ndi data was available for 82.9% (136/164) of patients having one - level cda and 70.4% (57/81) of patients having one - level acdf . The mean range of motion was maintained in the cda group in both flexion extension (10.3 6.8 degrees) and lateral bending (5.5 3.4 degrees) compared with baseline (8.2 4.5 degrees, 5.0 2.9 degrees, respectively). The mean range of motion in the acdf group was effectively eliminated in flexion extension (0.7 1.1 degrees) and lateral bending (0.7 1.0 degrees) compared with baseline (7.5 4.1 degrees, 5.4 3.2 degrees, respectively). For patients having one - level cda, severe heterotopic ossification (continuous bridging bone and <2 degrees angular motion) was present in 8.5% of treated levels . Patients with continuous bridging bone and <2 degrees angular motion demonstrated similar ndi headache scores compared with the other patients having cda . The baseline distribution of headache scores was similar between the groups with the most frequent headache response being 3 for both groups (fig . The percentage of patients reporting a score of 3 or more was 50.6% for patients having cda and 53.0% for patients having cdf . The baseline mean headache scores were also similar between the two groups . The baseline mean headache scores significantly improved from a baseline of 2.52 1.45 and 2.41 1.57 to 1.25 1.24 and 1.05 1.05 at 60 months for both the cda and acdf groups, respectively (p <0.0001). (a) the distribution of headache scores at baseline for patients having one - level cervical disk arthroplasty (cda) and patients having anterior cervical diskectomy and fusion (acdf). (b) the distribution of headache scores at baseline for the one - level group at 60 months . At 60 months, the distribution of headache scores shifted noticeably toward lower scores, indicating a significant reduction in pain for both groups . (c) the mean neck disability index (ndi) headache score of patients having cda and patients having acdf from baseline to 60 months' follow - up . (d) the distribution of changes in headache score from baseline to 60 months . Although the majority of patients experienced some degree of pain relief, a minority of patients experienced no change or an increase in headache score from baseline . Both the investigational and control groups experienced a significant improvement from the baseline headache scores at each point up to 60 months (p <0.0001). The distribution of headache scores shifted noticeably toward lower scores with the most common headache score being 1 for the cda group and 0 for the acdf group (fig . The percentage of patients reporting a score of 3 or more was 15.4% for patients having cda and 10.5% for patients having acdf, a nonsignificant difference (p = 0.5). The cda group had a mean headache score improvement of 1.24 1.53 from baseline, and the acdf group experienced a similar mean improvement of 1.26 1.43 . The mean improvement in headache scores was not statistically different between the investigational and control groups at each point from 6 months through 60 months (fig . The most common improvement in headache score was by 1 point for both groups (fig . Additionally, 11.8% (12/136) of patients having cda experienced an increase in headache score of 1 to 2 points more from baseline compared with 8.77% (5/57) of patients having acdf, though this difference was not statistically significant (p> 0.99). The mean improvement in headache score was not significantly different between the demographic groups stratified by age, gender, and bmi in both the single - level and two - level cohorts (tables 1 2). Abbreviations: acdf, anterior cervical diskectomy and fusion; bmi, body mass index; cda, cervical disk arthroplasty . Abbreviations: acdf, anterior cervical diskectomy and fusion; bmi, body mass index; cda, cervical disk arthroplasty . At 60 months, data was available for 85.3% (192/225) of patients having two - level cda and 70.5% (74/105) of patients having two - level acdf . As in the one - level cohort, the mean range of motion was maintained in the two - level cda group in both flexion extension (superior: 10.1 6.1 degrees, inferior: 8.4 5.0 degrees) and lateral bending (superior: 5.6 3.6 degrees, inferior: 5.1 3.5 degrees) compared with baseline (superior: 9.1 4.8 degrees, inferior: 7.4 4.3 degrees; superior: 5.8 3.4 degrees, inferior: 4.9 3.4 degrees). The mean range of motion in the acdf group was eliminated in flexion extension (superior: 0.3 0.3 degrees, inferior: 0.6 0.9 degrees) and lateral bending (superior: 0.8 1.9 degrees, inferior: 0.6 0.7 degrees), compared with baseline (superior: 9.3 4.9 degrees, inferior: 7.1 3.9 degrees; superior: 5.5 3.0 degrees, inferior: 4.8 2.9 degrees). For patients having two - level cda, severe heterotopic ossification (continuous bridging bone and <2 degrees angular motion) was present in 5.9% of superior levels and 5.4% of inferior levels . Patients with continuous bridging bone and <2 degrees angular motion demonstrated similar ndi headache scores compared with other patients having cda . The distribution of headache scores at baseline was similar between the groups with the most frequent headache response being scored 3 for the two - level cda group and 2 and 3 for the two - level acdf group (fig . The baseline mean headache scores significantly improved from 2.65 1.56 and 2.49 1.58 to 1.29 1.30 and 1.50 1.34 at 60 months for both the cda and acdf groups, respectively (p (a) the distribution of headache scores at baseline for patients having two - level cervical disk arthroplasty (cda) and patients having anterior cervical diskectomy and fusion (acdf). (b) the distribution of headache scores for the two - level group at 60 months . At 60 months, the distribution of headache scores shifted noticeably toward lower scores, indicating a significant reduction in pain for both groups . (c) the mean neck disability index (ndi) headache score of patients having cda and patients having acdf from baseline to 60 months' follow - up . Patients having cda demonstrated a greater mean improvement than patients having acdf at 6, 12, 24, 36, and 48 months . Asterisks denote a statistically significant difference in mean change from baseline (p <0.05). (d) the distribution of changes in headache score from baseline to 60 months for the two - level group . A minority of patients experienced no change or an increase in headache score from their baseline measurement . Both the two - level cda group and the two - level acdf group experienced a significant improvement from the baseline headache scores at each point up to 60 months (p <0.0001). As in the one - level cohort, the distribution of headache scores shifted significantly toward lower scores at 60 months' follow - up . At 60 months, the most common headache score was 0 and 1 for the cda group and 0 for the acdf group (fig . The percentage of patients with a score of 3 or higher decreased from 55.1 to 18.8% in the cda group and from 51.4 to 25.7% in the acdf group . The cda group demonstrated a statistically significant greater improvement from baseline compared with the acdf group at 6, 12, 24, 36, and 48 months (fig . 4c). At 18 and 60 months, patients undergoing cda experienced a greater mean improvement in headache scores that approached but did not reach statistical significance . At 18 months, the investigational group had a mean improvement in headache of 1.28 1.29 compared with 1.57 1.47 (p = 0.051). Similarly at 60 months, the cda group had a mean improvement in headache score of 1.37 1.68 compared with the acdf group 0.986 1.37 (p = 0.0824). The most common improvement in headache score was by 1 point for both the cda population and the acdf population (fig . 4d). Additionally, 8.85% (17/192) of patients having cda experienced headaches worsening by 1 to 4 points from baseline compared with 9.46% (7/74) of patients having acdf who experienced worsening headache scores of 1 to 2 points from baseline . As in the one - level cohort, the mean improvement in headache score was not significantly different between the demographic groups stratified by age, gender, and bmi (table 2). Female patients in the control group had nearly twice the improvement in headache scores than their male counterparts . However, this difference was not statistically significant (p = 0.17) and may be attributed to numerically higher baseline headache scores for female patients in the acdf group . Although male and female patients did not demonstrate significantly different improvement in headache scores, it is interesting to note that female patients in the cda and acdf populations for both one - level and two - levels of treatment reported more headache than their male counterparts at baseline and at 60 months . At 60 months, ndi data was available for 82.9% (136/164) of patients having one - level cda and 70.4% (57/81) of patients having one - level acdf . The mean range of motion was maintained in the cda group in both flexion extension (10.3 6.8 degrees) and lateral bending (5.5 3.4 degrees) compared with baseline (8.2 4.5 degrees, 5.0 2.9 degrees, respectively). The mean range of motion in the acdf group was effectively eliminated in flexion extension (0.7 1.1 degrees) and lateral bending (0.7 1.0 degrees) compared with baseline (7.5 4.1 degrees, 5.4 3.2 degrees, respectively). For patients having one - level cda, severe heterotopic ossification (continuous bridging bone and <2 degrees angular motion) was present in 8.5% of treated levels . Patients with continuous bridging bone and <2 degrees angular motion demonstrated similar ndi headache scores compared with the other patients having cda . The baseline distribution of headache scores was similar between the groups with the most frequent headache response being 3 for both groups (fig . The percentage of patients reporting a score of 3 or more was 50.6% for patients having cda and 53.0% for patients having cdf . The baseline mean headache scores were also similar between the two groups . The baseline mean headache scores significantly improved from a baseline of 2.52 1.45 and 2.41 1.57 to 1.25 1.24 and 1.05 1.05 at 60 months for both the cda and acdf groups, respectively (p <0.0001). (a) the distribution of headache scores at baseline for patients having one - level cervical disk arthroplasty (cda) and patients having anterior cervical diskectomy and fusion (acdf). (b) the distribution of headache scores at baseline for the one - level group at 60 months . At 60 months, the distribution of headache scores shifted noticeably toward lower scores, indicating a significant reduction in pain for both groups . (c) the mean neck disability index (ndi) headache score of patients having cda and patients having acdf from baseline to 60 months' follow - up . (d) the distribution of changes in headache score from baseline to 60 months . Although the majority of patients experienced some degree of pain relief, a minority of patients experienced no change or an increase in headache score from baseline . Both the investigational and control groups experienced a significant improvement from the baseline headache scores at each point up to 60 months (p <0.0001). The distribution of headache scores shifted noticeably toward lower scores with the most common headache score being 1 for the cda group and 0 for the acdf group (fig . The percentage of patients reporting a score of 3 or more was 15.4% for patients having cda and 10.5% for patients having acdf, a nonsignificant difference (p = 0.5). The cda group had a mean headache score improvement of 1.24 1.53 from baseline, and the acdf group experienced a similar mean improvement of 1.26 1.43 . The mean improvement in headache scores was not statistically different between the investigational and control groups at each point from 6 months through 60 months (fig . The most common improvement in headache score was by 1 point for both groups (fig . Additionally, 11.8% (12/136) of patients having cda experienced an increase in headache score of 1 to 2 points more from baseline compared with 8.77% (5/57) of patients having acdf, though this difference was not statistically significant (p> 0.99). The mean improvement in headache score was not significantly different between the demographic groups stratified by age, gender, and bmi in both the single - level and two - level cohorts (tables 1 2). Abbreviations: acdf, anterior cervical diskectomy and fusion; bmi, body mass index; cda, cervical disk arthroplasty . Abbreviations: acdf, anterior cervical diskectomy and fusion; bmi, body mass index; cda, cervical disk arthroplasty . At 60 months, data was available for 85.3% (192/225) of patients having two - level cda and 70.5% (74/105) of patients having two - level acdf . As in the one - level cohort, the mean range of motion was maintained in the two - level cda group in both flexion extension (superior: 10.1 6.1 degrees, inferior: 8.4 5.0 degrees) and lateral bending (superior: 5.6 3.6 degrees, inferior: 5.1 3.5 degrees) compared with baseline (superior: 9.1 4.8 degrees, inferior: 7.4 4.3 degrees; superior: 5.8 3.4 degrees, inferior: 4.9 3.4 degrees). The mean range of motion in the acdf group was eliminated in flexion extension (superior: 0.3 0.3 degrees, inferior: 0.6 0.9 degrees) and lateral bending (superior: 0.8 1.9 degrees, inferior: 0.6 0.7 degrees), compared with baseline (superior: 9.3 4.9 degrees, inferior: 7.1 3.9 degrees; superior: 5.5 3.0 degrees, inferior: 4.8 2.9 degrees). For patients having two - level cda, severe heterotopic ossification (continuous bridging bone and <2 degrees angular motion) was present in 5.9% of superior levels and 5.4% of inferior levels . Patients with continuous bridging bone and <2 degrees angular motion demonstrated similar ndi headache scores compared with other patients having cda . The distribution of headache scores at baseline was similar between the groups with the most frequent headache response being scored 3 for the two - level cda group and 2 and 3 for the two - level acdf group (fig . 55.1% of patients having cda and 51.4% of patients having acdf reported a score of 3 or more . The baseline mean headache scores significantly improved from 2.65 1.56 and 2.49 1.58 to 1.29 1.30 and 1.50 1.34 at 60 months for both the cda and acdf groups, respectively (p <0.0001). (a) the distribution of headache scores at baseline for patients having two - level cervical disk arthroplasty (cda) and patients having anterior cervical diskectomy and fusion (acdf). (b) the distribution of headache scores for the two - level group at 60 months . At 60 months, the distribution of headache scores shifted noticeably toward lower scores, indicating a significant reduction in pain for both groups . (c) the mean neck disability index (ndi) headache score of patients having cda and patients having acdf from baseline to 60 months' follow - up . Patients having cda demonstrated a greater mean improvement than patients having acdf at 6, 12, 24, 36, and 48 months . Asterisks denote a statistically significant difference in mean change from baseline (p <0.05). (d) the distribution of changes in headache score from baseline to 60 months for the two - level group . A minority of patients experienced no change or an increase in headache score from their baseline measurement . Both the two - level cda group and the two - level acdf group experienced a significant improvement from the baseline headache scores at each point up to 60 months (p <0.0001). As in the one - level cohort, the distribution of headache scores shifted significantly toward lower scores at 60 months' follow - up . At 60 months, the most common headache score was 0 and 1 for the cda group and 0 for the acdf group (fig . The percentage of patients with a score of 3 or higher decreased from 55.1 to 18.8% in the cda group and from 51.4 to 25.7% in the acdf group . The cda group demonstrated a statistically significant greater improvement from baseline compared with the acdf group at 6, 12, 24, 36, and 48 months (fig . 4c). At 18 and 60 months, patients undergoing cda experienced a greater mean improvement in headache scores that approached but did not reach statistical significance . At 18 months, the investigational group had a mean improvement in headache of 1.28 1.29 compared with 1.57 1.47 (p = 0.051). Similarly at 60 months, the cda group had a mean improvement in headache score of 1.37 1.68 compared with the acdf group 0.986 1.37 (p = 0.0824). The most common improvement in headache score was by 1 point for both the cda population and the acdf population (fig . Additionally, 8.85% (17/192) of patients having cda experienced headaches worsening by 1 to 4 points from baseline compared with 9.46% (7/74) of patients having acdf who experienced worsening headache scores of 1 to 2 points from baseline . As in the one - level cohort, the mean improvement in headache score was not significantly different between the demographic groups stratified by age, gender, and bmi (table 2). Female patients in the control group had nearly twice the improvement in headache scores than their male counterparts . However, this difference was not statistically significant (p = 0.17) and may be attributed to numerically higher baseline headache scores for female patients in the acdf group . Although male and female patients did not demonstrate significantly different improvement in headache scores, it is interesting to note that female patients in the cda and acdf populations for both one - level and two - levels of treatment reported more headache than their male counterparts at baseline and at 60 months . The mechanism of referred pain can be explained by nociceptive afferents from c1, c2, and c3 spinal nerves that synapse onto second - order neurons in the trigeminocervical nucleus . This convergence described by bogduk and govind can help explain the referral of pain to the frontoparietal and orbital regions of the head.3 additional studies have shown that stimulation of the rostral spine evokes referred pain to the occipital, frontal, and orbital regions of the head, whereas stimulation of the caudal cervical spine elicits pain mainly in the occipital region.3 18 the association between cervical nerve roots below c3 and headache is not as well understood . Cervicogenic headache arising from the lower cervical spine was first described by diener et al, who theorized that nociceptive afferents from the lower cervical roots also converge on the cervical trigeminal nucleus.2 persson et al analyzed headaches in patients with cervical radiculopathy from the lower cervical spine . Selective nerve root blocks of the pathologic level produced a 50% or more reduction in headache with 69% of these patients reporting complete relief of headache using the vas.19 this study lends support to the theory that cervicogenic headache has the potential to be relieved with diskectomy in the lower cervical spine (in addition to the upper cervical levels). Attempts have also been made to correlate cervicogenic headaches with a sympathetic phenomenon in patients with cervical spondylosis . Barre and lieou first described these sympathetic symptoms in 1926, which include vertigo, headache, dizziness, and hyperhidrosis.20 21 li et al noted an association between anterior cervical diskectomy and improvement in these sympathetic symptoms, including headache . They hypothesized that headache relief was mediated by the removal of sympathetic nerve fibers contained within the posterior longitudinal ligmament.21 in the one- and two - level clinical trials studied here, resection of the posterior longitudinal ligament was left to the discretion of the operating surgeon for both arthroplasty and arthrodesis treatment.2 as noted above, cervicogenic headaches improve after anterior cervical diskectomy procedures . Schofferman et al first demonstrated headache relief after cervical arthrodesis in the upper cervical levels c2c5.22 riina et al reported headache relief after either single - level arthroplasty or arthrodesis with 2-year follow - up, and patients having arthroplasty experienced even greater headache improvement compared with the patients having acdf.8 this result was different from another one - level analysis with 2-year follow - up, which showed no significant difference in headache relief between arthrodesis and arthroplasty.9 both single - level procedures, however, showed a significant improvement in headache when compared with baseline . The mechanism for headache relief may be multifactorial, and the putative mechanisms include neuroanatomic connections of the trigeminocervical nucleus with lower cervical roots; sympathetically innervated structures, which are then denervated by surgery; relief of cerebrospinal fluid obstruction during systole caused by focal spinal stenosis; and kinesthetic improvements in the cervical spine after surgery, either through improved spinal biomechanics or relief of reflex muscle spasm . To further investigate the role that improved spinal biomechanics may have in relief of headache after anterior disk surgery, we broadened our analysis to include patients undergoing two - level surgery and with follow - up out to 5 years . We assumed that the loss versus preservation of two contiguous motion segments may have a greater impact on spinal kinesiology compared with analysis at only a single level . Indeed, pimenta et al has shown that multilevel cervical arthroplasty when compared with single - level arthroplasty allows for a greater improvement in ndi and vas scores.14 cervicogenic headaches, however, were not specifically analyzed in the study by pimenta et al . We found a statistically significant difference in the headache scores between the two - level procedures at all points except for 18 and 60 months . There was no difference in headache relief between the groups in patients undergoing only a single - level surgery . Both the two - level and single - level data analysis showed an improvement in headache from baseline, with the patients having two - level cda experiencing a greater mean improvement in headache . Although the two - level cda group demonstrated statistically significant improvement compared with acdf at most points, there was no difference between the two groups at the last follow - up (60 months). It remains to be seen if the difference favoring the arthroplasty group will reemerge with even longer follow - up . Therefore, we are not able to draw conclusions on the clinical relevancy on the difference between cda and acdf . We suggest that an improvement in spine kinematics may improve headaches and that the effect is more apparent with multilevel arthroplasty . With multilevel cda, each vertebral segment remains biomechanically distinct, unlike with arthrodesis, which consolidates motion segments.7 14 15 with the longer lever arm of multilevel fusion, spine kinematics is more adversely affected than with single - level fusion . However, we are not able to make a definitive conclusion, as kinematic measures were not analyzed in this study and the difference was not statistically significant at all points . Additional studies that measure the biomechanical profile at the index level and adjacent levels up to 5 years after surgery and its effects on headache relief can be done to help elucidate these relationships . Importantly, both the fusion and arthroplasty groups had statistically significant headache relief at all points up to 60 months compared with baseline . A fundamental assumption of cervical disk arthroplasty is that loss of a motion segment is detrimental to spinal biomechanics and that preservation or restoration of motion at that segment is beneficial . Yet patients with two - level fusion still improved significantly with respect to headache scores compared with baseline . If one postulates that improvement in spinal biomechanics alone mediates headache relief, then one must assume that for fusion, the correction of deformity through the restoration of intervertebral height or lordosis improves spinal biomechanics independent of segmental motion preservation . Previously, we reported headache outcomes for patients having one - level fusion and arthroplasty through 24 months postsurgery . We used a mixed - models repeated - measures regression to assess the postoperative pain relief and to establish a significant trend of gradually worsening pain from 6 weeks to 1 year, followed by the maintenance of pain relief from 1 to 2 years . The model accounted for differences in treatment type as well as differences in operative level . Here, we have chosen not to construct a new model or update the previous model, but focus rather on the comparative outcomes of acdf and cda through 5 years, extending our analysis to include a separate assessment of patients having two - level treatment . Unlike the previous 2-year analysis, this assessment does not model trends in pain attenuation nor does it account for differences in operative level . However, we were able to demonstrate that both patients having acdf and patients having cda maintained significant improvement from baseline headache scores through 5 years (p <0.0001). Whether a trend in worsening pain exists as it did between 6 weeks and 12 months remains unclear, as does the effect of treatment level on long - term headache outcomes . Limitations of this study include the inability to definitively diagnose cervicogenic headache in our patient population based on the diagnostic criteria proposed by the ihs . These patients did not undergo a diagnostic blockade to provide objective evidence of a cervical source of headache . As stated in published studies using similar methodology,3 4 headache assessment using a component of the ndi only provides a very basic headache questionnaire, and thus specific headache characteristics are not captured more detailed prospective studies that precisely qualify cervicogenic headaches should be done to corroborate our findings, as this study was a post hoc analysis of prospectively collected data . At 5 years, patients who undergo single- or two - level anterior cervical diskectomy procedures benefit from improvement in their baseline headaches . For one - level surgery, the mean improvement in headache scores was not statistically different between the investigational and control groups at all time points . In contrast to the one - level cohort, patients receiving two - level cda showed a greater improvement in mean headache scores from baseline at all points except for at 18 and 60 months . We also show that headache scores were not significantly different between the demographic groups for either one - level or two - level surgery . Although headache was apparently relieved after either arthroplasty or fusion, headache was relieved by two - level arthroplasty more so than by two - level fusion, and this difference was not observed in the single - level arthroplasty trial . Improved kinematics after arthroplasty may play a role but headache alone should not be the indication for surgery.
It is known that 9095% of all cancers are caused by or closely associated with environmental factors and lifestyle . This includes diet (3035%), cigarette smoking (2530%), and alcohol consumption (46%). Cigarette smoking is an important risk factor for heart disease, chronic obstructive pulmonary disease, stroke, and acute respiratory diseases . In addition to all these noncancer diseases, it is also highly associated with human cancer development . The international agency for research on cancer (iarc) identified cigarette smoking as the cause of cancer in more organ sites than any other human carcinogens . These include cancers of the lungs, oral cavity, larynx, nasal cavity, esophagus, stomach, pancreas, liver, kidney, urinary bladder, uterine cervix, and bone marrow . There are over 5000 chemical compounds identified in tobacco and 62 of these have been evaluated by iarc as showing sufficient evidence for carcinogenicity in either animals or in humans [2, 3]. The major carcinogenic compounds include, but not limited to, radioactive polonium, n - nitrosamines such as 4-(methylnitrosaminao)-1-(3-pyridyl)-1-butanone (nnk), polycyclic aromatic hydrocarbons (pahs) (e.g., benzo[a]pyrene (bap)), and benzene . Multistep processes of genetic and molecular defects have taken place before the manifestation of cancer . Traditionally, there are three basic stages of carcinogenesis: initiation, promotion, and progression . Carcinogenesis process is usually accompanied by changes in structure and function of central genomic information coded in the dna leading to various oncogene activations and tumor suppressor gene inactivations . In addition, multiple signaling pathways may also be deregulated during the process of cancer development . Cancer growth also requires molecular changes that either affect the tumor cells themselves or alter the interaction between tumor cells and their surrounding stromal environment or the immune system . Cigarette smoke components have been reported to promote tumorigenesis by several mechanisms involving all three stages of carcinogenesis . Genotoxic agents in cigarette smoke induce dna damage through several mechanisms including gene point mutation, deletions, insertions, recombinations, rearrangements, and chromosomal aberrations . In addition to genotoxic effects, nongenotoxic effects of cigarette smoke are also extremely important . These effects can also act as modulators which alter cellular functions including cell proliferation and cell death . While synergistic effects of genotoxic carcinogens are known to occur, interaction between non - genotoxic (epigenetic) factors and genotoxic agents may also synergistically increase the risk for carcinogenesis . The genotoxicity leading to carcinogenesis has been extensively reviewed in recent years [911]. In this present review, aside from a brief overview on the genotoxic effects of cigarette smoke components, we will provide a more extensive review on the non - genotoxic mechanisms of carcinogenesis by cigarette smoke or its components . Step 1 (initiation of carcinogenesis)carcinogenesis may be the result of chemical or biological insults to normal cells through multistep processes that involves genomic changes (initiation of cancer development). Some of the cigarette smoke components can act directly on dna, but many require enzyme conversion before becoming carcinogenic [10, 11]. Most of such conversions involve metabolic changes via cytochrome p450s (p450s) such as p450s 1a2, 2a13, 2e1, and 3a4 to form the electrophilic entities that can bind to dna to form dna adducts . Such adduct formation is usually at the adenine or guanine sites of the dna and lead to mutations such as those observed in the kras oncogene in lung cancer or those in the tp53 gene in a variety of cigarette smoke - induced cancers [13, 14]. 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (nnk) and n-nitrosonornicotine (nnn) are the most potent tobacco - specific nitrosamines in tobacco products and cigarette smoke . These compounds are formed from tobacco alkaloids like nicotine during the curing process of tobacco and are important tobacco carcinogens that can affect different tissues depending on the specific nitrosamines or their metabolites involved [5, 10]. Nnn has been shown to be carcinogenic to esophagus, nasal cavity, and respiratory tract in laboratory animals . In humans, metabolites derived from nnk and the metabolites of nnk can also be identified in the smoker's urine .benzo[a]pyrene (bap), one of the pahs, is classified as a group 1 carcinogen to humans . It has been shown to have strong association and tumor - induction potentials in lungs, trachea, and mammary glands . The carcinogenic potency of bap has been demonstrated to be related to its metabolites which form dna adducts with site - specific hotspot mutation in the p53 tumor suppressor gene . Positive correlations of such adduct formation and tumor are indeed found in the lung cancer tissues of cigarette smokers . These findings indicate that dna mutations are increased in both tumor and nontumor bearing tissues of smokers . However, it must be pointed out that dna adduct formations induced by cigarette smoke still cannot fully represent all the risk factors for cancer development in cigarette smokers . For example, while there is higher incidence of pancreatic cancer in cigarette smokers than nonsmokers . Assays for nnk metabolites in pancreatic cancer tissues in humans showed no significant difference between smokers and nonsmokers . Thus, it is apparent that nnk - induced dna adducts alone are not solely responsible for the pancreatic cancers in cigarette smokers . Nevertheless, nnk and its metabolite, nnal (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol), are the only environmental carcinogens known to induce pancreatic cancer in animal models . Thus, the contribution of nnk to pancreatic cancer in cigarette smokers still cannot be ignored . Furthermore, it is suggested that, in addition to dna damage, synergistic interactions between dna reactivity and epigenetic actions such as increased cell proliferation induced by nnk or by other chemicals in cigarette smoke may be needed for actual cancer development in such patients [23, 24]. There is indication that cigarette smoke carcinogens or cocarcinogen, such as nicotine, may also play a direct role to enhance cancer promotion and progression in human cancers after cancer development . Such genotoxic mechanisms for cancer initiation and carcinogenesis by cigarette smoke components are well covered and discussed in several excellent reviews [5, 10, 11, 2628]., we will provide more information on the non - genotoxic (epigenetic) mechanisms involved in cancer promotion and progression via cigarette smoke . Carcinogenesis may be the result of chemical or biological insults to normal cells through multistep processes that involves genomic changes (initiation of cancer development). Some of the cigarette smoke components can act directly on dna, but many require enzyme conversion before becoming carcinogenic [10, 11]. Most of such conversions involve metabolic changes via cytochrome p450s (p450s) such as p450s 1a2, 2a13, 2e1, and 3a4 to form the electrophilic entities that can bind to dna to form dna adducts . Such adduct formation is usually at the adenine or guanine sites of the dna and lead to mutations such as those observed in the kras oncogene in lung cancer or those in the tp53 gene in a variety of cigarette smoke - induced cancers [13, 14]. 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (nnk) and n-nitrosonornicotine (nnn) are the most potent tobacco - specific nitrosamines in tobacco products and cigarette smoke . These compounds are formed from tobacco alkaloids like nicotine during the curing process of tobacco and are important tobacco carcinogens that can affect different tissues depending on the specific nitrosamines or their metabolites involved [5, 10]. Nnn has been shown to be carcinogenic to esophagus, nasal cavity, and respiratory tract in laboratory animals . In humans, metabolites derived from nnk and the metabolites of nnk benzo[a]pyrene (bap), one of the pahs, is classified as a group 1 carcinogen to humans . It has been shown to have strong association and tumor - induction potentials in lungs, trachea, and mammary glands . The carcinogenic potency of bap has been demonstrated to be related to its metabolites which form dna adducts with site - specific hotspot mutation in the p53 tumor suppressor gene . Positive correlations of such adduct formation and tumor are indeed found in the lung cancer tissues of cigarette smokers . These findings indicate that dna mutations are increased in both tumor and nontumor bearing tissues of smokers . However, it must be pointed out that dna adduct formations induced by cigarette smoke still cannot fully represent all the risk factors for cancer development in cigarette smokers . For example, while there is higher incidence of pancreatic cancer in cigarette smokers than nonsmokers . Assays for nnk metabolites in pancreatic cancer tissues in humans showed no significant difference between smokers and nonsmokers . Thus, it is apparent that nnk - induced dna adducts alone are not solely responsible for the pancreatic cancers in cigarette smokers . Nevertheless, nnk and its metabolite, nnal (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol), are the only environmental carcinogens known to induce pancreatic cancer in animal models . Thus, the contribution of nnk to pancreatic cancer in cigarette smokers still cannot be ignored . Furthermore, it is suggested that, in addition to dna damage, synergistic interactions between dna reactivity and epigenetic actions such as increased cell proliferation induced by nnk or by other chemicals in cigarette smoke may be needed for actual cancer development in such patients [23, 24]. There is indication that cigarette smoke carcinogens or cocarcinogen, such as nicotine, may also play a direct role to enhance cancer promotion and progression in human cancers after cancer development . Such genotoxic mechanisms for cancer initiation and carcinogenesis by cigarette smoke components are well covered and discussed in several excellent reviews [5, 10, 11, 2628]., we will provide more information on the non - genotoxic (epigenetic) mechanisms involved in cancer promotion and progression via cigarette smoke . Step 2 (cancer promotion)cancer promotion is characterized by deregulation of signaling pathways which control cell proliferation, apoptosis, and so forth, . It is believed that although there are various genetic pathways which may lead to cancer development or cancer behaviors, there are certain hallmark capabilities or mechanisms which are commonly shared by all tumors . In the following discussion cancer promotion is characterized by deregulation of signaling pathways which control cell proliferation, apoptosis, and so forth, . It is believed that although there are various genetic pathways which may lead to cancer development or cancer behaviors, there are certain hallmark capabilities or mechanisms which are commonly shared by all tumors . In the following discussion these signals are transmitted into cells by receptors that bind distinct signaling molecules . In cancer cells, receptor overexpression allows cancer cells to become hyper - responsive to low levels of growth factors that generally are not sufficient to trigger proliferation in normal cells . Nicotine, a major component of cigarette smoke, is known to be a chemical that plays an important role in carcinogenesis in cigarette smokers . Nicotine behaves like those growth factors which exert their biological functions mainly through the nicotinic acetylcholine receptors (nachr), -adrenoceptors (-ar) or epidermal growth factor receptor (egfr). The functions of these receptors are cell - type specific and the expression level and receptor sensitivity can be modified by nicotine . Obviously, alterations in either the receptor expressions or sensitivity play an important role in cigarette smoke - induced carcinogenesis [3436]. Recent study by lee et al . Reported that 9 nachr expression in human breast tumors is elevated in advanced stages of breast cancer and plays important roles in human breast carcinogenesis . Nicotine has been shown to mediate 9 nachr signaling and upregulate cyclin d3 expression in breast caner cells and breast cancer tissues . Furthermore, it is also found that activation of the expression of 9 nachr by nicotine is through akt signaling and activation of 9 nachr signaling would elevate the phosphorylation status of adhesion molecule which plays a role in cancer metastasis . This enhancement has been shown to be via 7 nachr with activation of erk1/2 cascade as well as induction of nf-b and bad phosphorylation . All these events eventually lead to inhibition of apoptosis and increase of cancer risk . These findings were further supported by wada et al . Who observed that nicotine promoted cell proliferation via 7 nachr mediated p44/p42-mapk activation . Moreover, in our own study, we also reported that nicotine induced human bladder cells proliferation through erk1/2 and stat3 signaling downstream of 7 nachr and -adrenoceptors (-ar). In sum, all these studies indicate that nicotine, an important ingredient of cigarette smoke, promotes cellular proliferation which plays a critical role in carcinogenesis . Other than nicotine, nitrosamines, such as nnk and nnn, also induced cancer cells growth through nachr . Nnk induced carcinogenesis by binding to nachr especially for 7 nachr, whereas the biological impact of nnn is mainly modulated by 4/2 nachr [8, 4446]. It has been demonstrated that nicotine or nnk stimulated lung cancer cell proliferation via 7 nachr with activations of pkc, raf1, akt, erk1/2, and transcription factors such as jun, fos, and myc [4749]. Question has been raised concerning the possibility that specific nachr subunit upregulated by nicotine or nnk may be tissue specific or dependent . For instance, with nicotine or nnk, 7 nachr is the primary nachr subunit which mediates tumorigenesis in lungs giving rise to pulmonary squamous cell carcinoma and mesothelioma . On the other hand, thus, the specific types of nachr expressed in cancer cells may be considered as useful molecular targets for potential clinical therapy . However, most of the nachr present in cancer cells are still not functionally characterized yet . Future study will be needed to understand the functions of different nachr subtype in cancer cells and the downstream signal pathways involved in tumorigenesis . In addition to nachr, a number of studies indicated that nicotine and nnk might also exert their biological activities through activation of receptors such as -adrenoceptors (-ar), egfr, or insulin - like growth factor receptor (igfr) or transactivation by nachr signaling . For example, nnk can stimulate ht-29 cell proliferation through -ar followed by cyclin amp elevation and cox-2 expression . Consistently, nnk stimulates the growth of pulmonary adenocarcinoma in vitro and in vivo via the release of arachidonic acid through cox-2 and 5-lipoxygenase (5-lox) pathways that are mainly regulated by -ar . In another study by schuller and cekanova, nnk is reported to stimulate 2-ar receptor pathway (including pka, camp, creb) and transactivate egfr pathway (such as raf-1/erk1/2 signaling) in the development of lung cancer . It has also been reported that antagonists of -ar can inhibit the development of nnk - induced lung adenocarcinoma . Such antagonists are also found to be effective in reducing the stimulatory effects of nicotine on pkc, erk1/2 activations, cox-2 expression, and gastric cancer cell proliferation . Elevation of noradrenaline by nicotine via 7 nachr up - regulation leading to significantly enhanced growth and angiogenesis in both gastric cancer and colon cancer has also been demonstrated . Various investigators have also shown increases in neurotransmitters lead to -ar activation, transactivation of egfr, and the release of egf [32, 54, 56]. Indeed, our recent investigation provided compelling evidence that chronic nicotine exposure induced release of noradrenaline via 4/2 nachr activation followed by -ar transactivation . Our study further demonstrated that blocking of -ar with antagonist reversed the nicotine - induced cellular proliferative and chemoresistance . Al - wadei et al . First reported that nicotine contributes to the development of smoking - related pancreatic ductal adenocarcinoma (pdac) with elevated levels of stress neurotransmitters (adrenaline and noradrenaline) and induction of camp, pcreb, and perk1/2, and inhibition of -aminobutyric acid (gaba). Gaba has been reported to possess tumor suppressor function suppressing both -ar stimulated pdac growth and migration in vitro . However, while gaba is suppressed in pdacs, noradrenaline, pka, p - creb, and perk1/2 in these tissues are overexpressed . These authors suggested that nicotine and nnk may contribute to the development of pdac in smokers by suppression of gaba with induction of stress neurotransmitters . Further proposed that nicotine induces the release of stress neurotransmitters through activation of 7 nachr and inhibits release of gaba via inhibition of 4/2 nachr . It is now believed that the stress neurotransmitter released via nachr activation plays an important role in smoking - associated tumorigenesis . However, the precise mechanisms involved in the regulation and the function of neurotransmitter released by nicotine and nnk are still uncertain . Future research on this area is encouraged . It has also been shown that nnk can promote -ar - mediated transactivation of egfr followed by erk1/2 phosphorylation leading to an increased proliferation in pancreatic cancer cells . Nnk is also reported to induce endogenous igfr which is associated with the development of lung tumors . Huang et al . Also indicated that both activation of thromboxane a2 (txa2) receptor and synthesis of txa2 play critical roles in nnk - promoted lung cancer cell proliferation . Txa2 activates the transcriptional factor creb through both erk and pi3k / akt pathways, which may also lead to pcna and bcl-2 overexpressions and cell proliferation . These studies provide valuable information on the mechanisms which involve in proliferative signaling stimulated by nicotine and nnk through activation of nachr, -ar and other growth factor receptors in cancer cells . Triggering such receptors by cigarette smoke would further lead to rapid cell proliferation, cellular migration, invasion, and metastasis . In short, these investigations on the nachr, and nachr transactivated with other receptors represent the pivotal role in regulating multiple cellular cascades in general cell functions and in carcinogenesis . Nicotine is also known to influence signal transducers and activators of transcription 3 (stat 3) which is an important signal transducer mediating signaling by numerous cytokines, growth factors, and oncoproteins . Findings from our own laboratories indicate that nicotine induces bladder cancer cells proliferation through 7 nachr, 42 nachr, and -ar followed by activation of erk1/2 and stat 3 . Stat3 signaling further enhanced nf-b activation, cyclin d1 overexpression, and cell cycle progression . Moreover, we also revealed that prolonged stimulation by nicotine upregulated 4/2 nachr and -ar followed with activation of stat 3 leading to significant increase in chemoresistance in cells from bladder cancers . In recent years the biological effects of pahs are mainly mediated via aryl hydrocarbone receptor (ahr). Through ahr, other pahs, such as benz(a)anthracene (baa), has also been found to increase dna synthesis and promote g1-s progression in serum deprived mcf-7 cells . Bap has been shown to increase incidence of tumors in estrogen - responsive rodents, suggesting that it may also affect er - mediated signaling . Some investigators believed that the estrogenic property of pahs may be responsible for the induction of cell proliferation . Bap and baa have been reported to act as estrogens that stimulate and initiate the er - mediated transcription and cell cycle progression and enhance er phosphorylation . On the other hand, there is also indication that the estradiol - dependent cell growth of mcf-7 cells can be inhibited by bap and baa [71, 72]. Thus, the actions of pahs on estrogen - dependent cell proliferation are still controversial . Further studies are needed to elucidate more on the roles of pahs in carcinogenicity . In normal tissues, antigrowth signals can block proliferation by forcing the cell cycle progression into the quiescent (g0) state . The cell cycle transition from g1 to s phase is the key regulatory step in the cell cycle and is mainly regulated by cdk4/6-cyclin d and cdk2-cyclin e complexes . Nicotine has been reported to induce binding of raf-1 to rb with activation of cyclins and cdks as well as inactivation of rb . Via activations of nachr and -ar, nicotine and nnk both exhibit mitogenic properties by inducing cyclin d1 overexpression leading to g1/s transition and increasing cell cycle progression [49, 75, 76]. Nnk can also stimulate normal human lung epithelial cells proliferation through nf-b and cyclin d1 upregulation in an erk1/2-dependent pathway . In our own laboratory, we have also demonstrated that nicotine - induced cyclin d1 overexpression is regulated via stat3, erk1/2, and nf-b - dependent pathways in bladder cancer cells . Other study also shows that pi3k / akt - dependent cellular proliferation is also enhanced in response to nnk . The pi3k / akt pathway is critical in cancer cells because it influences tumorigenesis, tumor growth, and therapeutic resistance . The pi3k / akt activation is documented in both nnk - treated a / j mice and in human lung cancers from smokers . It also plays a role in nnk - induced cell transformation, proliferation, and metastasis . It has been suggested that akt and nf-b may serve as key targets for nicotine or nnk stimulation in the development of lung cancer . West et al . Also reported that beas2b cells treated with nnk for eight - week period increased cellular proliferation through activation of pi3k / akt pathways . However, pi3k / akt activation does not always occur in all cancer cells induced by nicotine . Our previous study indicates that nicotine induced bladder cancer cell proliferation through stat3 and erk1/2 signalings instead of via akt pathway . All these investigations suggest that nicotine or nnk can activate erk1/2, stat3, or akt signaling to interrupt the antigrowth signals leading to enhanced cell cycle progression and cancer promotion . It is important to remember that cigarette smoke components other than nicotine or nnk may also impede on antigrowth mechanisms enhancing cancer development and promotion . Apoptosis plays an important role in controlling normal development, homeostasis, and immune defense via elimination of redundant or abnormal cells in the organism . Failure in cell elimination (reduction of apoptosis) may lead to undesirable cell survival and unchecked cell growths . Resistance to apoptosis is often seen in cancers where cancer cells tend to lose their proapoptotic potentials because of gene mutations . Nicotine has been shown to inhibit apoptosis induced by tumor necrosis factor (tnf), by ultraviolet (uv), radiation, or by chemotherapeutic drugs such as cisplatin, vinblastine, paclitaxel, and doxorubicin . This antiapoptotic action has been shown to be via pi3k / akt, raf / mekk / erk1/2, nf-b, bcl-2, bax, bad, or surviving [23, 8082]. West et al . Demonstrated inhibition of apoptosis and promotion of proliferation in human bronchial epithelium cells by nnk are induced via activation of 3/4 nachr followed by upregulation of akt, mitogen - activated protein kinase (mapk), and pkc pathways . Similar results are also observed by xu and coworkers showing that both akt and survivin pathways are involved in anticisplatin - induced apoptosis by nicotine . Indeed, drug - induced enhancements of p53 and p21 expressions are shown to be suppressed by nicotine . Our recent study also indicated that long - term nicotine treatment activated 4/2 nachr and -ar leading to reduction of apoptosis induced by cisplatin or paclitaxol . Consistently, zhao et al . Also reported that nicotine induced up - regulation of mcl-1 phosphorylation though erk1/2 via -ar activation with increased chemoresistance (anti - apoptosis) of human lung cancer cells . Other investigators also indicate that nnk can prevent cell apoptosis by modulating the anti - apoptotic bcl-2 and c - myc proteins . It is found to be associated with cellular proliferation and is elevated during the developments of certain malignant tumors such as gastric and thyroid cancers [1113]. Comparing the ho-1 in lung tissues of smokers and nonsmokers, li et al . Noticed that the expression of ho-1 is significantly increased in both tumor and nontumor tissues of smokers . These studies further revealed that nnk or its metabolites probably induce oxidative stress in lung tissues with elevation on stimulates the expression of ho-1 . Such event is through erk and nf-b activation and bad phosphorylation induction leading to eventual apoptosis inhibition [11, 85]. Cell proliferation and apoptosis can also be modulated by the peroxisome proliferator - activated receptors (ppars). Ppar/ is expressed in most tissues and has been reported to be associated with cancer growths, especially those in liver, colon, breast and lungs [8789]. Sun et al . Reported a novel mechanism that nicotine increases ppar/ expression through 7 nachr follow by pi3k / mtor activation leading to enhanced lung tumor cells proliferation . In contrast to ppar/, activation of ppar by its ligands induces apoptosis and inhibits cell proliferation . Thus, an intact ppar levels or its activation is needed to reduce cancer risk (anti - apoptosis and cell proliferation). Furthermore, a significant reduction in the transcriptional activity of ppar and its endogenous ligands, including 15-s - hydroxyeicosatertraenoic acid (15(s)-hete) and 3-s - hydroxyocatadecadienoic acid (13(s)-hode), are found reduced in lung tissues of nnk - treated mice . Indeed, lung tumors developed in these mice later . Further suggested that the reduction of 15(s)-hete and 13(s)-hode may enable lung cells to be more resistant to apoptosis by nnk and facilitate tumor development in the animals . In contrast to nicotine or nnk, pahs induce either apoptosis or antiapoptosis in mammalian cells [94, 95]. For instance, bap is known to induce signaling through igfr and increases cell survival through pi3k activation in human mammary epithelial cells . Reported that both akt and erk1/2 act as anti - apoptosis signals leading to bad phosphorylation . However, bap can also induce apoptosis through p53 and p21 signaling in the same model . The results suggest that bap is capable in stimulating both apoptosis and anti - apoptosis signals . Reported that light - irradiated bap (lbap) inhibited apoptosis through production of ros from degraded bap . This anti - apoptotic signal induced by bap in combination with dna damage would increase the possibility of cell survival and producing mutations . Thus, while the apoptotic signal of bap induces cell death (cytotoxicity), the anti - apoptotic signals of bap play an important role in cell proliferation and carcinogenesis . The anti - apoptosis mechanisms induced by components of cigarette smoke are obviously quite complex . It is evident that evading apoptosis plays a critical role in cigarette smoke - induced tumorigenesis and chemoresistance . New understandings on the molecular target regulating the apoptotic and anti - apoptosis machineries by cigarette smoke could provide novel strategies for drug development with substantial therapeutic benefits . When a cell population has progressed through a certain number of doublings (replications), they would normally stop growing and enter into a process called senescence . Tumor cells, however, appeared to have limitless replicative potentials (immortalization) during tumor progression . Telomeres, which define the end segments of chromosomes, consist of short, tandemly repeated dna sequences (ttaggg)n together with associated proteins . Small amount of these end dna sequences may be lost during each cell cycle as a result of incomplete dna replication . However, de novo additions of ttaggg repeats by the enzyme telomerase may compensate for this loss . Thus, telomerase plays an important role in the maintenance of the telomere ends in normal cells . Ectopic expression of telomerase would immortalize the cells . By using human tissue samples, yim et al . Reported that there are different distributions of the telomerase activity between smokers or ever - smokers and non - smoker . A strong correlation between telomerase activity and the number of packs years smoked can be established among these subjects indicating that there is an association between tobacco exposure and telomerase activity in the human bronchial epithelium . Increased telomerase activity would extend the lifespan of cells and put these cells to be at higher risks for malignant transformation and carcinogenesis . Similar finding is reported by targowski et al . That extensiveness of tobacco smoking correlated positively with increases in telomerase activity in tumor cells from patients with non small cell carcinoma of the lungs . All these studies point to the fact that enhancement of the telomerase activity by cigarette smoke certainly underlies the cancer promotion potentials of cigarette smoke . However, which components in cigarette smoke altered telomerase activity are still not known . Indeed, there are indications that certain protein synthesis and mitochondria play central roles in neoplastic transformation . It is well known that mtor and map kinase signaling pathways modulate the phosphorylation of transcriptional factors, stability of mrnas, and protein synthesis . Jin et al . Reported that both nicotine and its metabolite nnk can induce survivin mrna expression through akt - mtor and mediated de novo synthesis of survivin protein in normal lung epithelial cell hbe cells . This induced survivin expression has been claimed to play a role in the malignant transformation of hbe cells by stimulating the survival pathways . Cigarette smoke may damage respiratory chain function in mitochondria enhancing oxidative stress leading to mitochondria dysfunction [103, 104]. It has also been reported that nicotine exposure resulted in reduced pancreatic mitochondrial enzyme activity, degranulation of beta cells, elevated islet oxidative stress, and impaired glucose stimulated insulin secretion in rats . Continued exposure to ros and free radicals from such mitochondrial stress may lead to mitochondria dna (mtdna) mutation which may play an important role in carcinogenesis . Analyzing clinical samples, . Also showed that tumor cells with mtdna mutations grow faster then cells without mitochondrial mutation . Hence, it is apparent that cigarette smoke would induce oxidative damage to the mtdna leading to more aggressive tumor growths . Step 3 (cancer progression)the malignancy of a tumor is usually evaluated by its ability in invasion and metastasis as well as in the associated angiogenesis . There are ample evidence which indicate that cigarette smoke participates in the processes of angiogenesis, invasion, and tumor metastasis . The malignancy of a tumor is usually evaluated by its ability in invasion and metastasis as well as in the associated angiogenesis . There are ample evidence which indicate that cigarette smoke participates in the processes of angiogenesis, invasion, and tumor metastasis . Angiogenesis, the development of new blood vessels from endothelial cells (ecs), is a critical event which allows the cancer cells to receive adequate nutrients and oxygen . Angiogenesis involves mature vascular changes, including detachment of pericytes, degradation of extracellular matrix, endothelial cells remodeling, proliferation, migration, and formation of new endothelial cells into tubular structures . Survival and proliferation of vascular endothelial cells are often stimulated by tumor - derived mitogens, and vice versa . Tumor cells are known to activate angiogenesis by changing the balance of angiogenic inducers such as vegf (vascular endothelial growth factor) and bfgf (basic fibroblast growth factor), and by countervailing inhibitors such as thrombospondin-1 . It has been shown that nicotine can induce angiogenesis both in vitro and in vivo and contributes to the growth of tumors [30, 110]. Similar to the fgf, nicotine is found to have the ability to promote migration, proliferation, tube formation and nitric oxide (no) production of endothelial cells . No is a well - known vasodilator and angiogenesis mediator, and nicotine has been reported to enhance the expression of endothelial nitric oxide synthetase and promote no release . Nicotine is also found to induce expression of endothelial growth factors such as vegf, bfgf, pdgf, tgf-, and tgf- in endothelial cells and smooth muscle cells [112, 113]. Enhanced bfgf release and increases in metalloproteinase expression with degradation of ecm have been demonstrated with nicotine [114, 115]. Moreover, nicotine is found to induce secretion of prostacyclin which is a vasodilating molecule associated with endothelial cell proliferation, survival and migration . These effects are believed to be associated with cigarette smoke - induced hyperplasia of the intima in the blood vessels and other vascular wall lesions . 7 nachr is important in both physiological and pathological angiogenesis [110, 118]. 7 nachr in endothelial cells needs to be sensitized or activated by hypoxia or ischemia in order to induce angiogenesis . Indeed, specific antagonist of the 7 nachr (-bungaratoxin) is shown to inhibit nicotine - induced angiogenesis (new vascular tube formation from endothelial cells) [25, 114]. Interestingly enough, it is apparent that the akt pathway is found to be not involved in either angiogenesis or vegf release induced by nicotine . Suggested that inhibition of erk1/2, p38 mapk, and pi3k / akt can completely block and prevent endothelial tubule formation induced by nicotine - triggered 7 nachr activation . Consistent with heeschen's study, zhang and coworkers reported that nicotine apparently increases angiogenesis and invasion by activating pkc, pi3k / akt, erk1/2, mtor, and src in human nsclc . Excellent reviews on angiogenesis induced by nicotine were recently published [120, 121] and will not be further discussed here . Interaction between nachr and the growth factor - mediated angiogenesis occurs at signaling and transcription levels . Nicotine - induced expression of vegf has been shown to be regulated by egfr transactivation and via the erk1/2 pathway in smooth muscle cells . Phosphorylation of the vegf receptor kdr by nicotine activates vegf and increases its activity . Additionally, nicotine can also upregulate the expression of vegf receptor vegfr2 during angiogenesis in certain cancer cells . Recent study further indicated that nicotine can synergistically promote the proangiogenic effect of estradiol in nonsmall lung cancer . Induction of angiogenesis in colon cancer by nicotine via -ar followed by arachidonic acid pathway has also been reported [32, 125]. In sum, 7 nachr subtype has been linked to angiogenic process induced by nicotine leading to tumor vascularity, inflammation, and ischemia . Nevertheless, whether nicotine or nnk acts specifically via nachr or -ar receptors or both or whether it is controlled in a cell - specific manner needs further study . Other components present in cigarette smoke that may also contribute to angiogenesis remain to be identified . Several excellent reviews on the roles of nicotine and nachr in angiogenesis exist [117, 120, 121, 126]. The ability of invasion and metastasis allows cancer cells to escape from the primary tumor mass to new terrains in the body . The genetic and biochemical determinants as well as the molecular mechanisms involved are still poorly understood . Many evidence indicate that cigarette smoking not only increases proliferation of cancer cells but also promotes metastasis . Clinical and epidemiological studies suggest that smokers have more rapidly progressing tumors and cancer metastasis than non - smokers . These processes are now known to be dependent on cellular and stromal interactions and on extracellular matrix degradation . E - cadherin is a cell - to - cell interaction molecule expressed on epithelial cells . A loss of e - cadherin is seen in epithelial to mesenchymal transition (emt), which is a major pathologic event in cancer metastasis . Chronic treatment of nicotine downregulated the expression of ecm proteins such as e - cadherin and -catenin with concomitant increases of fibronectin and vimentin in lung cancer cells . Wei et al . Also indicated that nnk enhanced colon cancer cell migration with downregulation of e - cadherin . This author also found that the expressions of snail and zeb1, 2 major transcription repressors of e - cadherin, were also induced by nnk in colon cancer cell cultures . Its upregulation is significantly linked with tumor progression, metastasis and poor prognosis in lung cancer patients . It has been shown that nnk can upregulate contactin-1 via 7 nachr / erk activation and enhances invasiveness of lung cancer cells . Breakdown of the extracellular matrix (ecm) through a family of enzyme called matrix metalloproteinases (mmps) is needed for tumor cells to invade adjacent tissue and to metastasize . Reported that nicotine enhanced the invasiveness of esophageal squamous carcinoma cells (te-13) by up - regulating the expressions and activity of mmp-2, and cox-2 . Nicotine is found to enhance the activity of mmp-2, and mmp-9 as well as activation of plasminogen activators in a cox-2 and vegf - dependent manner . It serves as a good marker for pdac (pancreatic ductal adenocarcinoma) metastasis especially in cigarette smoking population . In a recent investigation, lazar et al . Demonstrated that nicotine contributes to pdac metastasis through the induction of mmp-9 and vegf mediated by opn . Pahs, including bap, are also found to play a role in the promotion of cancer metastasis . Through augmented cox-2 expression and pge2 production via activated ahr pathway, bap induces breast cancer cell invasions . Bap and pahs mixture has also been demonstrated to induce cancer cell invasions and metastasis through upregulating the expressions of mmps, proteinase - activated receptor-2, fibronectin, migration stimulating factor, and bcl-2 protein in lung adenocarcinoma . The importance of fgf-9 and its up - regulation by bap in lung cancer invasion and metastasis has been proposed . Indeed, recent study by ueng et al . Demonstrated that bap increases the invasive potential of lung cancer cells in vitro . Such process involves the up - regulation of fgf-9 mrna expression via the p38 and erk1/2 pathways . During metastasis, the cancer cells co - opt signals that control leukocyte trafficking and chemokines - mediated cell migration . Among these chemokines, cxcr4 and its natural ligand cxcl12 serve as key mediators for tumor migration and metastasis . Nicotine has been shown to increase the expressions of several cxc chemokines receptors such as cxcr2, cxcr3, and cxcr4 as well as ccl12 in sclc cells suggesting the nicotine would stimulate cancer cell migration and eventual metastasis . Although epidemiology studies have long demonstrated the relationship between smoking and cancer metastasis, the molecular mechanisms of metastasis influenced by cigarette smoke or its components remain very limited . In this paper, we have reviewed the recent investigations concerning cigarette smoke and cancer development, promotion and progression . While chemicals with carcinogenic potentials in cigarette smoke are many (over 62), most research efforts have been devoted to three components of cigarette smoke: nicotine, nnk, and pahs . While pahs are common chemicals in the environment, nicotine and nnk are considered to be tobacco specific . These three important components of cigarette smoke, especially nicotine and nnk, therefore, are targeted as the major compounds of focus in this review . Many previous reviews have devoted to the interrelationship between cigarette smoke and lung carcinogenesis or the genotoxicity of cigarette smoke or its components . In this review, we are focused on the mechanistic information on tumorigenesis, especially those involving epigenetic or non - gentoxic effects . Aside from lung cancer, it is our hope that this review will summarize the vast information cumulated in the literature and provide valuable reference resource for those who are interested in tobacco - related carcinogenesis . The overall mechanisms on carcinogenesis cancer promotion and progression are complex involving many molecular targets which include receptors, cell cycle regulators, mitogen - activated protein kinases, apoptosis mediators, angiogenic factors, and invasion, and metastasis mediators . Among the receptors, nachr, -ar, and ahr probably are the most important and have the closest association with cigarette smoke - induced carcinogenesis . Overexpression or activation of these receptors may result in the release of neurotransmitters and growth factors that participate in apoptosis inhibition, cell proliferation, angiogenesis, cancer cell invasion and metastasis . It should be noted that the importance of nachr in cancer may be cell - type - dependent or specific and their sensitivity and expression can be also be modified by various environmental factors such as insecticide organophosphates . As shown in figure 1, signaling pathways, pi3k / akt, stat3, and erk1/2 play important roles in the carcinogenesis processes . They are also common paths affected by the cigarette smoke components, including nicotine, nnk, and pahs . In addition, pkc, akt, erk, and cox-2 signaling pathways are involved in both promotion and progression stages by cigarette smoke . It is suggested that these molecules could be utilized the potential targets for future developments in cancer diagnoses or therapies . Avoidance of cigarette smoke remains to be the best way of prevention for cigarette - related cancer . However, in view that tobacco smoke is legalized and smokers are still abundant, understanding on the health impacts by tobacco smoking still constitutes important public health concern . Understanding the disease process and the mechanisms involved is the first step to solution . The emerging understanding on the molecular mechanisms in the development and progression of caners induced by cigarette smoke provides novel inspirations and approaches for potential measures on early diagnosis, reduction in progression and metastasis, and therapy of cancers . Many dietary supplements, foods, or herbal medicines might significantly attenuate the proliferative effects by cigarette smoke ., the natural compound pterostilbene could induce apoptosis and autophagy in chemoresistant bladder cancer cells derived from nicotine exposure . Future research on natural compounds may help to provide additional novel chemopreventive or chemotherapeutic possibilities in reducing cancer risks or other health impacts of cigarette smoke . This review has also discussed the various molecular mechanisms and paths involved in carcinogenesis induced by cigarette smoke . However, there are still many mysteries in the carcinogenic process by cigarette smoke ., most research efforts were focused on the proliferative and antiapoptosis mechanisms induced by cigarette smoking . As tumors are the results of multiple and interactive genetic abnormalities, study of cancers induced by cigarette smoke should assess more than one or two acquired alterations or paths . Explorations of other paths or mechanism other than those popular ones are needed . Those molecular pathways which are significantly activated by cigarette smoke are probably the most important ones involved in cigarette smoke - induced tumorigenesis . These pathways include nachr signaling (such as 7 nachr, 9 nachr, or 4/2 nachr), -ar signaling, pi3k / akt signaling, erk1/2 signaling, stat3 signaling, vegf, and mmps pathways, and so on . Targeting to modulate these pathways via dietary factors or therapeutic drugs may reduce cigarette smoking induced tumorigenesis significantly . Studies on the non - genotoxic (epigenetic) effects of cigarette smoke components are few and need more efforts . The epigenetic effects of cigarette component must be evaluated to include both upstream and downstream pathways . Carcinogenesis is often species or cell - type specific and can be influenced by many factors or cofactors . The same factor which is highly oncogenic to certain cell type or individuals may not be oncogenic to others . Moreover, some cell type may become susceptible to a carcinogen only in the presence of certain factor(s), co - factor(s), genetic predisposition, or immune depression . Specific mechanism for carcinogenesis for the same carcinogen may also vary in different tissues . Synergistic interaction between cigarette smoke components and other environmental toxicants or carcinogens, such as arsenic or dioxin, on cancer development has been demonstrated both epidemiologically and in animal studies [143145]. Traditionally, most past investigations focused only on single compound or one cigarette smoke component . The synergistic interaction between otherwise safe level of environmental chemical and low level of cigarette smoke or its component (via either active or secondhand smoking) for carcinogenesis raised novel public health concerns and challenging questions . We have provided an overview on the major concepts and insights on the molecular mechanisms involved in cigarette smoke - induced cancers . It is hoped that these mechanistic insights can be translated into practical applications for the prevention and treatment of cigarette smoke - related cancers . We also hope that these suggestions will be helpful to those who are interested in this area of cancer research.
Little is known about the relationship between good self - reported health and disability in connection with life- expectancy . To show empirical evidence of the significant statistical association between self - reported health status and disability using spanish survey data and to discuss implications for measures of life expectancy . We model jointly absence of disability and self - reported good - health status correcting for socio - demographic characteristics such as age, gender and years of education . More than 50% of the correlation existing between self - reported health status and disability cannot be explained . The proportion of years lived in disability or in good health with respect to the remaining life expectancy increases with age, but longevity is more related to disability than to self - reported bad - health . Women report to have good health less frequently than men, while men report disability more frequently . Joint factors other than basic socio - demographic indicators induce a significant association between a disability - free status and good self - reported health . Life - expectancy in good self - reported health is longer than life - expectancy in a disability - free condition.
The benefits of vats include decreased postoperative pain, improved cosmetic surgical wounds, decreased morbidity, and shorter hospital stays when compared to open thoracic procedures.13 pain associated with thoracic surgery is multifactorial and involves muscular pain, neuropathic pain, pleural irritation, and referred pain . While patients undergoing vats have been shown to have less acute pain compared to thoracotomy,2,4,5 the development of chronic pain is similar.610 furthermore, the degree of acute post - thoracotomy pain has been shown to predict the presence and significance of chronic pain related to these procedures,6 increasing the importance of acute postoperative pain control . Paravertebral blocks (pvbs) are a validated method of limiting postoperative pain for patients undergoing vats and other thoracic procedures.1117 our institution began providing ultrasound - guided pvbs for patients undergoing vats in the spring of 2011 with positive feedback from both patients and surgeons . Unlike many published descriptions of ultrasound - guided pvb, in which the operator approaches the pvb space in - plane to the ultrasound beam with the probe held in a transverse mediolateral fashion, we utilize an out - of - plane approach with the ultrasound probe in a parasagittal plane combined with intermittent low - volume hydrodissection to guide needle advancement . The aim of this study was to retrospectively evaluate the analgesic efficacy of the preoperative pvbs provided for patients undergoing vats . Following approval of an exemption application from the university of wisconsin health sciences institutional review board, we used a billing database to identify consecutive adult patients undergoing vats by a single thoracic surgeon at our institution between january 2011 and july 2012 . A retrospective review of these patients electronic medical records was performed . Patients who underwent additional procedures at the time of the vats, required postoperative care and continued mechanical ventilation in an intensive care unit, had chronic pain as defined as a baseline preoperative pain score of 5 or greater, were under the age of 18, or who presented with injuries sustained as part of trauma were excluded from our analyses . Additionally, two patients were excluded secondary to postoperative death attributed to long - standing hepatic disease and postoperative stroke attributed to the patient s history of cardiovascular disease . Of the 78 eligible patients, 49 patients received a pvb prior to their planned surgical procedure . All patients on the operating room schedule during the day were considered for a nerve block . Reasons for exclusion of patients would include hemodynamic instability, patient intubated and sedated already, coagulopathy, extreme obesity, prior back surgery, and a patient with active infection . The block occurred in a dedicated holding room and was performed by either a resident or a fellow, and a staff anesthesiologist assigned to the block team . Blocks performed by residents or fellows were done under the direct supervision of a staff anesthesiologist on the regional anesthesia team . Blocks were performed with minimal sedation using intravenous midazolam (12 mg) and fentanyl (50100 g). After assisting the patient into the sitting position, ultrasound guidance in the sagittal plane was used to identify transverse process, costotransverse ligament, and pleura on the side of the surgery at levels appropriate based on the surgeon s proposed port and chest tube sites . Once ultrasound visualization was adequate, the patient s skin was infiltrated with 2% lidocaine (13 ml), and a 22-gauge (50 mm) or 21-gauge (100 mm) uniplex nanoline regional anesthesia needle (pajunk medizin technologie, geisingen, germany) was inserted out - of - plane relative to the ultrasound probe . Hydrodissection with sterile saline was used as the needle was advanced until anterior pleural deflection was visualized on ultrasound . Bupivacaine (0.5%) with 2.5 g / ml epinephrine was injected in divided doses after negative aspiration . In total, 1020 ml of bupivacaine was injected at each level, with most patients receiving 10 ml or 15 ml per level . Total block volume from all levels was 2530 ml in most cases, although a single patient received a total of 40 ml . All patients received blocks at either two or three levels ranging from t34 to t67 on the side ipsilateral to the proposed procedure . Using patients electronic medical records, the amount of opioids administered intraoperatively, in the postanesthesia care unit (pacu), for the first 24 hours after discharge from the pacu and during the hospital stay were recorded . To facilitate comparisons, we converted all administered opioids to intravenous morphine equivalents using standard conversion factors as follows: morphine 10, hydromorphone 1.5, fentanyl 0.1, and meperidine 75.18 the use of additional analgesics, including nonopioid intravenous and oral analgesics, and intraoperative incision infiltration by the surgeon were also recorded . Once the patient was admitted to the floor, the patient reported numerical pain scores ranging from 0 (least pain) to 10 (most pain) that were recorded by nursing staff . The following patient - reported numerical pain scores were recorded or calculated using medical records: patients initial pain score on arriving to the pacu, highest pain score while in the pacu, mean pain score while in the pacu, pain score on initial assessment once the patient reached the floor, resting and dynamic pain scores at 24 hours after discharge from the pacu, and mean resting and dynamic pain scores for the first 24 hours following discharge from the pacu . Mean pain scores were computed using all - nurse recorded patient - reported numerical pain scores during the respective time frame . Pacu time was defined as the amount of time the patient was physically present in the pacu before meeting nursing discharge criteria . All patients undergoing vats procedures, with or without a preoperative pvb, received pain medications ordered by the surgery and anesthesia team in the perioperative period . Intraoperative opioids were administered at the discretion of the in - room anesthesia staff based on clinical signs of inadequate analgesia such as increased heart rate and blood pressure during the procedure . Opioids in the pacu were administered at the patient s request or based on the patient s verbal pain score at the discretion of the pacu nurses . Once admitted to the floor, pain medications were administered at the discretion of the surgical service and floor nurses . The standard pain medication regimen included a hydromorphone patient controlled analgesic pump with a nurse bolus available . Most patients were placed on scheduled oral acetaminophen unless contraindicated, and most received scheduled doses of either intravenous or oral nonsteroidal anti - inflammatory drug (nsaid) medication . Patients who received a rescue antiemetic in either the pacu or within the first 24 hours postoperatively were considered to have postoperative nausea . Our primary interest was whether pvbs led to improved pain scores and reduced opioid consumption . Intergroup comparisons (pvb vs no pvb) were performed using the student s t - test, fisher s exact test, and mann whitney u test for continuous, binary, and ordinal variables, respectively . An independent statistician performed statistical analysis of data . Patients were excluded from our analysis for the following reasons: baseline pain score of> 5 either from pain related to reason for surgery or from an unrelated chronic issue (n=17), remained intubated post - operatively (n=12), presented as a multiple injury trauma (n=4), deceased on postoperative day 1 (n=1), stroke on postoperative day 0 (n=1), and had a prolonged narcotic infusion lasting into pacu as part of a total - intravenous anesthetic (n=1). Of the 78 eligible patients, 49 received an ultrasound - guided pvb, while the remaining 29 did not . Chart review revealed that among the patients receiving a pvb, four received a block at three levels, while the remaining 45 patients received a block at two levels . Two three - level blocks were performed at levels t4, 5, and 6, one at t1, 3, and 5, and one at t2, 3, and 5 . Twenty - six patients received a two - level block at levels t4 and 6, nine at t3 and 5, five at t5 and 7, four at t3 and 6, and a single patient received a block at t1 and 5 . The two groups (pvb vs no pvb) were comparable in terms of patient age, sex (%), body mean index, and american society of anesthesiologists score (table 1). Our analysis showed that all patients received as - needed intravenous opioids, frequently in the form of patient - controlled analgesia . All but six patients in the pvb group and five patients in the control group additionally received scheduled acetaminophen once admitted to the floor . Results in table 2 show that mean intraoperative opioid consumption in morphine equivalents was significantly lower for patients who received preoperative pvb compared to those who did not (p=0.001). Mean opioid consumption in the pacu and for the first 24 hours after surgery appeared to be less in the pvb group compared to controls; however, this small difference failed to achieve statistical significance (p=0.246 and 0.467, respectively). Mean opioid consumption for the duration of hospital stay was significantly lower in patients who received pvb (p=0.011). Patient - reported numerical pain scores immediately upon arrival to the pacu were significantly lower in the pvb group (p=0.005; table 3). Peak pain scores in the pacu (p=0.097) and mean pain scores in the pacu (p=0.061) were lower in the pvb group but also failed to reach statistical significance . Pain scores on initial assessment once patients reached the floor were also significantly lower in patients receiving a pvb (p=0.031). Both dynamic and resting pain scores at the 24-hour time point after surgery were significantly lower in the pvb group (p=0.003 and p<0.001, respectively); however, mean dynamic and rest pain scores over this 24-hour period, while lower in the pvb group, failed to reach statistical significance (p=0.089 and p=0.078, respectively). Patients who received preoperative pvb had a lower incidence of nausea requiring treatment both in the pacu (4% vs 28%, p=0.004) and for the first 24 hours after pacu discharge (8% vs 43%, p=0.001) compared to the control group (table 4). Lastly, patients who received pvb spent significantly less time in the pacu before meeting discharge criteria compared to those who did not receive pvb (105 vs 125 minutes; p=0.025). We found that 40 of 49 (82%) patients in the block group received scheduled postoperative acetaminophen, while 25 of 29 (86%) patients who did not receive a block received scheduled acetaminophen . Thirteen of 49 patients (27%) in the block group received at least one dose of scheduled nsaid medication in the form of intravenous or oral ibuprofen, diclofenac, or ketorolac . Six of 29 patients (21%) who did not receive a block received nsaid medication . Lastly, 24 of 49 patients (49%) in the block group received intraoperative wound infiltration with 0.25% bupivacaine by the surgeon, while 24 of 29 patients (83%) in the control group received intraoperative local anesthetic wound infiltration . The findings of this study support the assertion that preoperative pvbs performed with an out - of - plane needle insertion technique improve analgesic outcomes in the immediate postoperative period and improve the quality of the anesthetic . Specifically, our patients had lower reported pain scores, lower perioperative opioid requirements, fewer incidences of treated postoperative nausea or vomiting, and shorter stays in the pacu . All analyzed outcomes showed a trend toward improved results in patients who obtained preoperative pvb compared to those who did not . Statistically, this fact is in itself significant and provides an objective correlation to the subjective positive feedback we have received from both surgeons and patients . The analgesic efficacy of pvbs in patients undergoing both video - assisted thoracoscopic procedures and open thorcatomies has been documented.1114,16,17 furthermore, the use of paravertebral catheters for postoperative pain control for thoracic procedures has also been investigated.12 classically, pvbs have been placed using landmark techniques with or without nerve stimulation, often by walking the needle off of transverse process and advancing an additional 1 cm or until the pop of the transverse costal ligament is felt.19 more recently, ultrasound guidance has grown in popularity with several different techniques being used for block placement, most commonly placing the ultrasound probe in the transverse plane with injection of local anesthetic using an in - plane technique.20,21 other ultrasound - guided techniques with out - of - plane needle placement rely on contact with the transverse process or loss of resistance to saline injection once the transverse costal ligament is passed to ensure block placement in the paravertebral space.22 the results of the present study demonstrate that using an out - of - plane ultrasound - guided method of single - injection pvb placement, we were able to improve our anesthetic via improved pain scores, less postoperative nausea, and less recovery time in the pacu . One limitation of the current study is a lack of documented dermatomal sensory blockade after block placement . It is not our practice to check sensory levels following placement of these blocks, and therefore, the rate of epidural spread and block failure is unknown . In addition, the retrospective nature of this study and the bias that was introduced due to the unblinded nature of this study introduces bias . Providers may have expected less pain in the block patients, and therefore, gave less medication . Despite these issues, these data do provide further evidence of the analgesic efficacy of pvb in patients undergoing vats procedures and introduce the possibility that a pvb catheter would add value by potentially prolonging our pacu results into postoperative day 1 . In this chart review, we cannot determine how long the single - injection blocks might have lasted . The literature supports a duration of single - shot pvb ranging from 6 hours postoperatively to 12 hours or even longer . We know that the block lasted long enough to get the patients through the pacu, so perhaps by placing catheters, we can extend the analgesia in a meaningful way for patients . If we get improved analgesia on the floor, then perhaps we begin to impact the ability of patients to ambulate and avoid respiratory complications . The out - of - plane approach utilized at our institution may put patients at elevated risk of misplaced injection of local anesthetic, and this needs to be carefully considered . Though no adverse events from the pvbs were noted in our chart review, we clearly did not have enough patients to draw any conclusions regarding block safety . In summary, the current study presents data suggesting that ultrasound - guided paravertebral blockade using an out - of - plane needle approach may offer a number of clinical benefits to patients undergoing vats procedures.
Colorectal cancer is rare in teenagers, especially without known risk factors such as inflammatory bowel disease, familial adenomatosis polyposis, and hereditary nonpolyposis colon cancer (hnpcc). Although the presenting features of colorectal carcinoma are similar between adolescents and adults, it is frequently overlooked in the differential diagnosis in young patients.1 many reports suggest that children are more likely than adults to have advanced - stage disease at presentation, unfavorable tumor histology, and poor outcome.2 in the pathological point of view, a mucinous histology is considerably more frequent in children and adolescents than in adults . Here, we report a case of mucinous adenocarcinoma of the colon in a 19-year - old male patient without any risk factors . We also review the clinical and pathological characteristics of young - age sporadic colorectal cancer (yscc). A 19-year - old man visited the gastroenterology outpatient department complaining severe left abdominal pain that developed 1 month ago . On physical examination, he was found to have a distended abdomen with severe tenderness on the left lower quadrant . 1a). Abdominal computed tomography scan was then immediately performed which revealed a distal descending colon mass causing mechanical obstruction (fig . He also had acute constipation and passed loose stool 1 to 2 times per week . He visited a private clinic in australia, and the physician gave a diagnosis of acute colitis with regard to his abdominal pain and loose stool . Despite medication, he did not feel any symptomatic improvement so he returned to south korea the previous day . The patient was sent to an endoscopy room, and an emergency colonoscopy showed a large, fungating mass obstructing lumen at 40 cm from the anal verge . Through - the - scope stent insertion was performed to decompress the gastrointestinal tract (fig . No other polyp or mass was seen on the sigmoid colon and rectum . On the second hospital day, his abdominal pain markedly improved . His serum carcinoembryonic antigen level was 3.85 ng / ml, and his erythrocyte sedimentation rate and c - reactive protein level were elevated (13 mm / h and 3.31 mg / dl, respectively). No other abnormal laboratory findings, such as anemia, or leukocytosis, were observed . Positron emission tomography scan revealed a hypermetabolic lesion on the descending colon without other metastatic lesions . The patient was transferred to the general surgery department and was subjected to left hemicolectomy under general anesthesia . The pathology was confirmed as mucinous adenocarcinoma (7.04.5 cm), penetrating the visceral peritoneum and with one regional lymph node involvement out of 47 (fig . Rt - pcr analysis did not detect microinstability in any of the markers tested (fig . The patient had no family history of cancer, including his parents, grandparents, and cousins (fig . He was transferred to other hospital for adjuvant chemotherapy according to his family's decision . It is generally considered to be a disease of older persons; more than 90% of colorectal cancer patients is above 55 years old.3 groups of young patients known to be at increased risk for colorectal carcinoma are those with inflammatory bowel disease, hnpcc, and polyposis syndromes of the gastrointestinal tract.4 apc gene mutations produce classic or attenuated familial polyposis coli . Mutations in mismatch repair (mmr) genes, principally msh2 and mlh1 but also pms1 and pms2, cause hnpcc.5 the amsterdam criteria, the most widely used diagnostic criteria for hnpcc, have the following requirements: 1) there should be 3 or more family members with histologically verified colorectal cancer, 2) with 1 member being a first - degree relative of the other two, 3) at least 1 case should have been diagnosed before the age of 50 years, and 4) 2 or more generations should be involved.6 most patients with clinically diagnosed hnpcc will have germline mmr gene mutations . Although identification of patients with germline mmr gene mutations in the absence of a family history is important, most young colorectal cancer patients without a family history of hnpcc are unlikely to have germline mmr gene mutations.5 both hnpcc and yscc are considered to arise through the mutator phenotype genetic pathway of colorectal carcinogenesis, although yscc may also be caused by hypermethylation of the mmr gene mlh1 instead of gene mutation itself.4 several observations have suggested that chromosome 14 could play an important role in microsatellite stable (mss) colon carcinogenesis . As loss of chromosome 14 is more frequent in aggressive tumors and in young patients, this gene location is likely responsible for tumor aggressiveness and hereditary predisposition.7 we reported this case as an yscc because the patient had neither family history of colon cancer nor any other malignancy, and showed mss without any germline abnormality . Yscc also presents features similar to adult colorectal cancer, such as abdominal pain, altered bowel habits, weight loss, and rectal bleeding.1 however, young patients present with more advanced - stage disease at diagnosis compared with older patients . This may be because of delayed diagnosis and poorer pathological findings.3 in young patients who develop colorectal carcinoma but have no known predisposing genetic risk factors, late diagnosis may result from the clinician's failure to consider the possibility of malignant disease in the differential diagnosis.4 another distinct feature of yscc is its pathological characteristics . It has a higher proportion of patients with multiple nodal metastasis, distant metastasis, and poorly differentiated tumors . Yscc also shows aggressive tumor biology, including a mucinous component, signet - ring cell carcinoma, and perineural, vascular, and lymphatic invasion . Mucinous adenocarcinoma is a distinct subgroup of adenocarcinoma and comprises about 10% to 15% of all colorectal carcinomas.8 however, an average of 28% of the lesions found in younger patients constituted mucinous tumors compared with an average of 5% for adults with colorectal carcinoma.9 mucinous adenocarcinoma shows higher frequencies of poor differentiation, advanced tumor stage, loss of mmr expression, and increased muc2 expression compared with non - mucinous adenocarcinoma . These discrepancies might vary across different subgroups of colorectal cancer patients . According to a recent report, hnpcc patients showed a similar incidence of loss of mmr expression, and yscc patients showed a similar proportion of advanced tumor stage between mucinous and non - mucinous adenocarcinoma . In addition, the tumor locations of mucinous adenocarcinoma are significantly different between hnpcc (predominantly in the right colon) and yscc (predominantly in the left colon and rectum).8 the diagnostic and treatment tools for yscc are same as those used for adult colon cancer . However, a report suggested that exploratory laparotomy yields a higher proportion of diagnostic result for colorectal carcinoma in young patients than in adults . Early colonoscopy is less frequently performed in young patients than in older patients.1 our case was also initially misdiagnosed as simple enteritis without any study during the first one month even though presenting significant symptoms . In a review of young - age colon cancer, studies comparing stage - for - stage survival found that young patients with dukes' stage a or b tumors appear to have better survival than older patients with similar - stage disease . Young patients with dukes' c or d lesions do the same or worse than older patients with the same disease stages.3 a large cohort study found that young patients showed poor outcome despite the similarity of their treatment to the standard therapy for adult colorectal carcinoma,10 reflecting the preponderance of advanced - stage disease in young patients . Other factors for the outcome besides advanced - stage were incomplete resection, mucinous histology and proportion of signet ring cells.1 a prospective national cohort study following standardized total mesorectal excision for rectal cancer in patients aged less than 40 also showed considerably lower survival rate . The authors suggested that biologically more aggressive cancer rather than diagnostic delay seemed to account for the advanced stage at the time of diagnosis, leading to poor survival rates . Thus, age may be a significant and independent prognostic factor determining metastasis and survival in rectal cancer . Patients younger than 40 years of age had 5 times higher risk of metastasis and 6 times higher risk of death compared with patients between 40 and 44 years of age . The youngest rectal cancer patients seem to have additional negative prognostic factors, not related to histopathologic findings or tumor stage.11 in conclusion, we reported a case of a 19-year - old male patient with colon obstruction due to sporadic colon cancer with a mucinous histology his cancer diagnosis was delayed by the physician's failure to consider malignancy in the differential diagnosis . Therefore, even in low - risk young patients, symptoms such as unexplained abdominal pain, rectal bleeding, or changes in bowel habits should be considered representative of significant colorectal lesions . This case warrants increased awareness and aggressive pursuit of symptoms in young patients without any risk factors.
Over the last decade there has been a rise in incidence of subfertility among the reproductive age group couples . However, modifications in laboratory techniques have helped to improve pregnancy rates in a corresponding manner . Yet new techniques are being continuously evaluated in order to prove their value both in achieving clinical pregnancies as well as being cost - effective for these couples . Intrauterine insemination (iui) has been used for the treatment of infertile couples with male factor, ovulatory dysfunction, cervical factor and unexplained infertility often as a first line therapy . The overall success of iui varies with pregnancy rates ranging from 5% to 20% per cycle . Several prognostic factors for iui outcome have been proposed, including the age of the women, endometrial thickness, duration of infertility, type of ovarian stimulation, number of inseminations and total number of motile sperm inseminated . Renewed interest in iui is definitely the result of better washing procedures, enhancing the quality of the initial sperm sample . These washing procedures are necessary to remove prostaglandins, infectious agents, antigenic proteins, non - motile spermatozoa, leukocytes and immature germ cells . Current techniques of sperm preparation for iui such as swim - up and density gradient require the use of centrifugation to separate the sperm from the seminal plasma . This processing technique provides better sperm quality regarding concentration and motility, which may lead to higher success rates after iui . Ever since the advent of sperm processing techniques there has been an ongoing debate pertaining to the maintenance of core temperature considering the natural location of testes . In this regard, came about modification of the conventional centrifuge to include a temperature controlled centrifugation process . The temperature controlled centrifuge has been designed to regulate and subsequently maintain the critical inner chamber temperature at 37c before, after and during centrifugation . Maintenance of this critical temperature throughout the entire sperm processing procedure helps the sperm to maintain their peak motility and enhanced linearity . Processing of human spermatozoa during therapeutic procedures usually involves cell exposure to room temperature for different periods of time . Sperm incubation at room temperature regulates cellular mechanisms involved in sperm capacitation and produces a temporary blockage of capacitation related events . Capacitation and acrosome reaction can be achieved in vitro by placing the spermatozoa under defined conditions, which include incubation at body temperature . Previous studies have shown the effect of temperature on cell motility and spontaneous acrosome reaction . Human sperm incubation at room temperature does not allow capacitation, although it does not affect human follicular fluid - induced acrosome reaction in capacitated cells . The present study has been designed to evaluate the efficacy of non - temperature and temperature controlled centrifugation on semen preparation techniques for iui and their possible effects on pregnancy outcome in unexplained infertility . The concentration of sperm along with the motility and morphological characteristics of the sperm recovered through the two centrifuges were assessed and compared . This retrospective study analyzed data of 671 patients of unexplained infertility, who underwent homologous artificial insemination at our fertility research center from the period of january 2007 to september 2012 . The couples were randomized into two groups namely, group a patients (n = 303) being treated with sperm prepared by using non - temperature controlled centrifuge (heraeus biofuge prima; thermo scientific, germany) and group b patients (n = 368) being treated with sperm prepared by temperature (37c) controlled centrifuge (spermfuge sf 800; shivani scientific industries, mumbai, india). All the female partners in this study had documented patent fallopian tubes either by hysterosalphingogram and or by laparoscopy . Unexplained infertility was defined as couples who had normal tubal patency, regular ovulation and no cervical factors with regular unprotected intercourse and at least 1 year duration of infertility . The semen parameters were determined by using makler counting chamber according to world health organization criteria (2000) of semen analysis . Couples were excluded from the study when the female partner was aged more than 35 years and if there was presence of other known causes of infertility such as male factor, anovulation or immunological factors . All patients in both groups underwent ovulation induction by clomiphine citrate with a daily dose of 150 mg administered for 5 days from 5 to 9 days of menstrual cycle . Simultaneously the patients were on aloes compound (twice a day) from 1 to 21 day of the cycle and t. progynova (once or twice a day) for 10 days starting from day 5 . On the 12 day of the cycle, transvaginal ultrasonography was carried out and 10,000 iu of human chorionic gonadotrophin (human chorionic gonadotropin [hcg]; life medicare and biotech, new delhi, india) was given when at least 1 - 3 follicles had reached a diameter of 20 mm . Semen samples for iui were collected by masturbation into a sterile wide mouth container after 3 days of sexual abstinence . After liquefaction, semen samples were evaluated using phase contrast microscope (olympus medical systems, haryana, india). Assessment of semen parameters included sperm concentration per milliliter, percentage of sperm motility and sperm morphology . The spermatozoa were prepared by the conventional swim - up technique . In both groups, the semen samples after liquefaction were diluted with whittingham t6 culture media (in - house prepared media supplemented with 10% heat inactivated fetal cord serum) in the ratio of 1:2 . The tubes were placed in two centrifuges and then the semen samples were centrifuged for 5 min at 428 g to separate the seminal plasma from the serum . The supernatant was discarded and the pellet was resuspended in 1 ml of t6 culture media . The sample was again centrifuged for 5 min at 285 g to remove the cell debris and the supernatant was removed again . The final pellet was gently loosened by resuspension and under - layered in 1 ml of t6 culture media without allowing the sperm pellet and media to mix and incubated at 37c in 5% co2 for 30 min . Finally 0.3 ml of the top layer containing highly motile fraction of spermatozoa was aspirated gently in a 1 ml syringe and sperm concentration and motility was ascertained once again . In both groups, the pre- and post - wash semen characteristics that were reported include sperm concentration, percentage of sperm motility and normal morphology . The speculum (cusco's) is gently inserted in the vagina to expose the cervix . The insemination cannula (vardhman medicare, new delhi, india) was attached to the 1 ml syringe containing the processed sample and the cannula was then gently introduced through the cervical canal . Almost in all the cases, two inseminations were performed on 2 consecutive days in the periovulatory period in order to increase the success rate of iui . A pregnancy test was performed if next mensus was delayed for more than 3 days after the iui procedure . Clinical pregnancy was confirmed by the presence of an intrauterine gestational sac by transvaginal ultrasonography . Categorical data's were analyzed by chi - square test, fisher exact test and was used to determine the predictive power of the sperm parameters (sperm concentration, motility and sperm morphology) between the temperature and non - temperature controlled centrifuge . The statistical analysis of the data was performed using statistical package for the social sciences version 14.0 software (chicago, il, usa). Categorical data's were analyzed by chi - square test, fisher exact test and was used to determine the predictive power of the sperm parameters (sperm concentration, motility and sperm morphology) between the temperature and non - temperature controlled centrifuge . The statistical analysis of the data was performed using statistical package for the social sciences version 14.0 software (chicago, il, usa). A total of 671 patients of unexplained infertility were assigned to two different groups: group a (n = 303) had their semen samples prepared by ntc and group b (n = 368) had their semen samples prepared by tc . The patient's demographic and semen characteristics (pre- and post - washing) of the study population between the two groups are shown in tables 1 and 2 . Demographic characteristics of the study population between the two groups comparison of sperm parameters in patients before and after sperm preparation the clinical pregnancy rates and their outcome according to the semen parameters after preparation are summarized in table 3 . The nature of this study was to compare the efficacy of non - temperature and temperature controlled centrifugation on semen preparation techniques for iui and their possible effects on pregnancy outcome in unexplained infertility . The comparison of two types of centrifugation on more or less the same semen count and motility is the only way to detect possible intrinsic differences between the two processes . However, our data showed no significant difference between the sample prepared by using non - temperature and temperature controlled centrifuge . Unexplained infertility was considered ideal since in the absence of female pathology, the impact of sperm parameters can be studied in a better model . In unexplained infertility, ovarian stimulation and iui appears to be effective . In a retrospective analysis carried out by, pregnancy rate after iui has been evaluated according to indication, age, and duration of infertility in some studies . In our study, there was no significant difference between the two groups with respect to age, duration of infertility and in terms of endometrial thickness . Sperm preparation techniques have added value, safety and also enhanced success rates in iui treatment cycles . Ovulation induction combined with iui seems to be the first line of treatment in idiopathic infertility . No significant difference in the pregnancy rate was observed between the non - temperature and temperature controlled centrifuge in terms of sperm concentration, motility and morphology . These results confirm that the sperm preparation technique either at room temperature or at 37c did not affect the semen parameters and their clinical pregnancy outcome . However, further studies would need to be conducted in order to establish the fact . To the best of our knowledge, this is the first study done specifically to compare the outcomes.
Weaning from mechanical circulatory support after long controversy about the clinical relevance of unloading - promoted recovery from chronic heart failure (hf) and about the feasibility of elective weaning of patients from their ventricular assist device (vad), taking into account the risk of early post - weaning hf recurrence, this topic has gained increasing interest over the past 10 years, especially after the publication of the first long - term weaning results in patients with chronic hf reversal after lvad implantation . Assessment of cardiac recovery temporary interruptions of mechanical unloading (off - pump trials) are mandatory for assessment of recovery, regardless of whether the assessment is performed at rest or during exercise . Short off - pump trials allow evaluation of the heart without mechanical support under the same circumstances that will exist after vad removal . However, whereas pulsatile vads allow optimal assessment of heart function during complete pump stops, complete stops of axial - flow pumps lead to retrograde flow into the lv followed by reduction of the diastolic arterial pressure that, by reducing the left ventricular (lv) afterload, can generate overestimations of lv systolic function . According to our observations, the misleading retrograde bloodflow into the lv during off - pump trials is a major negative factor that can interfere with successful vad explantation . Therefore, for such pumps, rotor - speed reduction to values resulting in close to zero flow in one cardiac cycle (3000 - 6000 rpm) is better than complete pump - stop . Before off - pump trials, heparin must be given (60 - 100 iu / kg according to the prothrombin time reported as inr) to prevent thrombus formation inside the pump . Patients with heparin - induced thrombocytopenia should receive argatroban (synthetic thrombin inhibitor) infusions (2g / kg / min)which should be started 1h before the off - pump trials . Additionally, completely stopped pulsatile devices should be allowed to pump at least once a minute, or 3 bursts of pneumatic hand - pumping should be intercalated every 15 to 60 seconds to prevent blood stagnation [2, 4, 5, 7, 8]. In patients with a biventricular assist device (bivad) it appeared appropriate to stop the right ventricular (rv) pump 30 seconds earlier than the lv pump; after both pumps are deactivated, evaluation of recovery can be performed . Duration of individual off - pump periodscan vary between 3 and 15 minutes . In patients with insufficient recovery for possible vad removal, as already shown during the first 3 minutes, there is no reason to further extend the off - pump trial . With appropriate caution, the risk of interruption of unloading is low [4, 6, 9, 10]. In weaning candidates it appears useful to conduct such trials weekly or every 2 weeks and to make the final decision for elective vad explantation only after cardiac improvement has reached its maximum (no further improvement in at least 2 consecutive off - pump trials). During recovery it appears useful to change the working mode of the pumps, in order to intensify the unloading if the ventricle size needs further reduction, or to exert moderate load on the ventricular myocardium after maximum improvement [1, 5, 7]. In addition to the retrograde blood flow into the lv during off - pump trials, which makes the evaluation of lv function very difficult, another factor that may impair successful vad explantation in the subgroup of patients receiving continuous vad is the usually great difficulty in maintaining the recovered heart in a state as unmolested as possible throughout explantation surgery . During vad removal, which was performed on the beating heart in all our weaned patients, we aimed to leave the intrathoracic part of both cannulas in place after they were reliably occluded [6, 9]. This was possible in 91% of patients with pulsatile pumps, but only in 57% of patients with continuous - flow pumps . Also, the heart - lung machine, which was used only in less than 10% of patients with pulsatile vads, was required for all continuous - flow - pump explantations . Nevertheless, in our patients with non - ischemic chronic cardiomyopathy, there were no significant differences in the 3-year post - explant freedom from hf recurrence between patients weaned from pulsatile and those weaned from continuous flow pumps . Cardiopulmonary exercise testing is also increasingly used by some centers [4, 8]. Echocardiography is the cornerstone for assessment of cardiac recovery allowing both selection of potential weaning candidates and decision - making in favor of or against vad removal . After vad implantation, repeated transthoracic echocardiography (tte) screenings with normal vad function are necessary for selection of potential weaning candidates, i.e. Patients with signs of relevant reverse remodeling who also seem to have their contractile function improved to levels deserving further evaluation . Such potential weaning candidates are those with lv end - diastolic diameter (lvedd) <55 mm (or 55 - 60 mm at bsa 1.8m)and fractional shortening (fs)>15%, no or grade i mitral and/or aortic regurgitation, no rv dilation and tricuspid regurgitation grade ii . Before the first off - pump trial, it is useful to perform stepwise pump - rate reductions under tte monitoring, to verify whether complete interruption of unloading is possible . Thus, if incomplete unloading interruption alreadyprovokes symptoms (dizziness, sweating, etc . ), complete interruption of unloading is senseless and risky . If the patient remains asymptomatic but the lvedd increases beyond 60 mm, a complete interruption is also senseless, because such a patient is not yet a weaning candidate . Echocardiographic assessment of recovery in weaning candidates is usually based on the results of repeated off - pump trials at rest [2, 4, 7, 9]. After months of intensive vad support, even short periods of physiological loading by interruption of unloading may represent a serious challenge for a possibly incompletely - recovered ventricle . It therefore appears reasonable to avoid (at least initially) any risk of myocardial exhaustion, which might interfere with possibly still ongoing recovery . For this reason, all 119 patients who were weaned since 1995 in our center from long - term vads primarily designed as a bridge - to - htx or as permanent therapy underwent assessment of heart function exclusively at rest [6, 9, 10]. Although at rest the conclusive value of assessments is limited by the lack of information about inotropic reserves and cardiac adaptation to stress, our weaning results appeared not to be relevantly affected by that, insofar as the results reported by groups who also used stress echocardiography and/or exercise testing were not better [4, 9, 11]. However, stress echocardiography can provide valuable information on inotropic reserves and adaptation to stress, which might be useful for weaning decisions . According to certain authors, an absolute ef increase by 5% during dopamine infusion indicates preservation of contractile reserve . A possible limitation of dobutamine stress echocardiographycan be, at least theoretically, the risk of myocardial exhaustion with negative impact on an ongoingmyocardialrecovery process . Further studies are therefore necessary to establish the real value of dobutamine stress echocardiographyfor weaning decisions . In principle, off - pump tte should be as comprehensive as possible, including tissue doppler and strain imaging . * rwted = [interventricular septum thickness + posterior wall thickness] / lvedd . * * product of time velocity integral measured with pulsed - wave doppler at the lv outflow tract (lvot) and cross - sectional area of the lvot . However, not all these parameters can be reliably measured in all patients because of poor image quality in some patients with vad support . Image quality was a serious limitation, especially for off - pump doppler measurements that had to be performed during rotor - speed reduction instead of complete pump stop . Off - pump right heart catheterization off - pump right heart catheterization (rhc) is also a cornerstone for recovery assessment [4, 9] and is necessary for final decisions in favor of or against vad explantation . A final off - pump trial of 15minutes in the operation room, with repeated measurements of hemodynamic parameters under continuous echocardiographic monitoring, is indispensablebefore the start of explantation surgery . Rhc appeared also necessary before any preliminary decision - making in potential weaning candidates with borderline tte data and/or relevant cardiac improvement only after> 6 months of unloading and/or long history duration (> 3 years) of hf . In patients with axial - flow pumps, such preliminary rhc appears more reliable if off - pump measurements are preceded by occlusion of the outlet cannula with a balloon . Normal and stable hemodynamics during off - pump rhc trials is a necessary condition for a decision in favor of vad removal, but not sufficiently predictive for long - term post - explant cardiac stability [6, 9]. It was observed that, unlike non - recovered patients, those with unloading - promoted recovery show a significant lvef increase in the 6-minute walking test (6mw). Non - recovered patients showed a significant correlation between heart rate (hr) and mean arterial pressure (map) after 6mw, suggesting that hr increase was compensatoryfor map reduction, whereas recovered patients showed after the 6mw no correlation between hr and map, suggesting that hr increase was independent of map change and therefore a true inotropic reserve response . Harefield hospital (uk) developed an algorithm for testing recovery that includes a 6mw test with repeated measurements afterwards to determine the inotropic reserve [4, 8]. The same group also uses cardiopulmonary exercise testing with oxygen uptake (vo2) as a parameter for weaning decisions . Decision - making in favor of vad explantation our weaning criteria that have evolved during nearly 20 years of weaning experience are summarized in table 2 . Main left ventricular assist device (lvad) explantation criteria * * thesecriteria used at present at the deutsches herzzentrum berlin have evolved from a weaning experience of 18 years after explantation of 100 long - term vads, primarily designed as bridge - to - htx or permanent therapy . * * measurements at rest, without inotropic support . Since 2005 also speckle - tracking derived 2d - strain imaging parameters (global longitudinal strain and strain rate, intra - ventricular synchrony and synergy, including pre - explant stability of all these parameters after maximum improvement and during the final off - pump trial) were taken into consideration for weaning decisions . Essential criteria are, in addition to a stable off - pump lvef 45%, normal and stable lv size and shape, as well as high stability of hemodynamic parameters measured during the off - pump right heart catheterization (rhc) trials [6, 9]. Even in patients with good recovery, it appears useful to explant vads only after the recovery has reached its maximum (no further improvement during the next 2 - 4 weeks). If a relevant lvef reduction and/or lv enlargement occurs during the next 2 - 4 weeks after maximum cardiac recovery, it can be risky to explant the vad, even if the lvefdid not fall below 45% and thelvedddid not increase beyond55 mm . The weaning criteria used by the harefield group include fewer tte parameters; instead they usethe 6mw - test and the peak oxygen consumption (vo2)(optimal:> however, it is important to emphasize that they also do not explantlvadsin patients with lvef <45% [4, 6, 9]. For more reliable evaluation of cardiac recovery, since 2005 we have also used tissue doppler and speckle - tracking - derived 2d - strain imaging [12, 13]. The potential usefulness of tissue doppler wall motion velocity measurements is shown in figure 1 . Usefulness of left ventricular (lv) peak systolic wall motion velocity (sm) measurements at the basal posterior wall by pulsed - wave tissue doppler toassess the stability of lv contractile function during and between off - pump trials and for detection of further cardiac improvement after left ventricularejection fraction (lvef) has reached its peak value . At the time when lvef reached its maximum value (55%), sm was 9cm / s during full left ventricular assist device (lvad) support (a) and remained stable during the off - pump trial (b). Although the lvef remained unchanged, the sm values with (c) and without(d) lvad support increased during the next 3 weeks of unloading up to 11cm / s (+ 22%). The advantages of strain imaging are its ability to differentiate between active and passive movement of myocardial segments, the angle independency of deformation - velocity measurements, and the possibility to quantify intra - ventricular asynchrony and dyssynergy and to evaluate components of myocardial function, such as longitudinal myocardial shortening, thatcannot be visually assessed . Unfortunately, as 2d - strain imaging is a new method, the available off - pump data are insufficient for reliable assessment of their predictive value for long - term post - explant cardiac stability . However, in borderline cases, off - pump data on deformation velocity as well as on intraventricular synchrony and synergy of contraction, including their stabilityafter maximum improvement, can be useful for weaning decisions . Figures 2 and 3 show the potential usefulness of 2d - strain imaging for assessment of recovery after lvad implantation . Usefulness of speckle - tracking derived 2d - stain imaging for assessment of cardiac recovery after left ventricular assist device (lvad) implantation . The comparison of recordingsobtained before lvad implantation (a and b) and several months later before lvadexplantation (c and d) showed a striking improvement of systolic global longitudinal strain (myocardial longitudinal shortening) and strain - rate (velocity of myocardial longitudinal shortening) which increased during lvad support from 4% (a) and 0.3/s (b), respectively to 17.5% (c) and 9.6/s (d), respectively . The global early diastolic strain rate (early relaxation velocity) also increased from 0.7/s (b) to 1.6/s (d) and image d also shows a normalization of early / late strain - rate ratio . Speckle - tracking derived 2d - strain and strain rate off - pump recordings showing further cardiac improvement during left ventricular assist device (lvad) support after off - pumpleft ventricularejection fraction(lvef) reached its maximum value of 50%.at the time of maximum lvef improvement (images a and b) longitudinal global systolic strain (sl) and strain - rate (srl) were slightly reduced (17% and 1/s, respectively) and the global early / late diastolic strain - rate ratio(srle / srla) was high . After few weeks of further lvad support, although the lvef remained unchanged, both slandsrl increased, reaching normal values (22% and 1.25/s, respectively) and there was also improvement in intraventricular synchrony of contraction (images c and d). The 35% shortening of the time to peak strain rate (tpsr) additionally indicates improvement of left ventricular (lv) systolic function . The early relaxation velocity increase (from 1.7/s to 2.1/s) and the srle / srla ratio reduction (from 3.5 to 2.1) off - pump lvef 45% plus normal lvedd at rest are basic weaning requirements and their stability after maximum improvement over the next 2 to 4 weeks during and between additional off - pump tests is predictable for long - term post - explant patient outcome . Tissue doppler andstrain imaging data are useful for decision making especially if their stability after maximumremission is also considered . After maximum improvement, stable size, geometry and function also during moderate loading of theventricle by pump speed reduction for several days can be helpful for weaning decisions . Stable normal off - pump hemodynamics is necessary for weaning decision, but insufficiently predictive for long - term cardiac stability aftervad removal . Cardio - pulmonary exercise testing (with vad support and during off - pump trials) provides information on inotropic reserve and can be useful for weaning decisions.
Village indigenous birds are constantly exposed to immunosuppressive conditions such as aflatoxicosis and infectious bursal disease virus . In addition, management and ecological factors such as confinement, climatic and seasonal fluctuations, poor feeding, and worm infestations have been associated with stress and reduced immune response . Stressful factors have been reported to cause functional and morphological changes in chickens . In tanzania, it was observed that newcastle disease (nd) was a greater problem in villages with ducks . Earlier reports indicated that newcastle disease virus (ndv) persisted for a long time in a flock of ducks in a village situation in indonesia . However, the factors leading to shedding of the virus by the carrier ducks are not well documented . It was hypothesized that immunosuppression of immunised carrier ducks does not influence persistence of ndv in these birds . In this experiment, thus, the aim of the present study was to determine the effect of immunosuppression on the viral persistence and immune status of ducks . It was designed to simulate field situation where ducks that have varying levels of ndv antibodies undergo immunosuppression in the presence of high ndv challenge . One - day - old indigenous ducklings were hatched from the duck flock maintained at the university of nairobi premises . All the birds were reared in isolation and transferred to experimental units at one year of age . They were wing tagged, tested, and confirmed to be free of ndv and respective antibodies . Water and food were provided ad libitum . A kenyan virulent newcastle disease virus isolate (vndv) was obtained from the repository maintained at the university of nairobi and characterized by standard methods . The inactivated vaccine was prepared by mixing 40% formalin and allantoic fluid with a titer of 2 of vndv in a ratio of 1: 40, that is, formalin to virus . The reparation was kept at room temperature (24c to 26c) for 24 hours before use . All the ducks were vaccinated via an initial dose of 1 ml of the vaccine intramuscularly on the thighs and a booster of 0.5 ml of the same vaccine 16 days later . The live virulent kenyan newcastle diseases virus isolate, previously characterized by standard methods, was later used to challenge vaccinated and naive ducks . Dexamethasone (dexamethasone sodium phosphate and sodium methyl hydroxybenzoate, coophavet, france) was used to stress ducks in this study . The respective groups of ducks were injected intramuscularly with the dexamethasone, following the protocol of corrier et al . Modified as follows: the dosage was given at the rate of 2 mg per kilogram of body weight per day for 4 days continuously, then the ducks were rested for 2 days and the injections resumed at the same dosage for 2 more days . Sixty - four ducks were vaccinated with 1 ml of inactivated nd vaccine intramuscularly and 14 days later, they were bled from the brachial vein and sera prepared . They were later boosted with a single dose of 0.5 ml of the nd inactivated vaccine and bled 7 days later . Seven days after the booster dose, the ducks were divided into two groups, each with 32 birds, namely, low antibody level group (1: 32) and medium antibody level group (1: 64). Each group of 32 ducks was further subdivided into 4 minigroups, as follows: (i) immunosuppressed and challenged (1a, 2a), (ii) immunosuppressed only (1b, 2b), (iii) challenged only (1c, 2c), and (iv) not challenged nor immunosuppressed (1d, 2d). Another group (group 3) of 30 nonimmunized ducks were subdivided into 4 groups . Groups 3a and 3c had 12 ducks each while 3b and 3d had 3 birds each . Immunosuppression was done before respective groups were inoculated intranasally with 0.2 ml of undiluted amnioallantoic fluids of vndv having a titer of 1: 1024 . Five birds from each of the challenge groups and all the 3 ducks from each control group were sampled throughout the experimental period (28 days). The samples were taken on days 0, 1, 4, 8, 14, and 28-after inoculation (p.i . ). Blood for serum was sampled each time from the five ducks in each challenge group, and the three ducks from each of the controls . Further, two ducks from each of the ndv challenged groups were killed serially and brain, kidney, lung, cecal tonsils, liver, and spleen collected separately from each bird . The tissues were processed for nd viral recovery using chicken embryo fibroblasts, while serum samples were tested for ndv - specific antibodies by hemagglutination inhibition (hi) test . Virus recovery from the tissues was carried out in primary chicken embryo fibroblasts as described by oie . Analysis of variance was performed using sas software (sas institute inc ., cary, nc, usa, 2002 - 2003) to determine the treatments' main effects and the interaction between time (days) and treatment, on various responses . Immunosuppressed virus - challenged ducks (group 1a) had low mean antibody levels (5.0) up to day 4 after - inoculation (p.i .) Compared with day 0 (4.5). Thereafter, there was marked increase (from 4.5 to 7.0) in antibody titers up to 14 days p.i . After day 14 p.i ., there was a slight decrease (6.9) in antibody levels up to 28 days p.i . Although the levels were still higher than any period between day 0 and 8 p.i . The nonimmunosuppressed virus - challenged group (1c) had a moderate increase (5.0 to 6.0) in antibody levels from day 1 up to day 14 p.i ., after which there was a decrease to day 0 level titers by day 28 p.i . The immunosuppressed group (1b) had marked decrease in antibody titers from day 1 to 4 and gradual decrease (5.0 to 3.8) up to day 28 p.i . The immunosuppressed virus - challenged group for the medium antibody level ducks (2a) had a gradual decline (6.0 to 5.7) in antibody titers up to day 4 followed by an increase in antibody titers (6.9) up to day 14 p.i . This was followed by a marked decrease (6.1) and by day 28 p.i . The nonimmunosuppressed virus - challenged group (2c) showed a slight decrease (from 6.0 to 5.9) in the antibody titer followed by a gradual decrease and then an increase up to the end of the experiment . From day 1 up to day 4 after - inoculation, the immunosuppressed, immunised noninfected (2b) group showed a more rapid decrease (6.0, 5.2, 4.8, 3.7, 2.3, and finally 2.0) in antibody levels as compared (6.0, 4.7, 4.5, 3.8, 2.7, and finally 2.2) to the nonimmunosuppressed controls (2d). In general, all the nonchallenged, but immunized control ducks showed decrease in antibody titers with time (figure 1(b)). The immunosuppressed virus - challenged group (3a) had a gradual antibody response (from 0.0 to 6.5) up to the end of the experimental period . The nonimmunosuppressed virus - challenged group (3c) showed a massive increase (0.0 to 6.6) in antibody levels similar to immunosuppressed virus - challenged group 3a . The group 3c also had a marked decrease (from 6.6 to 4.6) in antibody titres after day 14 p.i . And by 28 days p.i ., the titers were quite low . Negative control ducks (3b and d), sampled at the same time, were negative for ndv antibodies (figure 1(c)). For days 4, 8, 14, and 28 p.i . Antibody titres of the following groups were found to be significantly different (p <0.05). When considering the antibody levels elicited in the different duck - groups, with respect to their initial antibody levels, both sets (low - antibody group and medium - antibody group), and both the immunosuppressed and non - immunosuppressed ducks showed higher responses in ducks that were challenged with virulent virus than in those that were not challenged . All the control naive ducks (groups 3b and 3d) did not sero - convert . Immunosuppressed medium - antibody - level, challenged with ndv ducks (group 2a) versus immunosuppressed medium - antibody - level, nonchallenged (group 2b), was lowest in the latter group . Nonimmunosuppressed low antibody level, challenged with ndv ducks (group 2c) versus 2d, the latter group had lower levels of antibodies . In addition, antibody titres of group 1a versus 1d were significantly different (p <0.05) on day 14, being lower in the latter . All the control nave (groups 3b and 3d) birds did not seroconvert . On day 1 after inoculation, ndv titers were recorded in liver tissues of group 1a (low - antibody - level group, immunosuppressed and challenged with vndv) ducks only . On day 4, high titres of the ndv were recorded in the kidneys (figure 2(a)). On day 8 p.i ., ndv was isolated in the liver, kidneys, caecal tonsils, and lungs of all treatment groups (figure 2(b)). The highest ndv titers were recorded in the liver and kidney tissues of immunosuppressed medium (2a) and nonimmune (3a) challenged ducks and nonimmunosuppressed, low - antibody - level challenged ducks (1c). No ndv was isolated by day 14 p.i . In the brain and spleen from any of the groups . High ndv titres were recorded from the liver tissues of ducks in all treatment groups . However, titres were recorded in the cecal tonsils only in group 2a on day 14 p.i . And in groups 1a and 1c at day 28 p.i . In addition, the immunosuppressed ducks of groups 1a (low - antibody - level group, immunosuppressed and challenged with vndv) and 2a (medium - antibody - level group, immunosuppressed and challenged with vndv) yielded the highest ndv titres as compared to other treatment groups . Newcastle disease virus was recovered from the brain and lung on day 28 p.i from immunosuppressed ducks only (figure 2(d)). There have been comparatively few sequential virological studies on the pathogenesis of nd in ducks and the reported studies involved only fully susceptible chickens [8, 9]. Reports in other studies have documented frequent isolation of virulent ndv from captive caged birds, healthy wild birds, and village chickens [1012]. In some cases, the ducks expressed clinical nd as a result of confinement that was understood to have induced stress . Our findings indicate that ndv carrier status in nonnatural hosts such as ducks would possibly, under stress, recrudescence virulent virus from sequestered sites in the kidney, liver, and caecal tonsils, leading to virus release in faecal and respiratory exudates . The excreted virus would set up an infectious index case in chickens, thus maintaining ndv endemicity . Immunosuppression induced by injection of dexamethasone in the three treatments influenced the pattern of antibody response and the ndv recovery rate . The immunosuppressed ducks that had low and medium antibody level showed a decrease in antibody titers up to day 4 after challenge with ndv . The nonimmunosuppressed virus - challenged ducks of low - to - medium antibody level had an increase in antibody titres up to day 14 p.i . The nonimmunized ducks manifested increased antibody titres after day 4 p.i . And had a massive increase in antibody levels as compared to immunosuppressed challenged group . In the present study, the number of immunosuppressed ducks that yielded the nd virus was higher compared to the nonimmunosuppressed . The prechallenge antibody titers may therefore play a significant role in shedding off the virus as well as clinical manifestation of the disease . . Noted that a few hours of treatment with low concentrations of synthetic glucocorticoid (analogue dexamethasone) are sufficient to inhibit the synthesis of interferon, a virus inhibitor . This may, in part, explain the observation that treatment with glucocorticoids increased virus yield and lethality in mice infected with coxsackie virus . Our present study using ducks concurs with those of asdell and hanson who showed that prior treatment of chickens with dexamethasone lead to massive nd virus multiplication . There was significant difference in geometric mean antibody titers between the immunosuppressed ducks of group 1a and nonimmunosuppressed counterparts (group 1d) and also between immunosuppressed ducks of group 3a and nonimmunosuppressed group 3d . This means that whereas dexamethasone seems to have an effect on immune system of ndv - infected ducks, the prechallenge titres also play a major role in the immune response of immunosuppressed birds in that immunosuppression of ducks with high viral titers allows virus multiplication making ducks better carriers . The effect of immunosuppression with dexamethasone in ducks appears to be the same as that induced by aflatoxin in chicks with ndv infections . Chickens fed on aflatoxin produced lower antibody levels when compared to the non - aflatoxin - treated ones . The nonchallenged preimmunized ducks had a progressive decrease in antibody levels suggesting that if they were to be exposed to the virus, they could come down with the nd or if the antibody titers were within the protective levels (2 to 2), they might not develop clinical disease but instead may remain as virus carriers . The fact that the ducks in these experiments had high levels of antibodies may not necessarily prevent subclinical infection and excretion of virulent virus as supported by other studies elsewhere . Based on these results, it is possible to assume that immunosuppressed ducks carrying ndv are more likely to shed virus under stress than nonimmunosuppressed ducks . Furthermore, the ducks showed progressively declining antibodies titres to very low levels making them more susceptible to infection by challenge nd virus, thus leading to clinical disease and virus excretion . The excreted virus would contaminate the birds' environment and be transferred to susceptible chickens and other birds . Thus, immunosuppression seems to have epidemiologically significant effects on ndv carrier ducks having varying antibody titer levels.
A questionnaire cum feedback form was circulated to ophthalmology residents in west maharashtra (appendix 1). Students were asked to gauge didactic lectures, lectures with power point presentations, seminars, journal club, case presentations, and wet lab on a scale of 0 (most unsatisfactory) to 4 (best). They were asked how many surgeries they had seen, how many they needed to assist and to perform to be considered confident and trained in that technique . There were open - ended questions regarding the library facilities, teaching basic techniques of examination of patient, outreach camps, eye banking, wet laboratory and other diagnostic aids . The form was developed at the community eye care department of the hospital and piloted on the residents of the same institute . It was administered to the resident doctors by giving it to them and asking for the completed form after three days . If they had not filled it, the request was repeated after a week . The request was made through a social worker or a fellow resident, but was not directed through the department head or the postgraduate teacher . The residents were free not to put their name at the end, but had to fill in details of age, sex and number of years of residency completed . The average age was 25 years (range 2234 years, 95% ci 21 to 29). The minimum number of residents in a program was three, and the maximum 18 . The distribution of residency programs was government medical college (one), private medical college (two) tertiary private general hospital (one) and eye hospitals (three), two of them being community eye care centers . The residents graded lectures with power point presentations (on a scale of 0 to 4) with median as 4 and didactic lectures (without power point) with median as 2 . Thirty - eight out of 67 (56.7%) gave the maximum 4 points to power point presentation lecture, while only 4/67 (6%) gave it for the didactic lecture . The median of seminar, case presentation with discussion, wet lab and journal club was 3, 4, 3 and 3 respectively . Twenty - seven out of 67 (40.3%) gave full marks to the seminar . Fifty - seven out of 67 (85.1%) gave the maximum points (4) for case presentation . Twenty out of 67 were unaware of wet lab facilities, but 29/47 (61.7%) who were aware of it gave full marks to wet lab . Twelve (17.1%) were unaware about the journal club and three institutions did not have it as a method of teaching . Twenty- seven of the 55 were aware of it (49.1%), gave the maximum points (4) to a journal club . All (67/67) residents were in favor of regular objectively structured clinical examination (osce) tests or viva voce examination . Thirty - eight out of 67 wanted to be tested every six months, 10/67 monthly, while 4 /67 were for weekly assessments . Only three institutions (all eye hospitals) had more than 100 books on ophthalmology in their library . All had internet facilities but 42/67 (61.2%) of students complained of lack of access to it . Residents also desired to be taught separately to handle perimetry, b - scan, neodymium yttrium argon garnet (nd: yag) laser and ultrasonic biomicroscopy . Thirty - two out of 67 wanted to learn more about eye donation and enucleation . The surgeries observed and those actually performed by the resident doctors are shown in table 1 along with the number they perceived they ought to assist and independently perform to gain mastery in it . The figures are of surgeries performed by second- and third - year residents only during their residency tenure . Most students wanted basic ophthalmology and refraction to be taught more with emphasis on squint and neuro - ophthalmology . They wanted more hands - on experience in using basic diagnostic aids like 90d, 78d, indirect ophthalmoscope, gonioscopy, applanation tonometry and perimetry . The case presentation in which a resident doctor presented a patient history and examination before a professor or a tutor in the presence of other residents was the most popular form of teaching for the trainees . The discussion regarding lacuna in history - taking, examination and possible investigations and management that followed the presentation was considered most rewarding . The resident doctors were not enamored by didactic lectures which are common in most medical schools, especially for undergraduates and they need to be supplemented with more audio - visual aids . The seminar in which one or more resident doctors made audiovisual presentations about a certain topic, with a tutor as facilitator, was popular . An indian study reported that almost one - third of the institutions had a wet lab facility.2 the journal club in which a resident doctor summarized or read an article from a peer - reviewed journal in presence of a tutor and other residents was graded an average of 3.14 . A structured journal club was found to be a useful tool for practice - based learning in the us.3 an indian study showed that only 51/128 (41.4%) institutions subscribed to more than two international journals.2 an austrian study showed three - dimensional animated teaching to be a useful adjunct to surgical videos, especially for female medical students.4 virtual reality training as used by trainee pilots has been proposed as a method to teach surgical competence in the us.5 a three - dimensional computer animation technology was shown to significantly increase the quality and efficiency of education and demonstration of complex topics in ophthalmology in austria.6 but only three eye hospitals in our study had video libraries . Though all the institutions had internet access, the majority of the students (42/67, 61.2%) in all except two institutions complained about lack of adequate access . Erratic connections, limited library hours and restrictions on trainee doctors using the web were cited as reasons for lack of access . As more and more information is available on the internet, this seems paradoxical and unfortunate . None of the teaching programs had structured curriculum for basic skills like history - taking and ocular examination . But a us study had reported worrisome erosion of acquired skills of eye examination amongst their students.7 there is a huge variation in the actual number of surgeries performed [table 1]. But the variation is still large even after allowing for the exceptions . A study in 2002 had shown that an average surgery per trainee in an institution per year was 111 cataract surgeries, 4.8 glaucoma surgeries, 2.2 squint surgeries, 4 lid surgeries, 2.2 keratoplasty and 0.8 dacryocystorhinostomy surgeries.2 even though there seems to be an adequate number of surgeries being performed annually per student in an institution, the large range and median of zero for most surgery types denotes the enormous difference between various institutions . And the resident doctors perceptions differ significantly from what was reported by the heads of ophthalmology . Half the residents had not performed any kind of surgery other than cataract surgery . The students expectations in the number needed to perform to be considered adequately trained were not met, even after considering that few had unrealistic expectations . Non - cataract surgical training is neglected and this shows the training program in poor light . Except two, none of the teaching programs had any emphasis on surgeries other than cataract . Nearly one - third of the residents enrolling for ophthalmology resident programs do not graduate into eye surgeons in the us but continue as medical ophthalmologists.8 some are unable to improve their surgical skills even after repeated training for surgical skills and 12% were asked to leave the residency . One study promotes the use of an aptitude test to assess basic surgical inclination / competence like the american dental associations dental (sample) admission test.9 a larger turnover of cases and easier acceptance of complications in the indian setting may be responsible for most of the trainees metamorphosizing into eye surgeons . Clinical audit should be employed to monitor and improve surgical outcome of ophthalmology trainess.10 preferred practice patterns put forth by the american academy of ophthalmology can be modified and used in indian settings . It is concluded that the residency program in ophthalmology is not up to the expectations of postgraduate students in many teaching centers in most of maharashtra . A study conducted by thomas et al . Revealed that despite provision of adequate resources to funded medical colleges, their quality of residency training was inadequate.11 the new curriculum and manual proposed by the national board of examination, new delhi addresses many of these issues . The national board has strived to streamline and standardize postgraduate medical education all over india . We hope that other universities in the country shall also follow a similar pattern.
Patient information was obtained over a six year period from 2003 and 2009 and included a description of the specific trauma episodes, surgical records, x - rays and mri of all patients with avulsion fracture of calcaneal tuberosity . Twenty patients with an avulsed calcaneal tuberosity out of a total of 764 cases of calcaneal fractures (2.6%) were identified including fourteen men and six women . Adequate x - rays and clinical notes were available for each patient included in the study . Other data included demographic information, the specific mechanism of injury and the anatomical variance of the achilles tendon according to the findings of both the mri and surgery . The mechanism of injury was recorded and defined as tripping, falling or a direct blow . Anatomical variances of the achilles tendon were previously described as differences in the level of insertion (more or less extensive) of the achilles tendon into the calcaneus.6) therefore, anatomical variances of the achilles tendon were divided into the infrabursal insertion, which is the usual anatomy and more extensive insertion or higher than what is normally described . A previous report by beavis et al.3) proposed a type i to iii classification scheme for avulsion fractures of the calcaneal tuberosity . We modified this specific study through the addition of another avulsion type iv fracture (fig . 1). The type i fracture is a' simple extra - articular avulsion' fracture (8/20 cases). Type ii is the' beak' fracture in which there is an oblique fracture line running posterior from just behind bohler's angle (5/20 cases). Type iii is the infrabursal avulsed fracture by superficial fibers from the middle third of the posterior tuberosity . In the type iv fracture there is the' beak', but a small triangular fragment is separated by deep fibers only from the upper border of the tuberosity (3/20 cases). All of the included calcaneal avulsed fractures were classified using the aforementioned criteria (figs . Statistical significance was determined using the mann - whitney u - test; categorical variables were examined with the chi - square or fisher's exact test when cell counts were less than or equal to five . Patient information was obtained over a six year period from 2003 and 2009 and included a description of the specific trauma episodes, surgical records, x - rays and mri of all patients with avulsion fracture of calcaneal tuberosity . Twenty patients with an avulsed calcaneal tuberosity out of a total of 764 cases of calcaneal fractures (2.6%) were identified including fourteen men and six women . Adequate x - rays and clinical notes were available for each patient included in the study . Other data included demographic information, the specific mechanism of injury and the anatomical variance of the achilles tendon according to the findings of both the mri and surgery . The mechanism of injury was recorded and defined as tripping, falling or a direct blow . Anatomical variances of the achilles tendon were previously described as differences in the level of insertion (more or less extensive) of the achilles tendon into the calcaneus.6) therefore, anatomical variances of the achilles tendon were divided into the infrabursal insertion, which is the usual anatomy and more extensive insertion or higher than what is normally described . A previous report by beavis et al.3) proposed a type i to iii classification scheme for avulsion fractures of the calcaneal tuberosity . We modified this specific study through the addition of another avulsion type iv fracture (fig . 1). The type i fracture is a' simple extra - articular avulsion' fracture (8/20 cases). Type ii is the' beak' fracture in which there is an oblique fracture line running posterior from just behind bohler's angle (5/20 cases). Type iii is the infrabursal avulsed fracture by superficial fibers from the middle third of the posterior tuberosity . In the type iv fracture there is the' beak', but a small triangular fragment is separated by deep fibers only from the upper border of the tuberosity (3/20 cases). All of the included calcaneal avulsed fractures were classified using the aforementioned criteria (figs . 1 and 2). Statistical significance was determined using the mann - whitney u - test; categorical variables were examined with the chi - square or fisher's exact test when cell counts were less than or equal to five . Twenty avulsion fractures of the calcaneal tuberosity were selected and reviewed out of 764 calcaneal fractures (2.6%). The type i fracture was the most frequently observed (40%) and was found more likely to occur in elderly women (table 1). However, type ii, iii, and iv fractures were observed more often in relatively younger male patients . This difference in age and gender were significant when comparing type i fracture with the three other types of fractures (p = 0.010, p = 0.018). The fracture patterns were then compared according to the mechanism of injury (table 2). However, the occurrence of type ii, iii, and iv fractures were usually observed in incidents involving more severe trauma (p = 0.019). A direct blow to the bone was the dominant cause of type ii fractures and falling for type iii and iv fractures . Extensive insertion of the achilles tendon was found for all types of fractures analyzed (15/20) (table 3). However, the involvement of fibers of the achilles tendon varied according to each fracture pattern . All the fibers of the achilles tendon were involved in type i and ii fractures, but only the superficial fibers were involved in type iii fractures and the deep fibers in type iv fractures respectively (fig . These observations imply that the insertion pattern was not related to the fracture type, but rather to the specific fibers that were involved . Several studies have suggested that avulsion fractures of the calcaneal tuberosity have an osteoporotic origin.7,8) these fractures have also been observed with increased frequency in diabetic patients due to insufficiency fractures associated with peripheral neuropathy.9,10) however, this description can only account for the classification of type i fractures within our study and cannot explain the other fracture patterns detected in young male patients . We examined this problem by examining the anatomical variations of the achilles tendon insertion and the different mechanisms of injury . Although the true prevalence of extensive insertion of the achilles tendon is not well established, a recent study by lowy6) demonstrated this anatomical variant in two of ten dissected specimens . In our study, extensive insertion was found in fifteen out of a total of twenty cases of avulsed calcaneal fractures . We hypothesize that individuals that possess extensive insertion are at increased risk for potential avulsion fractures.3) one potential explanation includes the direct force provided by the wide and broad insertion of the achilles tendon in these individuals and may result in an increased risk of avulsion fractures . However, we found that avulsion fractures could occur through partial involvement of the fibers of the achilles tendon as according to our surgical and mri analysis (fig . 2). Specifically, type iii and iv avulsion fractures occurred in patients with extensive insertion but only if the fibers are partially involved with an intact achilles tendon mechanism . It is interesting that a mechanism to explain partial avulsion fractures was already suggested by lowy.6) a potential explanation is that a few deep fibers of the tendon, and possibly the fibers arising from the soleus, may tilt the fragment while the rest remain distally attached . When the knee is in a flexed position with the gastrocnemius released, the contraction of the soleus muscle to the heel may induce the type iv type of fracture . We can hypothesize that the mechanism of injury for the type iii fracture is similar . The type iii fracture is believed to occur when the superficial fibers of the tendon are involved (figs . 2 and 3). The type i fracture is more likely to occur as a result of minor trauma such as tripping . However, type iii and iv fractures are likely to occur through more severe trauma such as falling down . This indicates that the type i fracture is an insufficiency fracture, however, type iii and iv fractures mainly occur due to strong muscular contraction with the heel fixed to the ground . If the strong force of muscular contraction is combined with a direct impact injury, then type ii fractures usually occur . Through the course of our study, we found several limitations that need to be addressed . First, this report is retrospective and therefore contained biases less likely to occur in a prospective study . In addition, our sample number was small due to the low incidence of avulsion fractures of the calcaneal tuberosity . However, most of the literature regarding the avulsed calcaneal fracture involves case reports and explanations of the fracture patterns which are heavily dependent on the opinion of individual authors . Our results demonstrate that the fracture pattern is determined by bone quality, the mechanism of injury and the fibers of the achilles tendon that transmit the force . We believe that our novel classification system is useful because the treatment options will depend on the specific fracture (fig . 3). Screw fixation is a good choice for treatment of type i and ii fracture because the bony fragment remains of sufficient size for this treatment.4) in type ii fractures, however, the posterior skin of the heel must be evaluated quickly, and if it is observed as tented or branched, the fracture must be reduced and fixed . These patients are at risk for skin necrosis of the posterior heel if they are not immediately treated.11) type iii and type iv fractures can be treated conservatively due to the preserved function of the achilles tendon . However, since these fractures often occur in young individuals, surgical treatment is recommended for maintaining athletic ability . Suture anchor fixation provides superior treatment to screws due to the presence of small bony fragments for type iii.5) in two patients included in the study, surgery was completed utilizing suture anchors type iv injuries . The surgery was difficult in the approach to deep fibers and in the management of fragments involved, leading to inferior treatment for the type iv as opposed to type iii fractures . Based on our observations, we recommend conservative treatment for type iv fractures . In summary, we observed four types of avulsed calcaneal fractures and developed criteria for further classification . The fracture patterns are created through osteoporosis, the mechanism of injury and fibers of the achilles tendon that transmit the force.
Due to early discharge of infants from the hospital, readmission has been increased (1). Therefore, early diagnosis of jaundice and timely actions are necessary . The measurement of bilirubin level (2) and alpha fetoprotein (3) in cord blood have been used for this purpose . For the first time aysin nalbantoglu et al . Used alkaline phosphatase (alp) level 6 hours after birth as a marker for determining hemolysis and hyperbilirubinemia (4). Alkaline phosphatase is a hydrolase enzyme and responsible for removing phosphate from many types of molecules (5). Alkaline phosphatase is found in almost all body cells, including red blood cells (4 - 6). Therefore, we hypothesized that it can be used as a marker for early diagnosis of hyperbilirubinemia . This study was conducted to evaluate the alp level as a marker for early diagnosis of neonatal jaundice . In this prospective study, between october and december 2013, infants who were born at the babol - clinic hospital in babol, northern iran, were selected . A total of 105 healthy term infants with gestational age between 37 and 42 weeks, weighing more than 2500 g born to healthy mothers were studied . Five milliliter cord blood was taken after birth and sent for determination of alp level . Serum alkaline phosphatase was measured with auto analyzer (sinnowa - ds301, china, 2013). Infants who were born to mothers with diseases such as eclampsia, diabetes, bone, kidney and liver diseases, and infants who were found suffering from other diseases except jaundice were excluded from the study . Three cases were lost to the study, so 102 cases were monitored for the emergence of clinical jaundice based on clinical observation by parents or physicians up to 10 days after birth . Infants with clinical jaundice were recalled and serum bilirubin level was measured and treated based on american academy of pediatrics (aap) protocols . These were assigned as the treatment group and neonates without clinical jaundice were assigned as the non - jaundiced group . In treatment group, to determine the cause of hyperbilirubinemia, work - up including complete blood count, reticulocyte count, estimation of blood group in mother and neonate, peripheral blood smear, evaluation of g6pd level and coombs test, was done . Demographic information of all infants including gestational age, birth weight and apgar score was recorded . The remaining 102 cases consisted of 50 (49%) males and 52 (51%) females . Ninety eight (96%) infants were born by cesarean section and 4 (4%) by vaginal delivery . The mean gestational age was 38.7 weeks and the mean birth weight 3649.59 grams (table 1). The rate of need for treatment was 9.8% (10 cases), of which 5 cases were abo incompatible, one case rh incompatible, 2 cases g6pd deficient and in 2 cases the cause of jaundice remained unknown . Hct levels and reticulocyte count were in normal range and coombs test was negative in these cases . There was no difference between groups with regard to gestational age, birth weight and apgar scores, but the comparison of cord blood alkaline phosphatase levels revealed a significant difference between the two groups (p value = 0.041) (table 2). Values are presented as mean sd . Values are presented as mean sd . Comparison of cord blood alkaline phosphatase levels between non - jaundiced group and jaundiced newborns in whom bilirubin level had reached 10 mg / dl, revealed a significant difference (p value = 0.016) (table 3). Values are presented as mean sd . Comparison of the non - jaundiced group with neonates who required treatment according to aap protocol (the treatment group) showed a significant difference in cord blood alkaline phosphatase levels (p value = 0.040) (table 4). A comparison of the roc curves of the alkaline phosphatase levels between the non - jaundiced and treatment groups (figure 1) revealed that a cord blood alkaline phosphatase level> 314 iu / l was the most suitable cutoff value for predicting severe jaundice (that needs treatment). (alp) at the treatment group, area under the curve is 0.730 . Of 60 neonates whose cord blood alkaline phosphatase measured less than 314 iu / l, only two neonates needed treatment . Thus, the negative predictive value of cord blood alkaline phosphatase for occurrence of hyperbilirubinemia was 96.6% . To our knowledge this is the first clinical study that examines cord blood alkaline phosphatase level as an indicator to predict severe neonatal jaundice . The results of our study indicate that measurement of cord blood alkaline phosphatase may be a predicting marker for neonatal jaundice that can exceed 10 mg / dl and necessitates treatment in the first week of life . Cord blood alkaline phosphatase level with sensitivity and specificity of 80% and 63% respectively in cutoff level> 314 iu / l predicts a need for treatment . They found that alp levels were significantly higher in patients with hyperbilirubinemia requiring treatment, either with phototherapy or exchange transfusion (p value 0.0001) (4). In our study, there was a significant difference in the levels of cord blood alkaline phosphatase between the non - jaundiced and clinically jaundiced newborns, and it was significantly higher in patients with hyperbilirubinemia requiring treatment . Moreover, the alp levels were significantly higher in newborns whose serum bilirubin level reached a level 10 mg / dl . One of advantages in our study was the site of sample collection, which was taken from cord blood . In addition, the neonate may not be lost to follow - up because of early discharge . Chou et al . Measured cord blood hydrogen peroxide level for prediction of neonatal hyperbilirubinemia . The cord blood hydrogen peroxide signal level of 2500 counts/10 seconds was an appropriate cutoff for predicting severe hyperbilirubinemia with sensitivity and negative predictive value of 76.2% and 93.3%, respectively (7). Our study showed that the alkaline phosphatase level of 314 iu / l was associated with sensitivity and negative predictive value of 80% and 96.6%, respectively . The rate of need for treatment of jaundice in our study was 9.8% (10 cases). Of these, 5 cases were abo incompatible, one rh incompatible, 2 g6pd deficient and 2 cases were of unknown etiology . According to these findings, it seems that the alkaline phosphatase level has a higher validity in disclosing hemolytic processes . (8) used the cord blood bilirubin level to predict the need for phototherapy . By a cord bilirubin cut - off level of 30 mol / l this revealed a sensitivity of 70.3% and a negative predictive value of 65.6% . Our study had a sensitivity and negative predictive value of 80% and 96.6%, respectively . The measurement of end tidal carbon monoxide (9) at the cutoff level of 1.8 /l (ppm) showed a negative predictive value 97%; our study showed a negative predictive value of 96.6%, which is comparable to this expensive and low accessible test . The first day serum bilirubin (10) and sixth hour serum bilirubin (11) have been used for prediction of hyperbilirubinemia . These tests require venous blood sampling (in many countries as in ours), and so are not suitable for screening all neonates . In our study found the mean level of cord blood alkaline phosphatase 159 49 iu / l (12). Another local study by abbasian et al . In shahrood, iran showed that mean cord blood alkaline phosphatase level was 314.34 122.42 the average level of cord blood alkaline phosphatase in iranian newborns seems to be higher than in other populations . Cord blood alkaline phosphatase level is a useful indicator in predicting subsequent jaundice in healthy term newborns.
One of the main objectives of orthodontic treatment and/or orthognathic surgery is to improve facial harmony and beauty . Although the perception of these characteristics varies according to cultures, socioeconomic status, and era, clinicians have traditionally measured the distance, angulation, and proportion of hard and soft tissues to evaluate the facial type and profile.12345678 in terms of facial profile, the preferred chin projection has been well investigated because it has a strong impact on the harmony between the upper, middle, and lower face; the shape and contour of the lower lip; and the mentolabial sulcus.56910 although the chin can be placed in a normal position by orthognathic surgery, some patients prefer a slightly more retrusive or protrusive chin than a normal chin . The reasons for the difference in preferred chin projection among patients might be the following: first, the final goal of chin projection is different for clinicians and patients; and second, the effect of chin projection on frontal facial appearance has not been properly assessed . The assessment of frontal facial type (fft) is important because patients usually evaluate their face by using a mirror before and after orthodontic treatment and/or orthognathic surgery . The most commonly used index, which can describe the fft, is the facial index.41112131415 it represents the ratio between the height of the middle and lower face (soft - tissue nasion soft - tissue gnathion) and the interzygomatic width (soft - tissue zy on the right side soft - tissue zy on the left side) (figure 1), and is classified as euryprosopic, mesoprosopic, or leptoprosopic.411121415 although numerous two - dimensional methods, including silhouettes, photographs, and digitally modified images, have been used to investigate the preferred chin projection,216171819 these methods have some limitations in terms of evaluating the esthetic chin position, such as unrealistic impression and distortion of the facial images.52021 recently three - dimensional (3d) morphing programs have been introduced to predict soft - tissue changes related to orthodontic treatment and/or orthognathic surgery.2223 these 3d morphing techniques can modify the extent of chin projection without distorting images of facial areas, thereby resulting in a more realistic impression of diverse facial types and profiles . The perception of facial harmony and beauty can also vary according to the occupation and age of the evaluators, sex and age of the subjects, as well as other conditions.41617181920212223 therefore, to avoid bias, the experimental conditions should be standardized as follows: 1) the occupation of the evaluators should be the same; 2) the age of the evaluators should preferably match that of the patients to reflect trends in society (e.g., twenties and early thirties); 3) the same subjects should be evaluated by both male and female evaluators; and 4) the facial images should be realistic despite the extent of modification of chin projection . Although numerous previous studies have investigated the preferred chin projection in the profile view by using samples from various ethnicities and evaluators from different professions,222324252627282930 few studies have explored the relationship between an esthetically preferred chin projection and fft . Therefore, the purpose of this study was to investigate the effects of fft and sex on esthetically preferred chin projection in 3d facial images, especially in the korean population . The null hypothesis was that there would be no significant difference in esthetically preferred chin projection in terms of fft and sex . The fft was determined using the facial index, which is the ratio between the height of the middle and lower face (soft - tissue nasion soft - tissue gnathion) and the interzygomatic width (soft - tissue zy on the right side soft - tissue zy on the left side).14 the facial indices in each sex are enumerated in table 1 according to the martin - saller scale.15 six 3d facial images (three of men and three of women with class i skeletal and dental relationships; and with the following three ffts for each sex: euryprosopic [eury - fft], mesoprosopic [meso - fft], and leptoprosopic [lepto - fft]) were acquired using a 3d facial optical scanner (morpheus 3d scanner; morpheus co., ltd ., the actual height of the middle and lower face, interzygomatic width, and facial indices of the six 3d facial images are enumerated in table 1 . Using the morpheus 3d aesthetic solution software (ver . 2.0; morpheus co., ltd . ), the normal chin projection in each 3d facial image was set to 10 of the soft - tissue facial profile angle (glabella subnasale - pogonion) based on the findings of previous studies on korean adult facial profiles.678 the chin projections were then morphed by gradations of 2 from the normal projection (10) (moderately protrusive [6], slightly protrusive [8], slightly retrusive [12], and moderately retrusive [14]). For each chin position, the survey participants were shown images in the 45 angled and 90 lateral views (figures 2, 3, 4, 5, 6, 7). The survey participants were 75 dental students from the school of dentistry, seoul national university, seoul, korea (48 men and 27 women; mean age, 28.9 3.3 years), who were asked to rate the five chin projections (6, 8, 10, 12, and 14) from the most to least esthetically preferred in each 3d image . This study was reviewed and approved by the institutional review board of seoul national university school of dentistry, seoul, korea (s - d20140028). The kolmogorov - smirnov test, kruskal - wallis test, and bonferroni correction were performed for statistical analyses to determine the effect of fft and sex on the preferred chin projection by using ibm spss statistics for windows ver . The fft was determined using the facial index, which is the ratio between the height of the middle and lower face (soft - tissue nasion soft - tissue gnathion) and the interzygomatic width (soft - tissue zy on the right side soft - tissue zy on the left side).14 the facial indices in each sex are enumerated in table 1 according to the martin - saller scale.15 six 3d facial images (three of men and three of women with class i skeletal and dental relationships; and with the following three ffts for each sex: euryprosopic [eury - fft], mesoprosopic [meso - fft], and leptoprosopic [lepto - fft]) were acquired using a 3d facial optical scanner (morpheus 3d scanner; morpheus co., ltd ., the actual height of the middle and lower face, interzygomatic width, and facial indices of the six 3d facial images are enumerated in table 1 . Morpheus co., ltd . ), the normal chin projection in each 3d facial image was set to 10 of the soft - tissue facial profile angle (glabella subnasale - pogonion) based on the findings of previous studies on korean adult facial profiles.678 the chin projections were then morphed by gradations of 2 from the normal projection (10) (moderately protrusive [6], slightly protrusive [8], slightly retrusive [12], and moderately retrusive [14]). For each chin position, the survey participants were shown images in the 45 angled and 90 lateral views (figures 2, 3, 4, 5, 6, 7). The survey participants were 75 dental students from the school of dentistry, seoul national university, seoul, korea (48 men and 27 women; mean age, 28.9 3.3 years), who were asked to rate the five chin projections (6, 8, 10, 12, and 14) from the most to least esthetically preferred in each 3d image . This study was reviewed and approved by the institutional review board of seoul national university school of dentistry, seoul, korea (s - d20140028). The kolmogorov - smirnov test, kruskal - wallis test, and bonferroni correction were performed for statistical analyses to determine the effect of fft and sex on the preferred chin projection by using ibm spss statistics for windows ver . 21.0 (ibm corp ., armonk, ny, usa). A value of p <0.05 was considered statistically significant no significant difference was observed between the male and female evaluators in the distribution of the preferred chin projections in each fft (table 2). The normal chin projection angle (10) was the most preferred without any sex difference (n = 25/75 in male 3d images and n = 27/75 in female 3d images; table 3). However, in eury - fft, the slightly protrusive chin projection angle (8) was favored in male 3d images (n = 37/75), but the normal chin projection angle (10) was preferred in female 3d images (n = 25/75) (table 3). In lepto - fft, the normal chin projection angle (10) was favored in male 3d images (n = 32/75), whereas the slightly retrusive chin projection angle (12) was favored in female 3d images (n = 38/75) (table 3). The means of the preferred cp angles in each fft were as follows; eury - fft: male, 8.7 and female, 9.9; meso - fft: male, 9.8 and female, 10.7; and lepto - fft: male, 10.8 and female, 11.4 (table 3). A significant difference was observed in the preferred cp angle according to fft (eury - fft male <[meso - fft male, eury - fft female, meso - fft female, lepto - fft male] <[meso - fft female, lepto - fft male, lepto - fft female]; p <0.001; table 3). No significant difference was observed between the male and female evaluators in the distribution of the preferred chin projections in each fft (table 2). In meso - fft, the normal chin projection angle (10) was the most preferred without any sex difference (n = 25/75 in male 3d images and n = 27/75 in female 3d images; table 3). However, in eury - fft, the slightly protrusive chin projection angle (8) was favored in male 3d images (n = 37/75), but the normal chin projection angle (10) was preferred in female 3d images (n = 25/75) (table 3). In lepto - fft, the normal chin projection angle (10) was favored in male 3d images (n = 32/75), whereas the slightly retrusive chin projection angle (12) was favored in female 3d images (n = 38/75) (table 3). The means of the preferred cp angles in each fft were as follows; eury - fft: male, 8.7 and female, 9.9; meso - fft: male, 9.8 and female, 10.7; and lepto - fft: male, 10.8 and female, 11.4 (table 3). A significant difference was observed in the preferred cp angle according to fft (eury - fft male <[meso - fft male, eury - fft female, meso - fft female, lepto - fft male] <[meso - fft female, lepto - fft male, lepto - fft female]; p <0.001; table 3). Several studies have investigated the preferred projection of the lip and chin in various ethnicities by using facial profiles.71819222331 coleman et al.7 reported that caucasian orthodontists and laypersons preferred a retrusive chin position in the normal profile . Based on their studies on chinese participants, soh et al.18 indicated that laypersons, dental students, and dental professionals preferred normal or bimaxillary retrusive facial profiles . Chong et al.19 also suggested that chinese individuals preferred more retrusive chin positions than did caucasian individuals . Similarly, mantzikos31 reported that japanese participants preferred normal, bimaxillary retrusion, bimaxillary protrusion, mandibular retrusion, and mandibular protrusion in the decreasing order of preference . On the basis of these studies, it can be assumed that a normal or slightly retrusive chin projection is preferred in diverse ethnicities . However, few studies have assessed the relationship between ffts and preferred chin projection . In this study, we found a significant difference in the distribution of preferred chin projection according to ffts and sex (eury - fft male <[meso - fft male, eury - fft female, meso - fft female, lepto - fft male] <[meso - fft female, lepto - fft male, lepto - fft female]; p <0.001; table 3). The age of the survey participants is an important factor in determining the preferred chin projection . Park et al.32 reported that young adults (2039 years old; mean age, 26 years) favored a straight facial profile, which was in accordance with the findings of this study . A normal chin projection (10) was preferred in most facial types except for a slightly protrusive chin projection in the eury - fft male (n = 37/75; mean preferred angle, 8.7) and a slightly retrusive chin projection in the lepto - fft female (n = 38/75; mean preferred angle, 11.4) (table 3). These results might be attributed to the similar age of the survey participants in this study (mean age, 28.9 3.3 years) and those in the study by park et al.32 however, they demonstrated that middle - aged (4054 years; mean age, 47 years) as well as older individuals (5570 years; mean age, 62 years) preferred a slightly retrusive chin as an esthetically pleasing one . In addition, shimomura et al.20 suggested that as one's age passes the thirties, one tends to prefer a slightly retrusive chin to a protrusive one . The ratio between middle and lower facial height and interzygomatic width of the eury - fft was smaller than that of the meso - fft (less than 0.85 vs. between 0.85 and 0.89; table 1). If the chin is retruded in the eury - fft, the interzygomatic width might be viewed as being relatively wider than that in the meso - fft . Therefore, a normal or slightly protrusive chin projection can be considered more esthetically acceptable than a retrusive chin projection . In the present study, a slightly protrusive chin projection (8) n = 37/75; table 3) and the second most acceptable for female eury - fft 3d images (n = 23/75; table 3). The ratio between middle and lower facial height and interzygomatic width of the lepto - fft was greater than that of the meso - fft (greater than 0.87 vs. between 0.84 and 0.87; table 1). When the chin is protruded in the lepto - fft, the height from the soft - tissue nasion and soft - tissue gnathion can appear relatively longer because of the increased height of the lower third of the face . If the chin is retruded in the lepto - fft, the height of the lower face can appear shorter than its actual height and, as such, the retrusive chin projection may be preferred . In the present study, a slightly retrusive chin (12) was the most preferred one for female lepto - fft 3d images (n = 38/75; table 3) and the second most preferred for male lepto - fft 3d images (n = 20/72; table 3). For example, in the male eury - fft 3d images, a slightly protrusive chin projection (8) was the most preferred (eury - fft male <[meso - fft male, lepto - fft male]; p <0.001; table 3). This finding suggested that if orthognathic surgery or adjunctive esthetic surgery were planned for male patients with class iii relationships and a eury - fft, it would be better to establish a slightly protrusive chin projection, rather than a retrusive one, for ensuring the patient's satisfaction with the esthetic facial change . In addition, this finding may be related to a mature and responsible impression that is conveyed by a normal or slightly protrusive chin projection in males . In the female lepto - fft 3d images, a slightly retrusive chin projection (12) was considered the most esthetically pleasing ([eury - fft female, meso - fft female] <[meso - fft female, lepto - fft female]; p <0.001; table 3). For female patients with class ii relationships and a lepto - fft, a slightly retrusive chin projection, rather than a protrusive one, might be an esthetically appropriate treatment goal . This finding may be related to a feminine facial impression that is conveyed by a slightly retrusive chin projection in females . A limitation of this study is that the survey was completed only by young adults who were dental students . In addition, the number of male survey participants was almost double that of female participants, and this might have influenced the results . Therefore, future studies should include survey participants with various occupational backgrounds and from different age groups with a similar proportion of sexes . Since the preferred chin projection varied according to ffts and sex, the null hypothesis was rejected . These findings might serve as guidelines for setting the preferred chin projection according to fft and sex.
We report a case of a patient visiting africa for the first time presenting with malaria and arf . Malaria is an italian word composed of mala and aria, derived from malus (bad) and aeris (air). It is a disease caused by a protozoan parasite of the genus plasmodium, namely, p. falciparum, p. vivax, p. malariae, and p. ovale, while p. knowles commonly affects nonhuman primates . The disease remains to be the major cause of morbidity and mortality in many tropical developing countries where it is mostly caused by plasmodium falciparum . It is estimated that the latter causes around 600,000 deaths annually and the vast majority of such deaths occur in sub - saharan african countries . The high mortality rates caused by p. falciparum are to a great extent attributed to the parasite's ability to induce severe malaria associated with life - threatening complications such as cerebral malaria (cm), acute renal failure (arf), severe anemia, acidosis, respiratory distress, jaundice, and acute respiratory distress syndrome (ards). Plasmodium falciparum malaria presenting symptoms and mortality pattern vary considerably according to geographical distribution, parasite's transmission intensity, and host's immunity to the parasite . In areas with high and stable malaria transmission, for instance, severe malaria is common in children under 5 years of age and commonly presents as severe anemia, while adults with acquired immunity to the parasite do not usually suffer severe malaria . In areas with moderate malaria transmission intensity likewise, in low unstable malaria transmission intensity, severe malaria occurs in all age groups and can manifest as, cm, renal failure, severe jaundice, and/or pulmonary edema . However, arf has been reported to be one of the most common complications of falciparum malaria in nonimmune adults . Malarial acute renal failure (marf) can occur as an isolated complication or as a component of multiorgan involvement . Indeed, an association of arf and cm has been reported and found to cause relatively higher mortality rates than cm alone . Marf should be suspected when urine output falls to less than 400 ml/24 h or 20 ml / h despite adequate rehydration and the diagnosis is confirmed when serum creatinine exceeds 3 mg / dl (260 mol / l). The availability and provision of renal replacement therapy (rrt) as well as appropriate antimalarial chemotherapy has been shown to reduce marf associated mortalities and enhance the restoration of renal function . We are reporting a case of a 55-year - old male caucasian referred from morogoro regional referral hospital to the university of dodoma haemodialysis unit, with a 3-days history of acute onset of high - grade fever associated with episodes of nonprojectile vomiting with the vomitus being mostly comprised of recently eaten food . The fever was also associated with chills, myalgia, and loss of consciousness, with episodes of generalized tonic few hours following the fever onset, the patient developed shortness of breath with no cough . On the second day, while in the ward, he developed anuria (decrease in urine output), with 50 ml collected over 24 h that was soon followed by hiccups . The patient was visiting morogoro, tanzania for the first time and he had never been to any malaria endemic area before . He denied having chest pain, palpitations, diarrhea, bleeding from any site, and declared that he had not been exposed to sick contacts . At the morogoro regional referral hospital, he was given two doses of 600 mg quinine in 600 ml of 5% dextrose infusion . He was referred to the university of dodoma haemodialysis unit due to his nonimproving renal functioning (anuria). He had smoked one pack of cigarette per day for 30 years and he drank alcohol occasionally . However, he denied illicit or any kind of drug use, and he had no known allergies . Physical examination revealed that he was obtunded (gcs = 9/15), jaundiced, dyspneic, pale . Vitals: his blood pressure was 90/60 mm hg with no postural hypotension, his heart rate was 102 beats per minute . Pertinent findings on chest examination included fine crackles at the lung bases, with decreased vocal fremitus . His cardiovascular examination showed normal first and second heart sounds, with no jugular venous distention, murmurs, rubs, or gallops . Abdominal examination revealed normal bowel sounds without distension, tenderness to palpation, or organomegaly . Rectal examination showed normal rectal tone, heme - negative stool, and no masses . Additionally, an electrocardiogram was performed and results were remarkable for sinus tachycardia . Furthermore, laboratory analyses including complete blood cell count (cbc) with differential, complete metabolic panel, finger - stick blood glucose, blood slide (bs) for malaria parasites (mps), malaria rapid diagnostic test (mrdt), bilirubin levels, urine analysis, and lumbar puncture for cerebral spinal fluid (csf) were carried out . The cbc revealed a normal white blood cell (wbc) count without a left shift . He was found to be anemic, with a hemoglobin level of 11.9 g / dl, mean corpuscular volume of 87 fl, and an elevated erythrocyte sedimentation rate (esr) of 35 mm / h . The initial renal workup revealed serum creatinine of 1.33 mg / dl (117.24 mol / l), high serum urea of 30.66 mmol / l, serum potassium of 5.1 meq / l, and serum sodium of 140 meq / l . Mol / l, while direct bilirubin was 30 mol / l and random blood glucose was 8.8 . Microscopic examination of bs for mps showed 1200 trophozoites/200 wbc (4800 mps per l) and mrdt was positive for p.falciparum . Regarding urine analysis, sedimentation revealed muddy brown granular casts, specific gravity of 1.010, and sodium content of 42 (mmol / d). Csf analysis revealed normal findings and abdominal ultrasound showed normal - sized kidneys with no features of chronic kidney disease . On the basis of the foregoing findings, the case was diagnosed as complicated malaria with arf, pulmonary edema, cm and anemia, and the patient was scheduled for emergency dialysis . Double lumen catheter was inserted on the right femoral vein of the patient and hemodialysis was started and ran for 4 h on day one . The process was facilitated by pump pressure of 100 and no fluid removal from the body was allowed . The patient was also subjected to intramuscular (i / m) injection of artemether 160 mg bolus then 80 mg od 4/7, i / v frusemide 80 mg bid 3/7, i / v normal saline alternating with ringers lactate 6 l/12 h, and po paracetamol 1 g tid 5/7 . After three sessions of consecutive hemodialysis of 4 h per day, the patient was conscious, able to sit upright without support, and had urine output of 2400 ml/24 h. his serum creatinine, urea, and potassium were 0.43 mg / dl (37.8 mol / l), 4.72 mmol / l, and 3.5 meq / l, respectively . He was closely followed up after 2 weeks and 1 month and found to have normal and stable renal function . Marf is almost always caused by p.falciparum infection although renal involvement has also been reported in p. malariae, and recently in p. vivax infections . P. malariae has been associated with chronic progressive renal failure, while p. falciparum is usually associated with arf as in our patient . The incidence of marf worldwide is 14% and may be as high as 60% among nonimmune adults from nonendemic regions visiting endemic regions of the world as it is the case in our patient . In severe malaria, arf has been shown to be an important cause of mortality . In most cases, marf manifests clinically and pathologically as acute tubular necrosis as is reported in this case . The jaundice is usually biphasic with both conjugated and nonconjugated bilirubin elevated due to cholestasis and hemolysis, respectively . It has been shown elsewhere that early initiation of hemodialysis speeds recovery of renal function in the management of a patient with marf, . Hemodialysis has been shown to be more effective than peritoneal dialysis which is often readily available in sub - saharan africa . Hemodialysis is considered adequate when there is postdialysis decrease of blood urea nitrogen (bun) and serum creatinine by 50% from baseline . Our patient underwent three consecutive cycles and demonstrated complete recovery of renal function and postdialysis bun and serum creatinine more than 50% less than baseline values . The foregoing report conclusively outlines the importance of rapid recognition, proper case assessment, and early initiation of repeated sessions of hemodialysis in addition to fluid replacement and appropriate antimalarial therapy for early recovery of renal function in marf . A copy of the written consent is available for review by the editor - in - chief of this journal.
Over the past few decades, the advancement in breast screening and management has increased the overall survival in women with breast cancer . But this in turn, exposes them to the risk of developing new primary malignancies or metastases . The metastatic involvement of gastrointestinal (gi) tract is rare and that of the rectum even rarer . Even though lobular carcinoma constitutes only 10% of breast cancers, it is the commonest breast cancer metastasizing to the colon and rectum (1). These metastatic lesions can lead to diagnostic challenge for clinicians as they can mimic primary colo - rectal cancer due to the lack of diagnostic signs . We report a rather unusual case of metastatic invasive ductal carcinoma masquerading as primary rectal cancer . A 60 year old woman presented with a suspicious lump in her left breast in early 1996 and underwent open diagnostic surgical excision . She was found to have a moderately differentiated invasive ductal carcinoma measuring up to 20 mm in its greatest diameter extending less than 1 mm from the diagnostic excision margin . Subsequently, she underwent left mastectomy and axillary node clearance which failed to show any residual invasive cancer . However, there was residual high grade cribriform dcis present adjacent to the diagnostic surgical excision cavity in the mastectomy specimen . There was no evidence of vascular invasion and none of the seven lymph nodes excised contained any metastatic carcinoma [g2t1n0mx]. Since her cancer was oestrogen receptor (er) positive, she was started on tamoxifen post - operatively . She presented 2 years later with localised pain in her right knee and bone scan revealed an isolated bony metastasis in the right proximal tibia . This was managed using palliative radiotherapy to control the symptoms and her adjuvant hormonal therapy was switched to anastrazole (astrazeneca plc, london, united kingdom). She neither developed any loco - regional recurrence nor had any progression of her metastatic disease for the next 9 years . During this period, she underwent annual mammographic and clinical follow - up with her adjuvant hormonal treatment being stopped in 2005 after clinical consultation . In early 2007, she presented with ongoing history of fresh rectal bleeding, alternating bowel habit and mucus discharge per rectum . Abdomen was soft, non - tender without any organomegaly and digital rectal examination revealed a palpable tumour at 6 o'clock position . Fungating rectal tumour on colonoscopy she underwent colonoscopy, which revealed a fungating tumour at 10 cm from the anal verge in the rectum and rest of the colon was normal (figure 1). The histopathological examination of the biopsy revealed a poorly differentiated invasive adenocarcinoma (figure 2). Tumour overlying the large bowel mucosa with the line demarcating the tumour edge with arrow pointing at the body of the tumour (magnification4). Considering her previous history of invasive breast cancer, pathologist performed immunohistochemistry of the colonic biopsies . The tumour cells were strongly positive for cytokeratin (ck) 7 with focal positivity for er but were negative for both carcino embryonic antigen (cea) and ck 20 . The above immunohistochemistry profile with previous history of breast cancer favoured a metastatic breast origin over a primary colonic cancer even though the clinical and colonoscopic findings were not typical . The staging ct scan did nt show any evidence of metastasis and the mri scan revealed a locally advanced tumour with involvement of mesorectal facial margins with possible involvement of the peritoneum locally (figure 3). The bone scan failed to reveal any evidence of active metastatic disease . Mri scan . Arrow pointing to the localized tumour in the rectum with no evidence of widespread metastasis after discussing in the multi - disciplinary team meeting (mdt), it was decided to treat the tumour with a short course of pre - operative radiotherapy . The histopathological examination of the resected tumour showed poorly differentiated metastatic adenocarcinoma with predominantly solid, but with focal glandular growth pattern and large areas of necrosis . The tumour involved full thickness of the bowel but the serosal surface and the resection margins were free of cancer . There was no evidence of metastatic cancer in any of the twenty two lymph nodes excised . Immunohistochemistry showed tumour cells to be negative for cea and ck 20, but was strongly positive for ck7 (figure 4 & 5). The tumour also showed focal positivity for er and was negative for both progesterone receptor (pr) and human epidermal growth factor receptor 2 (her2). After comparing the morphological and immunohistochemistry charachteristics of the primary breast cancer and rectal tumour, the pathologist concluded that the features were consistent with a primary breast cancer metastasising to the rectum . She underwent an uneventful post - operative recovery and remains disease free during her follow - up until now . Ck 20 immunohistochemistry positive normal large bowel mucosa with the adenocarcinoma being negative (magnification 4). In the largest clinical series till date, only <1% (17 out of 2604 patients followed over 18 years) of the patients with breast cancer were found to have gastro - intestinal [gi] metastasis (2). Information gathered from autopsy series shows small intestine being the commonest site of metastasis (28%) followed by oesophagus (25%), stomach (25%), colon (19%) and rectum (4%). The commonest route of metastasis to the gi tract is through the haematogenous route followed by lymphatic and peritoneal spread . Even though there are a few case reports of lobular carcinoma metastasizing to the rectum, there are only 3 case reports in the literature of invasive ductal carcinoma metastasizing to the rectum till date (3 - 5). As a clinician the main challenge lies in the differentiation of such metastatic lesions from primary colo - rectal cancer as the clinical presentation of metastatic lesion can be non - specific and mimic a primary cancer as in the present case . The radiological investigations like, ct scan and double contrast barium enema are helpful in localizing the lesion but cannot differentiate between them . On endoscopy, the colo - rectal metastasis appear as diffuse thickening of the colonic wall mimicking linitis plastica or like in crohn s disease with ulcerated or nodular areas rather than as a solitary, discrete mass seen in primary colonic lesion (1). But the differentiation was made more challenging in our case, as colonoscopy revealed a solitary fungating mass arising from the rectal mucosa mimicking a primary rectal cancer . Histopathological examination by itself may not be conclusive as the tumour invasion is primarily in the sub - serosa, so unless deep endoscopic biopsies are available the diagnosis will be made only after resection of the tumour . But immunohistochemical markers such as er, pr, gross cystic disease fluid protein (gcdfp-15), and differential expression of ck7 and ck20 can facilitate an accurate diagnosis as seen in the present report . Systemic treatment with chemotherapy and/or hormonal therapy is usually employed in patients with confirmed diagnosis of gastrointestinal metastasis (1). The role of surgery is limited to palliation or in patients presenting acutely with obstruction or perforation of the hollow viscus . The lack of concordance between the clinical, radiological and colonoscopic findings with that of the histological findings resulted in our patient being managed with pre - operative radiotherapy followed with low anterior resection . The prognosis of gi metastasis from primary breast cancer is poor with few patients surviving beyond two years, although survival up to nine years has been reported . Our patient remains well and disease free four years after treatment for her rectal metastasis . Given the increased survival of breast cancer patients due to the early diagnosis through breast screening and better management with current therapeutic regimens, more unusual presentations of metastatic disease, including involvement of the gastrointestinal tract should be anticipated . So the recognition of this rare entity (rectal metastasis from primary breast cancer) is important, as presentation resembles that of primary rectal carcinoma and differentiation is vital as different therapeutic modalities may be appropriate.
Early in human life, this tissue is populated by two cell types: notochordal cells (ncs) and np cells (npcs). Ncs naturally exist in clusters and contain large vesicles, whereas npcs are small, nonclustered, and chondrocyte - like cells . Despite however, ncs can be distinguished from npcs by elevated brachyury (t) and cytokeratin 8, 18, and 19 expression . In humans, ncs disappear before adolescence, and thereafter the onset of intervertebral disk degeneration, starting in the np, has been observed . Certain species keep their ncs throughout adulthood and are known to experience the incidence and onset of disk degeneration much later in life . Consequently, it has been proposed that ncs might be involved in the maintenance and regeneration of healthy np tissue . It has been shown that ncs can indeed increase the proteoglycan production by npcs in alginate coculture for up to 14 days . When npcs were cultured in medium conditioned by ncs (for 4 days), however, it is yet unknown if ncs can have a direct regenerative potential on npcs in their native environment . This environment is characterized by low ph, oxygen, and glucose levels, as well as a high osmolarity . These factors, but also the composition of the extracellular matrix (proteoglycans, collagens), can affect the cell response to an exogenous stimulation . The aim of this study was to investigate the effects of direct nc therapy in npc - populated bovine np explants, using an np culture model that was previously developed in our group . Porcine spines (4 donors; 8 weeks old) were obtained from the slaughterhouse, in accordance with local regulations (no approval from an ethical board required), and stored overnight at 4c . The thoracic and lumbar intervertebral disks were transversally opened to harvest the np tissue under sterile conditions . The tissue was pooled per donor and digested in 0.1% pronase protease (calbiochem, darmstadt, germany) in high glucose (4.5 g / l) dulbecco's modified eagle's medium (hgdmem; invitrogen, bleiswijk, the netherlands) + 1% penicillin / streptomycin (pen / strep; lonza, basel, switzerland) for 1 hour at 37c and in 0.025% collagenase p (roche, basel, switzerland) in hgdmem + 10% fetal bovine serum (fbs; invitrogen) + 1% pen / strep overnight at 37c . After filtration on a 40-m cell strainer (bd biosciences, breda, the netherlands), cells and cell clusters 40 m were stained with 5 m of carboxyfluorescein diacetate succinimidyl ester (cfda - se) according to the manufacturer's protocol (vybrant, invitrogen) for cell tracking for the duration of the experiment . Bovine tails (12 donors; 24 months old) were obtained from the slaughterhouse in accordance with local regulations (no approval from an ethical board required). Thirty - six np samples (c2c5) were harvested, as described previously by van dijk et al . Samples of different levels were equally distributed among the native and culture groups (n = 12 per group). The np samples from the culture groups were individually put in dialysis tubing (15-kda molecular weight cut off, spectra - por, rancho dominquez, california, united states) and subsequently injected into the middle of the tissue with a 23-gauge needle (hamilton, bonaduz, switzerland). The injection groups were: sham (10-l injection of phosphate - buffered saline [pbs]) and nc (10-l injection of 250,000 cfda - se - stained porcine ncs in pbs). After the injection, the dialysis tubing was replaced and closed with a custom - made clip . The np samples were cultured in hypertonic polyethylene glycol (peg) medium (13.3% w / v 20 kda peg [sigma, zwijndrecht, the netherlands]; 8.3 g / l dmem + 3.7 g / l sodium bicarbonate + 50 mg / l ascorbic acid + 15.9 mg / l phenol red [all sigma] + 1 g / l glucose + 10% fbs [both invitrogen] + 2% l - glutamine + 1% sodium pyruvate + 1% pen / strep [all lonza]; ph 7.4) under hypoxic conditions (37c, 5% o2, 5% co2). Samples were cultured for 42 days, and the medium was changed three times a week . Native samples (n = 12 donors) were harvested at day 0, and each injection group was analyzed after 42 days of culture (n = 12 donors for each group). Samples were cut in half, and for each of the four assays (cell viability, biochemical assays and water content, gene expression, and histology), six half samples were used (corresponding to six donors for each assay). Np samples were incubated in 10 m calcein blue - am and 10 m propidium iodide (both molecular probes, invitrogen) in pbs for 2 hours . The viable cells (= 730 nm), dead cells (= 535 nm), and porcine nc location and morphology (= 488 nm; cfda - se staining) were imaged with a confocal microscope (clsm 510 meta, zeiss, mnchen, germany). The viability was assessed both near the center and the outer regions of the explant . The np samples were weighed (wet weight) and stored at 30c until further use . After overnight lyophilization (freezone 2.5, labconco, kansas city, missouri, united states), the sample weights were measured (dry weight). The water content was calculated as (wet weight dry weight)/wet weight . The lyophilized samples were digested overnight at 60c in a papain solution (100 mm phosphate buffer, 5 mm l - cysteine, 5 mm ethylenediaminetetraacetic acid, and 140 g / ml papain, all from sigma). The amount of dna per sample was measured with a hoechst 33528 assay as described by cesarone et al, using a standard curve of double - stranded calf thymus dna (sigma). The amount of sulfated glycosaminoglycans per sample relates to the proteoglycan content and was measured with a 1,9-dimethylmethylene blue assay as described by farndale et al, based on a standard curve of chondroitin - sulfate from shark cartilage (sigma). The amount of hydroxyproline relates to the collagen content and was measured with a chloramine - t assay as described by huszar et al, based on a standard curve of trans-4-hydroxyproline (sigma). Bovine np samples were snap - frozen in liquid n2 and stored at 80c for rna isolation . Briefly, samples were disrupted with a mikro - dismembrator (sartorius, goettingen, germany). Rna was isolated from the disrupted samples using trizol (invitrogen) and purified on a spin column (rneasy mini kit, qiagen, venlo, the netherlands). The rna concentration and purity were measured on a spectrophotometer (nd-1000, isogen, de meern, the netherlands). Of each sample, 75 ng of mrna was reverse transcribed to cdna (superscript vilo kit, invitrogen), and gene expression was determined by quantitative polymerase chain reaction (cfx384, biorad, veenendaal, the netherlands). 18s was selected as the endogenous reference gene because it was found to be more stable than gapdh and rpl13a in all groups . The genes of interest were collagen type i (col1a1), collagen type ii (col2a1), and aggrecan (acan; table 1). Relative quantification was determined with the comparative 2 method, with the expression of the gene of interest relative to the expression of the reference gene 18s . List of primers for real - time quantitative polymerase chain reaction primer design ltd (southampton, united kingdom). Designed with beacon designer software (premier biosoft, palo alto, california, united states) and ordered from sigma (zwijndrecht, the netherlands). Compound (sakuratek, zoeterwoude, the netherlands), slash frozen in isopentane at 80c, and stored at 30c until further use . From each sample, 10-m thick cryosections were cut (microm hm 550, thermo fisher scientific, kalamazoo, united kingdom) and mounted on polysine adhesion slides (thermo scientific). The slides were stained with weigert's hematoxylin for cell nuclei and eosin for eosinophilic matrix . Images were taken with a bright - field microscope (observer, zeiss). With r - project software (version 3.0.2), levene test and shapiro - wilk test were used to test for homogeneity of variance and normality, respectively . The biochemical data, both homogeneous and normally distributed, was analyzed with a one - way analysis of variance followed by a bonferroni adjusted post hoc independent test . The gene expression data was found to be nonnormally distributed and was analyzed with kruskal - wallis test followed by a bonferroni corrected post hoc mann - whitney u test . Statistical significance was assumed for p <0.05 . Porcine spines (4 donors; 8 weeks old) were obtained from the slaughterhouse, in accordance with local regulations (no approval from an ethical board required), and stored overnight at 4c . The thoracic and lumbar intervertebral disks were transversally opened to harvest the np tissue under sterile conditions . The tissue was pooled per donor and digested in 0.1% pronase protease (calbiochem, darmstadt, germany) in high glucose (4.5 g / l) dulbecco's modified eagle's medium (hgdmem; invitrogen, bleiswijk, the netherlands) + 1% penicillin / streptomycin (pen / strep; lonza, basel, switzerland) for 1 hour at 37c and in 0.025% collagenase p (roche, basel, switzerland) in hgdmem + 10% fetal bovine serum (fbs; invitrogen) + 1% pen / strep overnight at 37c . After filtration on a 40-m cell strainer (bd biosciences, breda, the netherlands), cells and cell clusters 40 m were stained with 5 m of carboxyfluorescein diacetate succinimidyl ester (cfda - se) according to the manufacturer's protocol (vybrant, invitrogen) for cell tracking for the duration of the experiment . Bovine tails (12 donors; 24 months old) were obtained from the slaughterhouse in accordance with local regulations (no approval from an ethical board required). Thirty - six np samples (c2c5) were harvested, as described previously by van dijk et al . Samples of different levels were equally distributed among the native and culture groups (n = 12 per group). The np samples from the culture groups were individually put in dialysis tubing (15-kda molecular weight cut off, spectra - por, rancho dominquez, california, united states) and subsequently injected into the middle of the tissue with a 23-gauge needle (hamilton, bonaduz, switzerland). The injection groups were: sham (10-l injection of phosphate - buffered saline [pbs]) and nc (10-l injection of 250,000 cfda - se - stained porcine ncs in pbs). After the injection, the dialysis tubing was replaced and closed with a custom - made clip . The np samples were cultured in hypertonic polyethylene glycol (peg) medium (13.3% w / v 20 kda peg [sigma, zwijndrecht, the netherlands]; 8.3 g / l dmem + 3.7 g / l sodium bicarbonate + 50 mg / l ascorbic acid + 15.9 mg / l phenol red [all sigma] + 1 g / l glucose + 10% fbs [both invitrogen] + 2% l - glutamine + 1% sodium pyruvate + 1% pen / strep [all lonza]; ph 7.4) under hypoxic conditions (37c, 5% o2, 5% co2). Samples were cultured for 42 days, and the medium was changed three times a week . Native samples (n = 12 donors) were harvested at day 0, and each injection group was analyzed after 42 days of culture (n = 12 donors for each group). Samples were cut in half, and for each of the four assays (cell viability, biochemical assays and water content, gene expression, and histology), six half samples were used (corresponding to six donors for each assay). Np samples were incubated in 10 m calcein blue - am and 10 m propidium iodide (both molecular probes, invitrogen) in pbs for 2 hours . The viable cells (= 730 nm), dead cells (= 535 nm), and porcine nc location and morphology (= 488 nm; cfda - se staining) were imaged with a confocal microscope (clsm 510 meta, zeiss, mnchen, germany). The viability was assessed both near the center and the outer regions of the explant . The np samples were weighed (wet weight) and stored at 30c until further use . After overnight lyophilization (freezone 2.5, labconco, kansas city, missouri, united states), the sample weights were measured (dry weight). The water content was calculated as (wet weight dry weight)/wet weight . The lyophilized samples were digested overnight at 60c in a papain solution (100 mm phosphate buffer, 5 mm l - cysteine, 5 mm ethylenediaminetetraacetic acid, and 140 g / ml papain, all from sigma). The amount of dna per sample was measured with a hoechst 33528 assay as described by cesarone et al, using a standard curve of double - stranded calf thymus dna (sigma). The amount of sulfated glycosaminoglycans per sample relates to the proteoglycan content and was measured with a 1,9-dimethylmethylene blue assay as described by farndale et al, based on a standard curve of chondroitin - sulfate from shark cartilage (sigma). The amount of hydroxyproline relates to the collagen content and was measured with a chloramine - t assay as described by huszar et al, based on a standard curve of trans-4-hydroxyproline (sigma). Bovine np samples were snap - frozen in liquid n2 and stored at 80c for rna isolation . Briefly, samples were disrupted with a mikro - dismembrator (sartorius, goettingen, germany). Rna was isolated from the disrupted samples using trizol (invitrogen) and purified on a spin column (rneasy mini kit, qiagen, venlo, the netherlands). The rna concentration and purity were measured on a spectrophotometer (nd-1000, isogen, de meern, the netherlands). Of each sample, 75 ng of mrna was reverse transcribed to cdna (superscript vilo kit, invitrogen), and gene expression was determined by quantitative polymerase chain reaction (cfx384, biorad, veenendaal, the netherlands). 18s was selected as the endogenous reference gene because it was found to be more stable than gapdh and rpl13a in all groups . The genes of interest were collagen type i (col1a1), collagen type ii (col2a1), and aggrecan (acan; table 1). Relative quantification was determined with the comparative 2 method, with the expression of the gene of interest relative to the expression of the reference gene 18s . List of primers for real - time quantitative polymerase chain reaction primer design ltd (southampton, united kingdom). Designed with beacon designer software (premier biosoft, palo alto, california, united states) and ordered from sigma (zwijndrecht, the netherlands). Compound (sakuratek, zoeterwoude, the netherlands), slash frozen in isopentane at 80c, and stored at 30c until further use . From each sample, 10-m thick cryosections were cut (microm hm 550, thermo fisher scientific, kalamazoo, united kingdom) and mounted on polysine adhesion slides (thermo scientific). The slides were stained with weigert's hematoxylin for cell nuclei and eosin for eosinophilic matrix . Np samples were incubated in 10 m calcein blue - am and 10 m propidium iodide (both molecular probes, invitrogen) in pbs for 2 hours . The viable cells (= 730 nm), dead cells (= 535 nm), and porcine nc location and morphology (= 488 nm; cfda - se staining) were imaged with a confocal microscope (clsm 510 meta, zeiss, mnchen, germany). The viability was assessed both near the center and the outer regions of the explant . The np samples were weighed (wet weight) and stored at 30c until further use . After overnight lyophilization (freezone 2.5, labconco, kansas city, missouri, united states), the sample weights were measured (dry weight). The water content was calculated as (wet weight dry weight)/wet weight . The lyophilized samples were digested overnight at 60c in a papain solution (100 mm phosphate buffer, 5 mm l - cysteine, 5 mm ethylenediaminetetraacetic acid, and 140 g / ml papain, all from sigma). The amount of dna per sample was measured with a hoechst 33528 assay as described by cesarone et al, using a standard curve of double - stranded calf thymus dna (sigma). The amount of sulfated glycosaminoglycans per sample relates to the proteoglycan content and was measured with a 1,9-dimethylmethylene blue assay as described by farndale et al, based on a standard curve of chondroitin - sulfate from shark cartilage (sigma). The amount of hydroxyproline relates to the collagen content and was measured with a chloramine - t assay as described by huszar et al, based on a standard curve of trans-4-hydroxyproline (sigma). Bovine np samples were snap - frozen in liquid n2 and stored at 80c for rna isolation . Briefly, samples were disrupted with a mikro - dismembrator (sartorius, goettingen, germany). Rna was isolated from the disrupted samples using trizol (invitrogen) and purified on a spin column (rneasy mini kit, qiagen, venlo, the netherlands). The rna concentration and purity were measured on a spectrophotometer (nd-1000, isogen, de meern, the netherlands). Of each sample, 75 ng of mrna was reverse transcribed to cdna (superscript vilo kit, invitrogen), and gene expression was determined by quantitative polymerase chain reaction (cfx384, biorad, veenendaal, the netherlands). 18s was selected as the endogenous reference gene because it was found to be more stable than gapdh and rpl13a in all groups . The genes of interest were collagen type i (col1a1), collagen type ii (col2a1), and aggrecan (acan; table 1). Relative quantification was determined with the comparative 2 method, with the expression of the gene of interest relative to the expression of the reference gene 18s . List of primers for real - time quantitative polymerase chain reaction primer design ltd (southampton, united kingdom). Designed with beacon designer software (premier biosoft, palo alto, california, united states) and ordered from sigma (zwijndrecht, the netherlands). Bovine np samples were embedded in tissue - tek o.c.t . Compound (sakuratek, zoeterwoude, the netherlands), slash frozen in isopentane at 80c, and stored at 30c until further use . From each sample, 10-m thick cryosections were cut (microm hm 550, thermo fisher scientific, kalamazoo, united kingdom) and mounted on polysine adhesion slides (thermo scientific). The slides were stained with weigert's hematoxylin for cell nuclei and eosin for eosinophilic matrix . With r - project software (version 3.0.2), levene test and shapiro - wilk test were used to test for homogeneity of variance and normality, respectively . The biochemical data, both homogeneous and normally distributed, was analyzed with a one - way analysis of variance followed by a bonferroni adjusted post hoc independent test . The gene expression data was found to be nonnormally distributed and was analyzed with kruskal - wallis test followed by a bonferroni corrected post hoc mann - whitney u test . After 42 days of culture, the distribution and morphology of the npcs in both culture groups were similar to native tissue (fig . Viable ncs were observed after culture (figs . 1 and 2b), but the vacuolar structures were much smaller than before injection (fig . 2a). Staining of nucleus pulposus tissue on (a) day 0 and day 42 in the (b) sham or (c) notochordal cell injected group . Cytoplasm and matrix are stained pink (eosin), cell nuclei black (hematoxylin). Notochordal cells (ncs) (a) prior to injection and (b) in nucleus pulposus tissue after 42 days of culture . Cytoplasm of ncs is stained in green (cell tracker carboxyfluorescein diacetate succinimidyl ester (cfda - se)), cytoplasm of all cells in blue (calcein blue - am; only in b), and nuclei of dead cells in red (propidium iodide). As expected, dna content in the nc group was larger than in the native samples at day 0 due to the injection of ncs (p = 0.016), but that of the sham - injected group was not significantly different (fig . The collagen content remained unchanged, but gene expression data indicates that there was a shift in the type of collagen: col2a1 decreased in both groups (p = 0.003 nc, p = 0.004 sham), whereas col1a1 increased statistically only in the sham group (p = 0.01; fig . The water content (a) per wet weight, and (b) sulfated glycosaminoglycans (sgag), (c) dna, and (d) hydroxyproline (hyp) content per dry weight on day 0 (native) and day 42 (sham or notochordal cell [nc] injection). Values are mean standard deviation; n = 6 (sham group: n = 5). * p <0.05 . Gene expression of (a) aggrecan (acan), (b) collagen type ii (col2a1), and (c) collagen type i (col1a1) relative to 18s on day 0 (native) and day 42 (sham or notochordal cell [nc] injection). After 42 days of culture, the distribution and morphology of the npcs in both culture groups were similar to native tissue (fig . Viable ncs were observed after culture (figs . 1 and 2b), but the vacuolar structures were much smaller than before injection (fig . 2a). Staining of nucleus pulposus tissue on (a) day 0 and day 42 in the (b) sham or (c) notochordal cell injected group . Cytoplasm and matrix are stained pink (eosin), cell nuclei black (hematoxylin). Notochordal cells (ncs) (a) prior to injection and (b) in nucleus pulposus tissue after 42 days of culture . Cytoplasm of ncs is stained in green (cell tracker carboxyfluorescein diacetate succinimidyl ester (cfda - se)), cytoplasm of all cells in blue (calcein blue - am; only in b), and nuclei of dead cells in red (propidium iodide). As expected, dna content in the nc group was larger than in the native samples at day 0 due to the injection of ncs (p = 0.016), but that of the sham - injected group was not significantly different (fig . The collagen content remained unchanged, but gene expression data indicates that there was a shift in the type of collagen: col2a1 decreased in both groups (p = 0.003 nc, p = 0.004 sham), whereas col1a1 increased statistically only in the sham group (p = 0.01; fig . The water content (a) per wet weight, and (b) sulfated glycosaminoglycans (sgag), (c) dna, and (d) hydroxyproline (hyp) content per dry weight on day 0 (native) and day 42 (sham or notochordal cell [nc] injection). Values are mean standard deviation; n = 6 (sham group: n = 5). Gene expression of (a) aggrecan (acan), (b) collagen type ii (col2a1), and (c) collagen type i (col1a1) relative to 18s on day 0 (native) and day 42 (sham or notochordal cell [nc] injection). Values are mean standard deviation; n = 6 . * p <0.05 this study is the first to show that translating the results of in vitro coculture of ncs / npcs to in situ repair in tissue is not straightforward . Whereas previous studies have shown that ncs were able to stimulate extracellular matrix production and related gene expression by isolated npcs, this effect was not observed when ncs were injected in np explants . This discrepancy may be explained by two mechanisms: the different ratio of nc to npc used or the initial presence of a proteoglycan - rich extracellular matrix . First, the nc - to - npc ratio in the current study was approximately 20:80, whereas this ratio was 50:50 in previous direct cell cocultures . In a therapeutic perspective, however, a ratio of 50:50 is not realistic: due to the nutrient limitations of the intervertebral disk in vivo, multiplying the local cell density by 2 may lead to the death of both endogenous and exogenous cell populations . Moreover, a ratio of 30:70, close to what was used here, was also reported to have a positive, even if milder, effect on npcs . Second, in the in vitro studies, the npcs were isolated from their extracellular matrix prior to coculture in alginate . Because the matrix produced during culture is compared with the initial amount, which is small (or even nonexistent) in alginate cultures after cell isolation, production of proteoglycans will appear as large increases in matrix production . Contrarily, the initial amount of matrix in the explants is high, and the matrix production per cell has to increase to a great extent to change the total matrix content . Because of this difference, it is unclear how the matrix production rates by npcs in alginate cocultures relate to those in tissue . However, it has been observed that the expression of acan and col2a1 decreased significantly after npc isolation, but gradually increased in three - dimensional culture (agarose) until reaching native gene expression levels by day 25 of culture . This result indicates that the isolation procedure and three - dimensional culture of npcs have an effect on their phenotype and metabolism . The proteoglycan production by npcs in three - dimensional culture (alginate and agarose) also increased in a time - dependent manner, but was not compared with the native proteoglycan production rate . The native expression of brachyury (t), cytokeratins 8 and 18, acan, and col2a1 by ncs decreased significantly after 1 day of culture in alginate beads . In addition, ncs are sensitive to the culture conditions: ascorbic acid, physiologic osmolarity, medium type, and hypoxia contribute to maintenance of their phenotype, whereas a ph <7.2, both very low and high glucose concentrations, and fbs in the culture medium (unpublished data from our group) are detrimental to the phenotype . The presence of fbs in the medium during the 42 days of culture could explain the disappearance of nc vesicles by the end of the culture . However, the morphologic changes can also be attributed to the isolation of ncs from their natural environment and culture in a mature np . The change in morphology is probably coupled to a change in functionality of the ncs . If the morphology and gene expression profiles of ncs change gradually during culture, this result could explain that the regulatory effect of ncs is stronger in short - term (3 days) than in long - term (> 14 days) coculture . In this respect, the use of nc - conditioned medium may be a solution for long - term in situ cultures . Medium conditioned by ncs for 4 days has indeed been shown to increase the proteoglycan production by npcs in in vitro cultures, in levels even higher than in cocultures with direct cell cell contact . . The administration of soluble factors in fact could be more attractive than exogenous cell injection as smaller - gauge needles can be used . Needle punctures of 25 gauge (required for cell injection) have been shown to have a detrimental effect on disk biomechanics . The np explants were cultured in hypertonic peg, as described by van dijk et al . This culture system previously showed a stable biochemical content and col2a1 gene expression during long - term culture (42 days), but native acan and col1a1 expression were not maintained . In the current study, the expression of the latter genes did not decrease, which may be due to two differences in culture conditions: (1) injection of pbs prior to culture, or (2) the ph of the culture medium, which was not adjusted to 7.1 in the current study . It is possible that the injection of pbs in the tissue triggered a change in gene expression, but an anabolic response would not be expected . Furthermore, it has been described that the proteoglycan synthesis by npcs is higher at ph 7.1 than at 7.4, which is not reflected in the proteoglycan content and related acan gene expression in the current study compared with the previous one . Improved np culture systems are currently available for future experiments . Although the results from this study show that direct injections of ncs are not effective for np regeneration, this finding should be interpreted with care for translation to the human situation . First of all, the np tissue used in this study corresponded to human grade ii on the thompson grading scale . Although the mechanical loading of bovine caudal tissue may be more similar to that in the human cervical rather than lumbar spine, the biochemical composition and cell population of bovine and human grade ii np tissue have been shown to be similar . Although early stage regeneration would theoretically be ideal, the regenerative potential of these cells may be less than for cells of a higher grade of degeneration . Second, although the issue of interspecies size differences was avoided by using an np explant culture, such a system also introduced drawbacks . Interplay with the surrounding tissues was not possible, and certain physiologic factors (i.e., dynamic loading, limited nutrient supply) could not be simulated in the current system . Molecular - based regenerative therapies are aimed for treatment of nps with moderate degeneration . In this stage, the resident cells are still chondrocytic in nature, able to produce matrix, and the degenerative changes are not yet insurmountable . Cell - based regenerative therapies are considered applicable for more advanced degeneration, where the depleted, senescent, or dedifferentiated resident cell population would need additional help to replenish the extracellular matrix . However, the sensitivity of detection methods for moderate disk degeneration is currently insufficient, and more importantly, our inability to provide accurate prognosis for aggressive symptomatic disk degeneration is actually a challenge to introduce early preventive treatments . Certainly, as more sensitive diagnostic tools will be developed, the selection of patients for disk regeneration will become clearer . Contrary to alginate coculture, no anabolic response was observed when ncs were injected into np explants, which may be due to the observed changed morphology of the ncs during the 42 days of culture where they became more similar in appearance to resident npcs . Therefore, considering the results with nc - conditioned medium, it may be a more promising alternative for in situ stimulation of npcs.
Therefore, they continuously remain of interest for non - linear optics, nanoelectronics, biotechnology and many other fields of science . From the point of view of material science, interaction - induced electric properties for hydrogen - bonded systems however, systems that become relevant as novel materials are usually composed of more than several light atoms and therefore high - level calculations of their properties are prohibitively expensive . The great abundance of the various available dft functionals allows to extend the investigations to the systems of hundreds of atoms and the wide range of analyzed properties . It is known that the molecular electric properties can be reproduced by dft methods with good accuracy [112]. However, one should bear in mind that for several classes of systems of importance to the non - linear optics, for instance long -conjugated polymer chains as well as push - pull systems with significant charge transfer, such calculations lead to significant deterioration in the accuracy of the results [1315]. Still, not much care has been devoted to the interaction - induced increments to dipole moments and (hyper)polarizabilities calculated beyond the wave function theory ., various dft functionals have been tested for the series of hydrogen - bonded dimers: water dimer, hf dimer and h2 co hf . Although there is no unambiguous answer to the question which functional is best for interaction - induced electric properties, it is quite easy to reproduce the ccsd(t) results for investigated systems with dft methods with satisfactory accuracy . Regarding the gathered information, further studies seem necessary in order to generalize such an observation . The aim of the present study is to verify the applicability of the dft formalism also for the interaction - induced electric properties of the longer hydrogen - bonded chains . The hf linear chain, h2co crystal structure and h3n (hf)n complex are investigated up to five units in the system . The chosen structures were previously investigated at an angle of the many - body contributions to the (non)linear electric response with second order mller - plesset perturbation theory and the many - body interactions in these systems proved to be significant [21, 22]. The performance of the dft functionals is tested against the ccsd(t) data that provide the reference level . In order to keep the consistency with the previous study, the same exchange - correlation functionals have been applied here: b3lyp [2326] and pbe0 as conventional hybrids, lc - blyp as a long - range corrected functional and m06 - 2x hybrid meta functional . Additionally, this set was appended by the b3lyp long - range corrected modification (cam - b3lyp) for which we expect to improve the accuracy of the results toward reference data . The subject of the present study are hydrogen - bonded complexes, namely: linear hf(hf)n, crystalline h2co(h2co)n and linear h3n(hf)n, for which n = 14 . The structures of linear hf(hf)n and h3n(hf)n chains have been obtained based on gas - phase monomer geometries optimized at the mp2/6 - 31g(d, p) level of theory . Moreover, the intermonomer distance which corresponds to the separation in the linear dimer has been optimized at the very same level of theory . On the other hand, the geometry of the h2co(h2co)n chain refers to that found in the formaldehyde crystal . All calculations of the static electric molecular properties were carried out at the scf - hf, mp2, ccsd(t), b3lyp, pbe0, lc - blyp, m06 - 2x and cam - b3lyp level of theory . Values of the electric dipole (hyper)polarizability tensors have been obtained using the finite field (ff) method [33, 34] by numerical differentiation of energy with respect to external electric field (f). Relying on our previous research [21, 22] the electric field of the strength 0.001 a.u . Has been chosen for further calculations to ensure the numerical stability of the ff procedure . Interaction - induced properties p for the investigated systems were obtained in a supermolecular manner:1\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\delta p = {{p}_{{{12} \ldots n}}} - \sum\limits_{{i = 1}}^n {p_i,} $$\end{document}where p12 n and pi stands for the magnitude of the property for the n - body system and its constituents, respectively . In order to make the consistent comparison of electric properties for the whole set of studied systems, the following values of the electric properties are discussed:2\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\left\| \mu \right\| = \sqrt {{\mu_x^2 {+} \mu_y^2 {+} \mu_z^2,}} $$\end{document}3\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\alpha} _ 0} = \frac{1}{3}\left ({{{\alpha} _ {{xx}}} + {{\alpha} _ {{yy}}} + {{\alpha} _ {{zz}}}} \right), $$\end{document}4\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\beta} _ {\mu}} = \frac{{{{\beta} _ x}{{\mu} _ x} + {{\beta} _ y}{{\mu} _ y} + {{\beta} _ z}{{\mu} _ z}}}{{\left\| \mu \right\|}}, $$\end{document}where5\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta _ {i} = \frac{3} {5} \cdot {\sum\limits_{j = x, y, z} {\beta _ {{ijj}}}} $$\end{document}and the kleinman symmetry is satisfied . The values of static electric properties obtained within the supermolecular approach together with basis set of finite size might suffer from the basis set superposition error [35, 36]. Due to the fact that the first - order hyperpolarizability is the most sensitive to the choice of the basis set of all the properties analyzed here [35, 36], the correction for bsse has been included according to the site - site function counterpoise scheme . The total interaction - induced counterpoise - corrected electric property totalp has been decomposed into:6\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${{\delta} _ {{total}}}{{p}^{{cp}}} = \sum\limits_{{i <j}} {{{\delta} _ {{ij}}}} p\left ({1 \ldots n} \right) + \sum\limits_{{i <j <k}} {{{\delta} _ {{ijk}}}} p\left ({1 \ldots n} \right) + \ldots + {{\delta} _ {{1 \ldots n}}}p\left ({1 \ldots n} \right), $$\end{document}where the first summation includes all the two - body terms ijp(1 the relative error of the investigated properties in different methods has been estimated with respect to the ccsd(t) calculations via:7\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\delta \left [\% \right] = \frac{{\delta {{p}^{{method}}} - \delta {{p}^{{ccsd(t)}}}}}{{\delta {{p}^{{ccsd(t)}}}}} \cdot 100\% . $$\end{document} similarly, the relative error for the bsse - uncorrected data has been predicted as:8\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$bsse\left [\% \right] = \frac{{\delta {{p}^{{uncorr}}} - \delta {{p}^{{corr}}}}}{{\delta {{p}^{{corr}}}}} \cdot 100\% . $$\end{document} all calculations have been performed with gamess - us and gaussian09 program packages with the aug - cc - pvdz basis set [40, 41]. Tables 1, 2 and 3 present the values of the electric properties and their interaction - induced contributions for all the investigated systems in different approaches . The corresponding relative errors ([%]) for \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\delta \left\| \mu \right\|(0) $$\end{document}, 0(0), and (0) are plotted in figs . 2, 3 and 4, respectively . For comparison the electric properties of the isolated units (hf, h2co and h3n) 2the percentage relative errors of the approximation applied for estimation of the interaction - induced dipole moment with respect to the ccsd(t) resultsfig . 3the percentage relative errors of the approximation applied for estimation of the interaction - induced polarizability with respect to the ccsd(t) resultsfig . 4structure of investigated complexes of maximal lengthtable 1the values of the electric properties and their counterpoise corrected interaction - induced counterparts [a.u .] For the hydrogen - bonded linear hf(hf)n complexes . N, the absolute value of the static dipole moment for the whole chain \|\|(0), the total interaction - induced dipole moment \|\|(0) and its contributions: two- and many - body, static polarizability 0(0), the total interaction - induced polarizability 0(0) and its two- and many - body components, first order hyperpolarizability (0), the total interaction - induced hyperpolarizability (0) and its two- and many - body components\|\|(0)\|\|(0)0(0)0(0)(0)(0)ntotal2-bodym - bodytotal2-bodym - bodytotal2-bodym - bodyccsd(t)11.58580.17300.173010.0000.0480.04811.674.324.3222.49850.37890.36190.017015.1390.1770.1190.05811.6511.409.451.9533.42180.59520.55540.039820.3050.3340.1940.14010.6319.6414.794.8544.34940.81590.75100.064925.4820.5030.2700.2339.1628.3720.218.16mp211.59820.17390.173910.0480.0340.03410.974.184.1822.51760.38100.36400.017015.2010.1460.0900.05610.6111.289.451.8333.44770.59850.55890.039620.3790.2840.1490.1359.4319.3114.834.4844.38200.82030.75570.064625.5680.4340.2100.2247.8327.8020.297.51scf - hf11.69310.16770.16778.8930.0190.0199.613.173.1722.65270.36480.35090.013913.4320.0940.0550.03910.498.126.971.1533.62160.57110.53880.032317.9890.1880.0940.09410.8913.6210.852.7744.59420.78120.72860.052622.5550.2900.1340.15611.0619.3614.764.60b3lyp11.59760.18270.182710.4580.0960.09612.033.443.4422.52510.40180.38180.020015.8540.2770.2110.06610.4911.868.083.7833.46420.63230.58580.046521.2800.4900.3270.1637.1422.1712.719.4644.40790.86740.79180.075626.7200.7180.4450.2733.1733.2918.0315.26lc - blyp11.61520.18570.185710.8330.0810.0819.335.005.0022.55460.40870.38840.020316.4230.2590.1820.0775.9914.4011.552.8533.50600.64360.59610.047522.0460.4720.2870.1851.2425.4718.337.1444.46240.88340.80590.077527.6830.7020.3920.3104.1937.3025.2812.02pbe011.59890.17910.179110.2370.1010.10111.143.693.6922.52360.39340.37430.019115.5210.2830.2210.0629.7211.458.113.3433.45960.61890.57430.044620.8350.4970.3430.1546.7621.4113.398.0244.40010.84880.77620.072626.1630.7240.4660.2583.2031.8018.4213.38m06 - 2x11.60970.17520.17529.8700.0960.0968.973.093.0922.53630.38410.36610.018014.9580.2680.2140.0547.659.907.092.8133.47350.60350.56170.041820.0740.4690.3350.1345.0218.1011.696.4144.41510.82730.75930.068025.2020.6820.4580.2241.7626.9615.9910.97cam - b3lyp11.61610.18140.181410.3550.0790.07910.154.224.2222.55160.39840.37930.019115.6910.2430.1770.0668.1512.6310.062.5733.49850.62670.58210.044621.0550.4400.2780.1624.7622.6616.286.3844.45000.85950.78690.072626.4340.6500.3800.2700.7832.9421.2411.70table 2the values of the electric properties and their counterpoise corrected interaction - induced counterparts [a.u .] For the hydrogen - bonded linear h3n(hf)n complexes . N, the absolute value of the static dipole moment for the whole chain \|\|(0), the total interaction - induced dipole moment \|\|(0) and its contributions: two- and many - body, static polarizability 0(0), the total interaction - induced polarizability 0(0) and its two- and many - body components, first order hyperpolarizability (0), the total interaction - induced hyperpolarizability (0) and its two- and many - body components\|\|(0)\|\|(0)0(0)0(0)(0)(0)ntotal2-bodym - bodytotal2-bodym - bodytotal2-bodym - bodyccsd(t)11.72560.42940.429418.3660.6930.69339.358.388.3822.69840.69480.64430.050523.6400.4360.5380.10225.0314.2913.370.9233.63790.92730.84340.083928.8750.2140.4510.23717.0229.6622.137.5344.57141.15381.04100.112834.0760.0210.3720.35112.9141.1928.8112.38mp211.73680.42710.427118.4150.7700.77039.619.859.8522.71590.69300.64360.049423.6870.5450.6340.08926.2111.2911.390.1033.66200.92630.84410.082228.9280.3480.5620.21418.7925.6020.225.3844.60221.15381.04300.110834.1390.1770.4980.32114.8936.5326.969.57scf - hf11.79390.39870.398716.6550.6210.62123.7216.7716.7722.80310.64460.60450.040121.2820.4590.5320.07316.457.437.210.2233.78500.86360.79720.066425.8810.3250.4840.15913.2316.4713.173.3044.76241.07830.98890.089430.4610.2090.4410.23212.1023.5717.875.70b3lyp11.76040.45750.457518.9350.7210.72143.4811.4511.4522.75260.74070.68330.057424.4970.3820.4830.10118.9321.8423.241.4033.70940.98890.89300.095930.0230.0750.3500.2753.3444.5432.3912.1544.65981.23051.10150.129035.5000.1900.2560.4465.7461.2738.4322.84lc - blyp11.79990.45790.457919.1000.7500.75033.6113.7413.7422.80260.74360.68790.055724.8270.4400.5370.09715.0412.6615.182.5233.77140.99580.90130.094530.5330.1490.4160.2672.1732.2526.106.1544.73421.24201.11330.128736.2010.1100.3060.4166.4047.4734.4513.02pbe011.76390.45720.457218.5970.6410.64139.459.609.6022.75250.73460.67880.055824.0490.2970.3970.10017.4120.4621.901.4433.70590.97720.88420.093029.4550.0040.2600.2643.9141.0631.239.8344.65281.21351.08820.125334.8160.2650.1320.3973.9255.9037.6118.29m06 - 2x11.76790.44660.446618.0540.5940.59432.8511.9011.9022.75430.71460.66330.051323.2790.2860.3650.07916.5210.9012.871.9733.70770.95000.86410.085928.4670.0160.2270.2116.6526.4120.615.8044.65521.17951.06370.115833.6200.2230.1020.3250.7438.0225.3212.70cam - b3lyp11.78080.44930.449318.6020.6960.69634.7410.6710.6722.77780.72720.67310.054124.0780.3960.4970.10116.4615.2715.760.4933.74140.97210.88140.090729.5250.1240.3820.2584.7133.8625.558.3144.69911.21111.08840.122734.9320.1160.2750.3912.5347.7031.8415.86table 3the values of the electric properties and their counterpoise corrected interaction - induced counterparts [a.u .] For the hydrogen - bonded crystalline h2co(h2co)n complexes . N, the absolute value of the static dipole moment for the whole chain \|\|(0), the total interaction - induced dipole moment \|\|(0) and its contributions: two- and many - body, static polarizability 0(0), the total interaction - induced polarizability 0(0) and its two- and many - body components, first order hyperpolarizability (0), the total interaction - induced hyperpolarizability (0) and its two- and many - body componentsn\|\|(0)\|\|(0)0(0)0(0)(0)(0)total2-bodym - bodytotal2-bodym - bodytotal2-bodym - bodyccsd(t)12.03580.14320.143234.8680.3970.39793.099.409.4023.15400.31580.30270.013152.7320.9590.8460.113129.2323.5921.212.3834mp212.03960.14180.141834.9010.3910.39171.8412.3712.3723.15920.31310.29990.013252.7870.9560.8370.11995.6129.7727.722.0534.28810.49370.46280.030970.7281.5771.2970.280117.4949.0543.625.4345.42080.67810.62770.050488.6932.2231.7640.459138.5769.1259.949.18scf - hf12.46310.16510.165133.0780.3220.32293.101.821.8223.80930.36310.34920.013949.9540.7740.6890.085136.255.064.620.4435.16580.57130.53870.032666.8711.2681.0690.199178.808.947.611.3346.52650.78370.73070.053083.8051.7801.4550.325221.0613.1110.682.43b3lyp12.09930.15270.152735.7940.4390.439127.426.306.3023.25620.33710.32250.014654.1481.0440.9210.123176.6023.6616.387.2834.42310.53140.49740.034072.5571.7071.4160.291222.0944.7027.1417.5645.59410.72980.67430.055590.9902.3941.9170.477266.0267.3138.2829.03lc - blyp12.16470.15570.155735.8290.4440.44494.8110.2910.2923.35660.34370.32900.014754.2141.0630.9370.126129.6327.1823.883.3034.55870.54200.50750.034572.6561.7431.4450.298161.9046.6338.198.4445.76490.74450.68820.056391.1212.4471.9590.488193.0367.2252.7714.45pbe012.09490.14990.149935.2360.4530.453118.826.066.0623.24760.33070.31660.014153.3081.0680.9500.118165.9920.8615.135.7334.41000.52130.48820.033171.4331.7391.4600.279210.1338.6724.8313.8445.57640.71580.66180.054089.5812.4331.9750.458252.9857.7834.7922.99m06 - 2x12.13650.14730.147334.5930.4400.440100.177.747.7423.30830.32510.31140.013752.3251.0400.9250.115139.8321.3217.783.5434.48970.51250.48040.032170.1081.6941.4240.270177.4037.0128.408.6145.67500.70390.65160.052387.9132.3691.9280.441214.0953.5639.1114.45cam - b3lyp12.17130.15470.154735.2320.4170.417100.599.149.1423.36550.34110.32680.014353.2880.9990.8800.119139.1224.7521.293.4634.56970.53760.50400.033671.3971.6381.3570.281175.2942.7333.928.8145.77800.73820.68340.054889.5292.3001.8400.456210.3661.8346.8315.00table 4the values of the electric properties [a.u .] Of analyzed monomershfh3nh2co\|\|(0)ccsd(t)0.70590.59190.9470mp20.71180.59990.9496scf - hf0.76310.63501.1498b3lyp0.70630.59490.9740lc - blyp0.71290.62491.0053pbe00.70940.59930.9733m06 - 2x0.71680.60510.9954cam - b3lyp0.71610.61291.00900 (0)ccsd(t)4.94113.81517.152mp24.97113.89817.169scf - hf4.40912.65216.321b3lyp5.14514.19417.584lc - blyp5.33714.23117.595pbe05.03113.89317.307m06 - 2x4.85613.54217.005cam - b3lyp5.10313.91817.321 (0)ccsd(t)8.8922.2452.11mp28.0520.5642.98scf - hf6.9010.7148.60b3lyp8.7224.5566.95lc - blyp8.4913.7653.23pbe08.1821.4562.74m06 - 2x6.7215.9154.62cam - b3lyp8.3417.0855.43 structure of investigated complexes of maximal length the percentage relative errors of the approximation applied for estimation of the interaction - induced dipole moment with respect to the ccsd(t) results the percentage relative errors of the approximation applied for estimation of the interaction - induced polarizability with respect to the ccsd(t) results structure of investigated complexes of maximal length the values of the electric properties and their counterpoise corrected interaction - induced counterparts [a.u . N, the absolute value of the static dipole moment for the whole chain \|\|(0), the total interaction - induced dipole moment \|\|(0) and its contributions: two- and many - body, static polarizability 0(0), the total interaction - induced polarizability 0(0) and its two- and many - body components, first order hyperpolarizability (0), the total interaction - induced hyperpolarizability (0) and its two- and many - body components the values of the electric properties and their counterpoise corrected interaction - induced counterparts [a.u .] For the hydrogen - bonded linear h3n(hf)n complexes . N, the absolute value of the static dipole moment for the whole chain \|\|(0), the total interaction - induced dipole moment \|\|(0) and its contributions: two- and many - body, static polarizability 0(0), the total interaction - induced polarizability 0(0) and its two- and many - body components, first order hyperpolarizability (0), the total interaction - induced hyperpolarizability (0) and its two- and many - body components the values of the electric properties and their counterpoise corrected interaction - induced counterparts [a.u .] For the hydrogen - bonded crystalline h2co(h2co)n complexes . N, the absolute value of the static dipole moment for the whole chain \|\|(0), the total interaction - induced dipole moment \|\|(0) and its contributions: two- and many - body, static polarizability 0(0), the total interaction - induced polarizability 0(0) and its two- and many - body components, first order hyperpolarizability (0), the total interaction - induced hyperpolarizability (0) and its two- and many - body components the values of the electric properties [a.u .] Of analyzed monomers the careful analysis of the gathered data for the interaction - induced dipole moments reveals quite a good performance of the tested dft functionals . For most cases, the dft results are of better quality than scf - hf data . However, the exceptions are observed for linear hf and h3n (hf)nchains . In both cases lc - blyp performs noticeably worse than scf - hf approach with the relative error almost twice as large for the hf chain . Additionally, for the hf chain also the b3lyp functional and its long - range corrected version cam - b3lyp demonstrate slight aggravation with respect to the scf - hf approach . The relative error for the dft calculations in all the investigated cases does not exceed 9% . The worst data are obtained with lc - blyp, where the relative errors are between 7.5% and 8.5% and the best agreement with the ccsd(t) interaction - induced dipole moments is observed for the m06 - 2x functional (errors 1.5%-4%). It is worth mentioning that the relative error for the mp2 calculations does not surpass 1% . 2 shows that the relative error of the interaction - induced dipole moment remains approximately constant with the chain elongation for the hf and h2co complexes . On the other hand, the lengthening of the h3n (hf)n system leads to the improvement of the m06 - 2x and pbe0 results - the errors decrease from 4% to 2% and from 6.5% to 5%, respectively . The opposite behavior is observed for the lc - blyp data - the results worsen by more than 1% with increasing chain length . The decomposition of the total interaction - induced dipole moment for the inspected complexes does not disclose any abrupt demeanor of the dft results: the quality of the obtained two- and more - body components does not raise many reservations and therefore the dft functionals tested in this study can be recommended for the many - body analysis of the interaction - induced dipole moment of the investigated complexes . Due to the almost negligible non - additivity in the case of the polarizability of the analyzed molecular chains (interaction - induced polarizability less than 1 a.u . For linear chains and less than 2.5 a.u . For formaldehyde the dramatic increase of the error for the longest h3n (hf)n complex should not be taken into account, since the interaction - induced increment for the ccsd(t) calculations of the five - membered chain accounts for only 0.06% of the total polarizability value . Therefore, the interaction - induced polarizability within for instance pbe0 approach, that makes up 0.76% of the total pbe0 polarizability for the longest chain, appears to be in large error with respect to the ccsd(t) data; however this still gives a very good predictive power for the total polarizability values . The contribution from many - body terms increases with the chain elongation and for long chains it may exceed the two - body terms . In the case of the h3n (hf)n complexes, where the two- and many - body terms have opposite signs, the expansion of the chain leads from the negative interaction - induced polarizability that weakens the total response of the system, to the positive 0 for larger number of hf units, where the interaction - induced contribution additionally strengthens the total polarizability of the system . This tendency would be observed for all the investigated methods, however the applied dft functionals predict the length of the chain for which 0 changes sign as smaller than respective ccsd(t), mp2 or scf calculations . In other words, dft overestimates the many - body terms with respect to the two - body contributions . This is however not a general observation for dft functionals, since for the hf chains the propensity is opposite: dft seems to underestimate slightly the many - body terms in comparison to the two - body contributions, and in the case of the h2co chains the agreement between all the applied methods for the ratio of the many - body to two - body contributions to interaction - induced polarizability is very good . All the calculations lead to the conclusion that again the mp2 method gives good quality results . Additionally, the best - performing dft functional is cam - b3lyp . According to the author s expectations and experiences [20, 42] the worst quality results have been obtained definitely with the scf - hf approach - the errors ranging from 25% to 100% . For the formaldehyde complex the dft functionals in general perform better than mp2 and the only exception is the pbe0 interaction - induced hyperpolarizability of the dimer . Taking mp2 approach as the suitable method for the incremental values estimation, one can treat its largest error (32% for the formaldehyde dimer) as an indicator for the satisfactory performance of the dft functionals . For the linear hf chain all the investigated functionals achieve smaller errors than this limit . In the case of the formaldehyde complexes the addition of one unit to the investigated dimers has a significant influence on the quality of the obtained results . With the chain elongation for the hf complexes the relative errors of the dft interaction - induced hyperpolarizability increase and in the case of pbe0 and b3lyp changes sign for trimer . Similar behavior might be expected for the m06 - 2x functional for chains longer than investigated . In other words, these functionals underestimate for hf dimer and overestimate it for longer chains . The dramatic decrease of the relative error value is observed for the lc - blyp, m06 - 2x and cam - b3lyp functionals when going from h3n hf to h3n (hf)2 . Thus, b3lyp, cam - b3lyp and pbe0 give the overestimation of for all the analyzed lengths . Lc - blyp exhibits the underestimation of the value for the h3n (hf)2 and its overestimation besides . Moreover, m06 - 2x for three of four investigated h3n (hf)n complexes underestimates, however its relative errors could be expected to change sign again for the chains longer than investigated . Additionally the correct qualitative description of the total nonlinear response of the hf chain was achived by the dft calculations, which is in contrary to the scf - hf results (observe the increase of the to the absolute value). The decomposition of the interaction - induced hyperpolarizability into the two- and more - body components reveals imperfections of the commonly applied hybrid functionals: b3lyp and pbe0 . For all the investigated systems the many - body component calculated with these functionals are of poor quality with respect to the ccsd(t) reference data . Additionally, for the h3n (hf)n systems also the two - body terms are not reproduced well . On the other hand, dft reproduces well the sign change of the two - body terms in the h3n (hf)n chains with the system elongation . For the many - body terms dft (and mp2) also predicts the change of sign going from n = 2 to n = 3, however this effect is not present in the ccsd(t) reference results, when all the many - body terms are positive . Comparison of the ratio of the many - body to the two - body terms in the case of the interaction - induced hyperpolarizability indicates its overestimation for the hf and h2co chains and good correspondence in the case of h3n (hf)n complexes . Additionally, the mutual relations of the monomer properties and interaction - induced contributions causes the total hyperpolarizability to change signs for shorter hf and h3n the careful analysis of the gathered data for the interaction - induced dipole moments reveals quite a good performance of the tested dft functionals . For most cases, however, the exceptions are observed for linear hf and h3n (hf)nchains . In both cases lc - blyp performs noticeably worse than scf - hf approach with the relative error almost twice as large for the hf chain . Additionally, for the hf chain also the b3lyp functional and its long - range corrected version cam - b3lyp demonstrate slight aggravation with respect to the scf - hf approach . The relative error for the dft calculations in all the investigated cases does not exceed 9% . The worst data are obtained with lc - blyp, where the relative errors are between 7.5% and 8.5% and the best agreement with the ccsd(t) interaction - induced dipole moments is observed for the m06 - 2x functional (errors 1.5%-4%). It is worth mentioning that the relative error for the mp2 calculations does not surpass 1% . The comparison of the data presented in fig . 2 shows that the relative error of the interaction - induced dipole moment remains approximately constant with the chain elongation for the hf and h2co complexes . On the other hand, the lengthening of the h3n (hf)n system leads to the improvement of the m06 - 2x and pbe0 results - the errors decrease from 4% to 2% and from 6.5% to 5%, respectively . The opposite behavior is observed for the lc - blyp data - the results worsen by more than 1% with increasing chain length . The decomposition of the total interaction - induced dipole moment for the inspected complexes does not disclose any abrupt demeanor of the dft results: the quality of the obtained two- and more - body components does not raise many reservations and therefore the dft functionals tested in this study can be recommended for the many - body analysis of the interaction - induced dipole moment of the investigated complexes . Due to the almost negligible non - additivity in the case of the polarizability of the analyzed molecular chains (interaction - induced polarizability less than 1 a.u . For linear chains and less than 2.5 a.u . For formaldehyde the dramatic increase of the error for the longest h3n (hf)n complex should not be taken into account, since the interaction - induced increment for the ccsd(t) calculations of the five - membered chain accounts for only 0.06% of the total polarizability value . Therefore, the interaction - induced polarizability within for instance pbe0 approach, that makes up 0.76% of the total pbe0 polarizability for the longest chain, appears to be in large error with respect to the ccsd(t) data; however this still gives a very good predictive power for the total polarizability values . The contribution from many - body terms increases with the chain elongation and for long chains it may exceed the two - body terms . In the case of the h3n (hf)n complexes, where the two- and many - body terms have opposite signs, the expansion of the chain leads from the negative interaction - induced polarizability that weakens the total response of the system, to the positive 0 for larger number of hf units, where the interaction - induced contribution additionally strengthens the total polarizability of the system . This tendency would be observed for all the investigated methods, however the applied dft functionals predict the length of the chain for which 0 changes sign as smaller than respective ccsd(t), mp2 or scf calculations . In other words, dft overestimates the many - body terms with respect to the two - body contributions . This is however not a general observation for dft functionals, since for the hf chains the propensity is opposite: dft seems to underestimate slightly the many - body terms in comparison to the two - body contributions, and in the case of the h2co chains the agreement between all the applied methods for the ratio of the many - body to two - body contributions to interaction - induced polarizability is very good . All the calculations lead to the conclusion that again the mp2 method gives good quality results . According to the author s expectations and experiences [20, 42] the analysis of the dft functionals assesment appeared to be most complex . Figure 4 depicts the relative error for the interaction - induced hyperpolarizability . The worst quality results have been obtained definitely with the scf - hf approach - the errors ranging from 25% to 100% . For the formaldehyde complex the dft functionals in general perform better than mp2 and the only exception is the pbe0 interaction - induced hyperpolarizability of the dimer . Taking mp2 approach as the suitable method for the incremental values estimation, one can treat its largest error (32% for the formaldehyde dimer) as an indicator for the satisfactory performance of the dft functionals . For the linear hf chain all the investigated functionals achieve smaller errors than this limit . In the case of the formaldehyde complexes the addition of one unit to the investigated dimers has a significant influence on the quality of the obtained results . With the chain elongation for the hf complexes the relative errors of the dft interaction - induced hyperpolarizability increase and in the case of pbe0 and b3lyp changes sign for trimer . Similar behavior might be expected for the m06 - 2x functional for chains longer than investigated . In other words, the dramatic decrease of the relative error value is observed for the lc - blyp, m06 - 2x and cam - b3lyp functionals when going from h3n hf to h3n (hf)2 . Thus, b3lyp, cam - b3lyp and pbe0 give the overestimation of for all the analyzed lengths . Lc - blyp exhibits the underestimation of the value for the h3n (hf)2 and its overestimation besides . Moreover, m06 - 2x for three of four investigated h3n (hf)n complexes underestimates, however its relative errors could be expected to change sign again for the chains longer than investigated . Additionally the correct qualitative description of the total nonlinear response of the hf chain was achived by the dft calculations, which is in contrary to the scf - hf results (observe the increase of the to the absolute value). The decomposition of the interaction - induced hyperpolarizability into the two- and more - body components reveals imperfections of the commonly applied hybrid functionals: b3lyp and pbe0 . For all the investigated systems the many - body component calculated with these functionals are of poor quality with respect to the ccsd(t) reference data . Additionally, for the h3n on the other hand, dft reproduces well the sign change of the two - body terms in the h3n (hf)n chains with the system elongation . For the many - body terms dft (and mp2) also predicts the change of sign going from n = 2 to n = 3, however this effect is not present in the ccsd(t) reference results, when all the many - body terms are positive . Comparison of the ratio of the many - body to the two - body terms in the case of the interaction - induced hyperpolarizability indicates its overestimation for the hf and h2co chains and good correspondence in the case of h3n (hf)n complexes . Additionally, the mutual relations of the monomer properties and interaction - induced contributions causes the total hyperpolarizability to change signs for shorter hf and h3n (hf)n chains within the dft approaches than within the ccsd(t) calculations . The estimated bsse values for the investigated complexes are summarized in tables 5, 6 and 7 . It should be repeated that all the calculations have been carried out with the aug - cc - pvdz basis set of the medium size . The relative error (bsse[%]) arising from the lack of the bsse correction for the dipole moment is usually smaller than 1% and only in the case of the linear hf chain for the lc - blyp functional it reaches 2% . On these basis one can conclude that the counterpoise procedure has a negligible influence on the quality and quantity of the obtained interaction - induced dipole moment values for the studied complexes.table 5interaction - induced electric properties for the hydrogen - bonded linear hf (hf)n complexes with and without counterpoise correction and the basis set superposition error [%] n\|\|(0)0(0)(0)cp - corcp - uncorbssecp - corcp - uncorbssecp - corcp - uncorbsseccsd(t)10.17300.17390.52%0.0480.118145.83%4.326.1041.20%20.37890.38070.48%0.1770.31678.53%11.4015.0131.67%30.59520.59800.47%0.3340.54061.68%19.6424.9226.88%40.81590.81960.45%0.5030.77754.47%28.3735.2824.36%mp210.17390.17460.40%0.0340.106211.76%4.185.1222.49%20.38100.38230.34%0.1460.28897.26%11.2813.5219.86%30.59850.60050.33%0.2840.49574.30%19.3122.7517.81%40.82030.82310.34%0.4340.71364.29%27.8032.3916.51%scf - hf10.16770.16700.42%0.0190.075294.74%3.174.1831.86%20.36480.36360.33%0.0940.206119.15%8.1210.2025.62%30.57110.56940.30%0.1880.35488.30%13.6216.6922.54%40.78120.77890.29%0.2900.51176.21%19.3623.4220.97%b3lyp10.18270.18501.26%0.0960.16773.96%3.445.4157.27%20.40180.40621.10%0.2770.41850.90%11.8615.6632.04%30.63230.63891.04%0.4900.69842.45%22.1727.7425.12%40.86740.87641.04%0.7180.99338.30%33.2940.4321.45%lc - blyp10.18570.18941.99%0.0810.15895.06%5.007.6553.00%20.40870.41591.76%0.2590.41158.69%14.4019.4735.21%30.64360.65441.68%0.4720.69647.46%25.4732.7228.46%40.88340.89781.63%0.7020.99641.88%37.3046.6325.01%pbe010.17910.18010.56%0.1010.17472.28%3.695.2341.73%20.39340.39540.41%0.2830.42750.88%11.4514.8329.52%30.61890.62190.48%0.4970.70942.66%21.4125.9821.35%40.84880.85300.49%0.7241.00538.81%31.8037.7218.62%m06 - 2x10.17520.17620.57%0.0960.15763.54%3.094.4744.66%20.38410.38600.49%0.2680.38844.78%9.9012.5026.26%30.60350.60650.50%0.4690.64838.17%18.1021.8620.77%40.82730.83130.48%0.6820.91934.75%26.9631.8318.06%cam - b3lyp10.18140.18401.43%0.0790.14887.34%4.226.5354.59%20.39840.40341.26%0.2430.38056.38%12.6316.8733.57%30.62670.63421.20%0.4400.64245.91%22.6628.5926.17%40.85950.86971.19%0.6500.91741.08%32.9440.9124.20%table 6interaction - induced electric properties for the hydrogen - bonded h3n (hf)n complexes with and without counterpoise correction and the basis set superposition error [%] n\|\|(0)0(0)(0)cp - corcp - uncorbssecp - corcp - uncorbssecp - corcp - uncorbsseccsd(t)10.42940.42770.40%0.6930.39043.72%8.388.221.91%20.69480.69450.04%0.4360.05786.93%14.2914.984.83%30.92730.92820.10%0.2140.236210.28%29.6631.887.48%41.15381.15570.16%0.0210.4972466.67%41.1944.888.96%mp210.42710.42510.47%0.7700.45341.17%9.8511.0011.68%20.69300.69240.09%0.5450.15371.93%11.2910.447.53%30.92630.92670.04%0.3480.117133.62%25.6025.901.17%41.15391.15520.11%0.1770.358302.26%36.5337.853.61%scf - hf10.39870.39580.73%0.6210.40634.62%16.776.1163.57%20.64460.64200.40%0.4590.18859.04%7.438.058.34%30.86360.86080.32%0.3250.003100.92%16.4718.1710.32%41.07831.07520.29%0.2090.174183.25%23.5726.2011.16%b3lyp10.45750.45920.37%0.7210.40543.83%11.4510.2110.83%20.74070.74500.58%0.3820.012103.14%21.8423.065.59%30.98890.99560.68%0.0750.393624.00%44.5447.376.35%41.23051.23960.74%0.1900.725281.58%61.2765.176.37%lc - blyp10.45790.46210.92%0.7500.46837.60%13.7411.3617.32%20.74360.75191.12%0.4400.07982.05%12.6615.7024.01%30.99581.00781.21%0.1490.290294.63%32.2537.0614.91%41.24201.25771.26%0.1100.620463.64%47.4754.1113.99%pbe010.45720.45520.44%0.6410.32748.99%9.609.822.29%20.73460.73440.03%0.2970.094131.65%20.4620.400.29%30.97720.97830.11%0.0040.46811600.00%41.0642.102.53%41.21351.21580.19%0.2650.797200.75%55.9058.103.94%m06 - 2x10.44660.44610.11%0.5940.34541.92%11.9010.2214.12%20.71460.71580.17%0.2860.024108.39%10.9012.8317.71%30.95000.95230.24%0.0160.3552318.75%26.4129.4211.40%41.17951.18310.31%0.2230.652192.38%38.0242.0510.60%cam - b3lyp10.44930.45180.55%0.6960.41939.80%10.679.3212.65%20.72720.73270.75%0.3960.04688.38%15.2717.3013.29%30.97210.98030.84%0.1240.297339.52%33.8637.3910.43%41.21111.22190.89%0.1160.601418.10%47.7052.9711.05%table 7interaction - induced electric properties for the hydrogen - bonded crystalline h2co (h2co)n complexes with and without counterpoise correction and the basis set superposition error [%] n\|\|(0)0(0)(0)cp - corcp - uncorbssecp - corcp - uncorbssecp - corcp - uncorbsseccsd(t)10.14320.14180.98%0.3970.56341.81%9.4011.1218.30%20.31580.31300.89%0.9591.27532.95%23.5927.0914.84%34mp210.14180.14040.99%0.3910.56343.99%12.3714.1314.23%20.31310.31030.89%0.9561.28134.00%29.7733.3411.99%30.49370.48950.85%1.5782.05330.10%49.0554.4410.99%40.67810.67260.81%2.2232.84928.16%69.1276.3510.46%scf - hf10.16510.16360.91%0.3220.43635.40%1.824.10125.27%20.36310.36000.85%0.7740.99228.17%5.069.5488.54%30.57130.56670.81%1.2681.58825.24%8.9415.5974.38%40.78370.77770.77%1.7802.20223.71%13.1121.9267.20%b3lyp10.15270.15120.98%0.4390.62742.82%6.306.482.86%20.33710.33410.89%1.0441.39733.81%23.6624.262.54%30.53140.52700.83%1.7072.22230.17%44.7045.722.28%40.72980.72390.81%2.3943.07228.32%67.3168.742.12%lc - blyp10.15570.15411.03%0.4440.63943.92%10.2911.6513.22%20.34370.34070.87%1.0631.42934.43%27.1830.0510.56%30.54200.53750.83%1.7432.27630.58%46.6351.019.39%40.74450.73840.82%2.4473.14728.61%67.2273.118.76%pbe010.14990.14841.00%0.4530.62237.31%6.066.669.90%20.33070.32780.88%1.0681.38729.87%20.8622.256.66%30.52130.51700.82%1.7392.20426.74%38.6740.855.64%40.71580.71010.80%2.4333.04525.15%57.7860.745.12%m06 - 2x10.14730.14571.09%0.4400.58432.73%7.749.0617.05%20.32510.32210.92%1.0401.31126.06%21.3224.0212.66%30.51250.50800.88%1.6942.08823.26%37.0141.0710.97%40.70390.69790.85%2.3692.88821.91%53.5658.9910.14%cam - b3lyp10.15470.15330.90%0.4170.59141.73%9.1410.2612.25%20.34110.33840.79%0.9991.32532.63%24.7527.169.74%30.53760.53350.76%1.6382.11429.06%42.7346.428.64%40.73820.73280.73%2.3002.92527.17%61.8366.777.99% interaction - induced electric properties for the hydrogen - bonded linear hf (hf)n complexes with and without counterpoise correction and the basis set superposition error [%] interaction - induced electric properties for the hydrogen - bonded h3n (hf)n complexes with and without counterpoise correction and the basis set superposition error [%] interaction - induced electric properties for the hydrogen - bonded crystalline h2co (h2co)n complexes with and without counterpoise correction and the basis set superposition error [%] the analysis of the relative bsse for the linear hf and h3n (hf)n complex is unreasonable due to the very small 0 values . However, for the formaldehyde chains the obtained results become more stable owing to the larger interaction - induced polarizability values than in the case of the remaining systems . Here, the relative bsse changes from above 40% for short chains to almost 20% for longer chains . Nevertheless, again the influence of the interaction - induced bsse for the total value of the polarizability of the whole complex is negligible and the qualitative picture of the 0 changes with the chain elongation remains the same with or without the counterpoise correction . The changes of the relative bsse for the interaction - induced hyperpolarizability are parallel to these observed for the dipole moment, however here the absolute values of the errors decrease from 60% to 20% for hf chains and from 20% to 2% for formaldehyde chains with the chain elongation . Again, similarly as in the case of dipole moment and polarizability, the counterpoise procedure does not affect much the total qualitative picture . However, it cannot be neglected for the correct quantitative description of the interaction - induced hyperpolarizability of the investigated systems [35, 36]. The investigation of the electric properties of the hydrogen - bonded dimers has shown that the m06 - 2x functional performed worst mainly for hyperpolarizability (compare hf dimer). However in the case of longer chains, it presents a serious advantage over any other tested functionals . Therefore, m06 - 2x appears to be the best for the description of the influence of the mutual interaction on the electric properties in the hydrogen - bonded chains . Although these conclusions were verified in the present contribution only for one basis set of the medium size (aug - cc - pvdz), they should not differ dramatically for other basis set choices . The ordering of the functionals may vary, however the main conclusion of this work remain unchanged: dft functionals can be applied succesfully for the investigation of the interaction - induced electric properties . One should however keep in mind that the dft functional performance can also significantly depend on the nature of the intermolecular interactions and the conventional functionals are known to fail for systems governed by dispersion interaction [4346]. It is also of importance for the complex systems, that the basis set superposition error in the case of interaction - induced dipole moment and polarizability is negligible . What is more for the first - order hyperpolarizability bsse only has a noticable qualitative meaning, but the quantitative picture remains the same . This observation allows for instance to save a lot of effort in a rational design of novel materials . The earlier studies suggest the possibility of estimating the electric properties for the large molecular aggregates based on the accurate description of molecular properties and approximate descripton of the interaction - induced contributions [21, 47]. Current findings allow to expect that calculations of molecular properties on high theory level (mp2 or ccsd(t)) supplemented by the interaction - induced increments accounted for in dft could provide a relatively accurate and cheap alternative for regular post - hartree - fock predictions.
Super scan is a well described phenomenon on skeletal scintigraphy which is characterized by high skeletal to soft tissue ratio, uniform symmetrically increased bone uptake, and absent renal visualization . Sye, et al . Hypothesized that diseased bone shows increased uptake of radiopharmaceutical leading to reduced phosphate excretion and production of faint renal images on bone scan . We report a case of f-18 fluorodeoxyglucose (fdg) positron emission tomography (pet) super scan which shows features akin to super scan of skeletal scintigraphy and resolution of these features following initial phase of chemotherapy . A twelve - year old male presented with multiple joint pains, periorbital and sternal swellings of one - month duration . Contrast - enhanced computed tomography (ct) scan of the abdomen revealed poorly enhancing, hypodense bilateral renal masses ranging from 4 cm to 5 cm in size along with multiple hepatic and pancreatic space occupying lesions . A 4.3-cm exophytic mass was noted in the lower pole of right kidney [figure 1]. Ct - guided biopsy from this mass revealed feature of renal primitive neuroectodermal tumor (pnet)/ewing's sarcoma [figure 2]. Contrast - enhanced computed tomography scan of the abdomen revealed poorly enhancing, hypodense bilateral renal masses ranging from 4 cm to 5 cm in size along with multiple hepatic and pancreatic space occupying lesions . A 4.3-cm exophytic mass was noted in the lower pole of right kidney histology of renal biopsy specimen from the lesion (h and e staining, 10) showing sheets of malignant small round blue cells . (a) on immunohistochemistry, the tumor cells show strong and complete membranous positivity for mic-2 . (c) tumor cells were also negative for other markers in the round cell panel namely cytokeratin, leukocyte - common antigen, desmin, and synaptophysin (not shown here). The findings are those of primitive neuroectodermal tumor / ewing's sarcoma of the kidney thereafter, patient underwent a true whole body pet scan including bilateral limbs and skull region as per the standard institution protocol for pre - treatment staging of the disease . The images revealed extensive hypermetabolic areas in ct described renal (suvmax: 6.9), hepatic (suvmax: 7.2), and pancreatic lesions (suvmax: 5.4). In addition, intense and non - uniform uptake was seen in marrows of axial and appendicular skeletal system with distinct focus in palpable sternal swelling (suvmax: 4.5). A medium - sized focus was also seen in left posterior chest wall with extension into basal pleura of left lung . Interestingly, low background tracer concentration was observed along with very low f-18 fdg uptake in the brain (suvmax of 3.3 in occipital region and 2.9 in rest of the brain), skeletal muscles of limb, mediastinum, and bowel . Due to its resemblance with super scan of skeletal scintigraphy, these scan findings can be termed to represent pet super scan [figure 3]. True whole body pet scan including bilateral limbs and skull region shows extensive hypermetabolic areas in computed tomography described renal (suvmax: 6.9), hepatic (suvmax: 7.2), and pancreatic lesions (suvmax: 5.4). Intense and non - uniform uptake was seen in marrows of axial and appendicular skeletal system with distinct focus in palpable sternal swelling (suvmax: 4.5) and in left posterior chest wall with extension into basal pleura of left lung . Scan shows low background tracer concentration with very low f-18 fdg uptake in the brain (suvmax of 3.3 in occipital region and 2.9 in rest of the brain), skeletal muscles of limb, mediastinum, and bowel . Due to its resemblance with super scan of skeletal scintigraphy patient was subjected to six cycles of euro - ewing 99 protocol including vincristine, ifosfamide, doxorubicin, and etoposide chemotherapeutic regimen as per the institution protocol . Repeat pet scans were done after three and six cycles, latter being done after 4 months of the initial scan [figure 4a]. The images revealed marked treatment response with disappearance of super scan-like pattern, reduction in number, size, and metabolic activity of the lesions that were shown in the earlier scan . Suvmax of the occipital region of the brain increased from the previous value of 3.3 to 9.9 . For rest of the brain, the suvmax increased from 2.9 to 8.6 [figure 4b]. The comparison of femoral marrow activity in the pre- and post - treatment scans showed hypermetabolic areas of stimulation in post - treatment scan, which is clearly missing from the previously diseased marrow and gcsf - induced stimulated marrow being seen in previously uninvolved marrow . We have termed this pattern as marrow flip flop and it appears that areas showing marrow flip flop are the ones that demonstrate treatment response [figure 4c]. Repeat pet scans were done after three and six cycles, latter being done after 4 months of the initial scan the images show marked treatment response with disappearance of super scan-like pattern, reduction in number, size, and metabolic activity of the lesions that were shown in the earlier scan . Suvmax of the occipital region of the brain increased from the previous value of 3.3 to 9.9 . For rest of the brain, the suvmax increased from 2.9 to 8.6 the comparison of femoral marrow activity in the pre- and posttreatment scans shows hypermetabolic areas of stimulation in post - treatment scan, which is clearly missing from the previously diseased marrow and g - cs - finduced stimulated marrow being seen in previously uninvolved marrow termed as marrow flip flop it is an extremely rare malignant tumor with fewer than 50 cases in english literature . Renal pnet is still rarer in children and adolescents age group with only four cases being reported in the pediatric age group. [57] to the best of our knowledge, we are reporting fifth such case . It usually presents in advance stage, behaves more aggressively than pnet at other sites, and usually shows poor response to the chemotherapy . Similar characteristics of diffuse and intense hypermetabolism throughout the skeleton have been described in the earlier publications. [911] however, the scan in this case shows a very high contrast between the metastatic and non - metastatic organs with extremely low f-18 fdg uptake in brain, muscles of limbs, mediastinum, and bowel . Unique feature of this case is demonstration of reversal of aforementioned f-18 fdg avidity after initial chemotherapy in the follow - up scan . Another unusual feature of this case is the excellent chemotherapeutic response shown in this patient; pnet of kidney is known to respond poorly to chemotherapy and a functional imaging modality may serve as a useful tool to demonstrate early response in such rare cases . Marrow flip flop could be due to involved marrow being replaced by fibrosis as a treatment response and hence would not demonstrate the marrow stimulation effect of colony - stimulating factors on a pet scan done to assess therapeutic response . In conclusion, though uncommon, the reporting physician should be aware of findings of pet super scan as demonstrated in this patient . Reversal of these abnormalities on functional imaging following therapy guides the clinician to choose the best available regimen for this otherwise chemotherapy nave disease.
Historically, respiratory muscle weakness during mechanical ventilation was recognized a state of muscular fatigue . It was thought to be caused by prolonged increased work of breathing . Although first reports of diminished myofibre crosssectional area in diaphragms because of long term mechanical ventilation date back to the 1980s,1 direct harmful effects of mechanical ventilation on respiratory muscles were only thoroughly studied, e.g. In animal models in the last two decades . Currently, respective findings from animal models can partially be reproduced in human experiments and find their way to the clinical bedside.2 within the complex of critical illnessrelated weakness,3, 4, 5 the dysfunction of respiratory muscles, particularly of the diaphragm, represents a highly relevant and distinct clinical problem in intensive care units (icus). From a clinical perspective, diaphragmatic dysfunction contributes to difficult weaning or even failure to wean from mechanical ventilation in icu patients . Overall, data indicate that weaning failure may affect up to 25% of mechanically ventilated patients in icus today.6 ventilatorinduced diaphragmatic dysfunction (vidd) was previously defined as loss of diaphragmatic forcegenerating capacity specifically related to the use of mechanical ventilation.7 vidd may typically be observed after variable periods of controlled mechanical ventilation (i.e. Ventilation without spontaneous breathing)7, 8 while assisted ventilation modes (i.e. Ventilation with preserved diaphragmatic contractions) attenuate the detrimental effects of controlled mechanical ventilation on the diaphragm and seem protective in animal models9, 10, 11, 12 unless high levels of support are used.8 as preserved muscle activity is protective, it seems conceivable that diaphragmatic contractile inactivity may be the cause for the development of vidd.13 despite the terminology vidd, the condition does not exclusively affect the major respiratory muscle (i.e. The diaphragm), but may also involve the intercostal musculature to a minor degree.14, 15 the diaphragm, however, seems particularly prone to dysfunction under mechanical ventilation while auxiliary respiratory muscles, e.g. Pectorales muscles,16 or skeletal limb muscles (e.g. Soleus or extensor digitorum muscles) are typically spared.17, 18, 19, 20 while many of the pathophysiological aspects of vidd that were found in animals have also been successfully reproduced in humans, the proof of decreased force generation was so far only performed indirectly in mechanically ventilated patients.21 only very recently, direct force generation of isolated human muscle fibres was studied with ambiguous results.22, 23, 24 the pathophysiology of vidd was to a large extent elucidated in models of healthy animals . Under continuous controlled mechanical ventilation, the diaphragm partially loses its force and pressuregenerating capacity, classically assessed in intact animal models with transdiaphragmatic pressure under maximal phrenic nerve stimulation.25, 26, 27 data demonstrate that loss of respective force may reach up to 50% within a few days . Importantly, the onset and time course of vidd may vary between the respective species studied.25, 26, 27 this makes transfer of respective data to the human bedside particularly difficult . However, loss of force seems neither linked to age28 nor to changes in lung volumes26 nor to positive endexpiratory pressure (peep).27, 29 in addition, phrenic nerve signal transmission and signal transduction at the neuromuscular endplate in vidd appear normal . This is a distinct difference between vidd and polyneuropathic forms of icuacquired weakness (icuaw),5 as the latter often shows conduction abnormalities in electrophysiological studies . Moreover, loss of force is reproducible in isolated muscle specimens,18, 29 rendering the pathophysiological changes of vidd to a cellular level . Besides ultrastructural muscle fibre injury,14, 27, 30 the most common histopathological correlate is muscle atrophy.14, 18, 26, 31, 32, 33 this is especially the case in regard to type ii (fast twitch) muscle fibres within the early course of the disease.14, 19 remodelling processes with a histopathological increase of hybrid fibres at the expense of (slow twitch) type i fibres can be identified at later stages.31 however, it must be noted that atrophy alone does not explain the observed loss of force . When force is normalized for crosssectional area of isolated muscle strips (socalled specific force or tension), the loss is disproportionally larger than would be expected by atrophy alone.18, 27, 34 overall, protein synthesis in the diaphragm in established vidd seems dramatically decreased.35 in murine models, proteolysis is activated as early as 6 h after initiation of controlled mechanical ventilation36 and loss in myosin heavy chain synthesis of up to 65% may be observed.19, 35 a decrease in anabolic signalling including pathways via insulin like growth factor1 (igf1)32 and myogenin (myod) are found.37 proteolysis is significantly increased.19, 38 all four proteolytic systems of the mammalian cell are activated in animal models of vidd and later found induced in mechanically ventilated humans.39 proteolysis is mediated by the calpain 19 and caspase system,40, 41 as well as the ubiquitin lysosomal system.42 the biochemical changes of vidd, mainly examined in healthy murine models, may be augmented or even triggered by oxidative stress pathways,19, 43, 44, 45 nuclear factor kb (nfkb),46, 47 or jak stat signalling pathways.48 similar to patients with severe sepsis and septic shock, distinct cytokine cascades are upregulated . This includes interleukin (il)6, tumour necrosis factor, and il1 in rat diaphragmatic tissue exposed to mechanical ventilation47 and may further amplify major inflammatory signalling pathways including nfkb.47, 49 this humoral response is well known to promote systemic immune cellular consequences and may also result in immune phenotypic changes.50, 51, 52 there is experimental proof of diaphragmatic dysfunction directly induced by sepsis.53, 54, 55, 56, 57 this includes free radicals, mitochondrial dysfunction,54 calpain,56 and caspase activation.57 in addition, clinical evidence points to severe sepsis / septic shock as an important risk factor for diaphragmatic dysfunction.58, 59 interestingly, maes and colleagues have developed a lipopolysaccharide rat model of controlled mechanical ventilation . They could now show that controlled mechanical ventilation potentiates sepsisinduced diaphragm dysfunction, possibly because of increased proinflammatory cytokine production, autophagy, and worsening of oxidative stress.60 to our knowledge, this is the first study of vidd in a sepsis model that may indicate an important pathophysiological overlap . It should be clearly noticed that vidd and sepsis induced diaphragmatic dysfunction are distinctive pathological entities13 and the findings of overlapping pathophysiology are controversial, even more so as mechanical ventilation was shown to protect rat diaphragms in sepsis.61 nevertheless, this shared pathophysiology may offer therapeutic options in the future . Strategies to modulate the systemic cytokine and/or immune response by, e.g. Blocking respective cascades or by improving host defence, may in theory be beneficial.62, 63, 64 however, because of current lack of respective trials, this remains speculative at this point in time.65 recent data indicate a profound reduction in diaphragmatic blood flow in vidd.66 it is therefore speculated that reduced oxygen delivery may lead to formation of reactive oxygen species with consecutive triggering of proteolytic cascades and enhanced oxidative stress.67 angiogenetic factors change their expression profile under mechanical ventilation [downregulation of vascular endothelial growth factor (vegf), upregulation of hypoxiainducible factor (hif)1], but their role in vidd remains incompletely understood.47, 68 exercise training or a genetically high aerobic capacity interesting in the context of decreased blood supply may protect the diaphragm from vidd . Upregulation of heat shock proteins and improved antioxidant properties of a trained diaphragm with decreased activation of proteases and respective mitochondrial protection are among the postulated mechanisms.69, 70 nevertheless, mechanical ventilation may also enhance antioxidative pathways, potentially as a counterregulatory measure.68, 71 overall, this aspect of vidd may open new therapeutic avenues as the redox state of a cell might theoretically be influenced positively.71 recent data indicate protection from diaphragmatic weakness with nacetylcysteine via augmented autophagosome formation in mice.72 data from animal vidd models demonstrate a quick and complete recovery within hours if spontaneous breathing is resumed early . However, this needs to be interpreted before the background of limited times of mechanical ventilation (12 to 27 h.).73, 74, 75 moreover, the respective kinetics and time course of vidd are species dependent,7 and data may therefore not be easily transferrable between species . As pathophysiology of vidd was investigated mainly in models of healthy animals, it must be kept in mind that in critically ill patients, underlying comorbidities may contribute to diaphragmatic dysfunction . The bulk of evidence derives from mechanically ventilated brain dead organ donors . Only very recently levine and colleagues could show in a seminal study in 14 brain death organ donors that disuse atrophy may occur shortly after start of controlled mechanical ventilation.16 when compared to controls (i.e. Patients with thoracic surgery on 3 h of cmv), muscle biopsies of the costal diaphragm in organ donors (mechanically ventilated for 18 to 69 h) exhibit strongly decreased crosssectional areas of slowtwitch and fasttwitch fibres (57% vs. 53%, respectively). These changes were limited to the diaphragm and not reproducible, e.g. In pectorales muscles.16 findings were later confirmed and linked to upregulation of autophagic systems via transcription factors (e.g. Foxo1), activation of the ubiquitin proteasome complex, nfkb activity, and induction of the calpain system in mechanically ventilated human muscles and diaphragms.76, 77, 78, 79, 80 hooijman lately documented the activation of the ubiquitin proteasome complex in a mixed population of critically ill patients.22 while direct force loss (i.e. Loss of performance of isolated muscle strips) is extensively documented in animal models, the condition in humans was until recently only indirectly characterized by airway occlusion pressure responses to phrenic nerve stimulation.81, 82 in the last year, first reports of human muscle fibres appeared and finally documented a loss of specific force in mechanically ventilated human muscles.22, 23, 24 but a clear distinction of the studied populations should be noted . Hooijman initially found no evidence for diminished contractile performance in sarcomeres when studying specimens from brain death organ donors (mean duration of mechanical ventilation of 26 5 h),23 whereas the group later published data from mixed icu populations suffering from comorbidities like sepsis or trauma (duration of mechanical ventilation 14 to 607 h) with contractile weakness of human diaphragmatic muscle fibres.22, 24 it is unclear to what extent these underlying conditions have contributed to the documented weakness . So, speaking in strict terms, the formal proof of contractile weakness caused by mechanical ventilation is not yet established . . Mechanical ventilation may alter mitochondrial respiratory chain enzyme function (e.g. Cytochromec oxidase), may induce microdeletions within mitochondrial dna, and may decrease levels of mitochondrial scavengers for reactive oxygen species (ros) culminating in diaphragmatic lipid overload . A causative link between lipid overload and mitochondrial dysfunction is not firmly established.84 nevertheless, oxidative stress has been linked to activation of apoptic, proteasomal, and autophagocytic pathways,85 and expression of angiogenetic factors varies with the ventilation mode used.86 in conclusion, histopathological and biochemical changes of respiratory muscle dysfunction / vidd in animal models could now be reproduced to some extent in human experiments.2 loss of specific force in isolated human muscle fibres has finally been documented22, 24 in mixed icu populations with underlying comorbidities (but not in brain death organ donors23). With vidd animal models available, this may open up new options for development of specific therapeutic approaches in order to treat or prevent respiratory muscle dysfunction / vidd . Vidd is diagnosed by exclusion of other causes of weaning failure (e.g. Decompensated congestive heart failure) and other specific causes of diaphragmatic weakness such as electrolyte disturbances, malnutrition, drugs, central nervous system disorders, and distinct neuromuscular disorders.7 global tests of respiratory muscle strength like measurement of maximum inspiratory pressures87 or the rapid shallow breathing index (respiratory rate divided by tidal volume)88 serve as screening tools . Assessment of transdiaphragmatic pressure in combination with transdermal phrenical nerve (magnetic) stimulation may be considered the gold standard.82, 89, 90 however, this approach is invasive with the need for an oesophageal and gastric balloon . Alternatively, twitch tracheal airway pressure avoids balloon placement,81, 82, 91 but the accuracy of this approach is debated.92 the electrophysiological assessment of diaphragmatic electrical activity via a modified nasogastric tube (eadi)93, 94 is a new promising but almost unexplored technique for vidd . The electromyographic signal describes the summation amplitude of electrical activity and allows assessment of neuronal respiratory drive and estimation of neuroventilatory efficiency (tidal volume divided by electrical activity).94, 95, 96, 97 although there is lack of specific vidd studies, this approach may hold potential for respiratory monitoring as it incorporates the neuronal feedback loop of the respiratory drive.93, 94, 97 further studies are warranted . A noninvasive modality for patients with suspected diaphragmatic weakness is ultrasound, readily available at the bedside and most likely free from adverse effects (figures 1 and 2). With reference values established, ultrasound allows both reproducible assessment98 of diaphragmatic function and structure and may exclude alternative causes of weaning failure.94, 99, 100, 101, 102 the inspiratory downward motion of the diaphragm should be greater than one centimetre (figure 1). This was evaluated in ventilated patients during spontaneous breathing trials and predicts successful weaning with an accuracy similar to the rapid shallow breathing index.103 a thickening fraction (inspiratory minus expiratory thickness divided by expiratory thickness) of 30% was shown to exert a positive predictive value of 91% for weaning success104 (figure 2a and 2b). A sustained loss of diaphragmatic thickness was reported for patients undergoing prolonged mechanical ventilation, most prominent in the first three days.105 changes in thickness may be associated with diaphragmatic weakness.106 assessment of diaphragmatic motion (motion mode, phased array 3.55 mhz transducer). Subcostal position (midclavicular line, angle of> 70 in supine subjects) with visualization of the posterior diaphragmatic third . Diaphragmatic dome excursion at rest should be 1 cm (lower limit of normal in healthy controls).101, 108 inspiratory diaphragmatic position (solid line), expiratory position (dotted line), and excursion (arrow) are indicated . Assessment of (a) expiratory and (b) inspiratory diaphragmatic thickness (brightness mode, linear array high frequency transducer of> 10 mhz, zone of apposition: midaxillary line). Note in and expiratory variation (limits of normal may vary).102, 108 the thickening fraction [i.e. (inspiratory expiratory diameter) / expiratory diameter] can be assessed.143 maximum diameters are measured between diaphragmatic pleural line (*) and peritoneal line (#). The consequences of application of peep (inducing caudal diaphragm displacement) remain unknown.107 as ultrasound allows dynamic assessment of the diaphragm, it will mainly provide useful insight if performed during unassisted breathing, i.e. During spontaneous breathing trials.108 recent data indicate that the thickening fraction is also of use under mechanical ventilation, as thickening is caused by muscular contraction, but not by passive inhalation.98 despite a growing body of evidence for the use of ultrasound, it remains a pure imaging technique without direct assessment of ventilation or diaphragmatic force . Ultrasound may also be of limited value in the early course of critical illness, when controlled ventilation needs to be applied.94 for technical details, please refer to figure legends (figures 1 and 2). When choosing ventilatory modes, spontaneous breathing efforts during mechanical ventilation seem protective for vidd in healthy animals.12, 109, 110 as most studies on vidd were performed in healthy animals, questions remain about the applicability in the acute phase of critical illness.39 the complex and dynamic pathophysiology of respiratory support during critical illness asks for an individually tailored approach . Early in acute critical illness, controlled mechanical ventilation may often be mandatory, but an early switch to an assisted mode seems clearly desirable . Diaphragmatic inactivity is considered to initiate vidd.13 interventions like intermittent spontaneous breathing or respiratory muscle training might be of benefit . Diaphragmatic contractions via phrenic nerve pacing have been successfully used in tetraplegic patients111, 112 and during cardiothoracic surgery113 with documented increases in mitochondrial respiration.114 while short periods of intermittent spontaneous breathing had little effect in a rodent model,12 adding a resistive inspiratory load in order to train the inspiratory muscles has shown promising results for patients in small series115, 116, 117 and improved weaning outcome in a randomized trial.118 the period of controlled mechanical ventilation should be kept short.82, 119 ventilation modes with sustained patient effort should be introduced early,39 as can be inferred from various animal experiments . Assist control ventilation modes (controlled mechanical ventilation with the possibility to trigger additional breaths) and pressure support ventilation (patient's breathing efforts supported by a preset pressure) may attenuate vidd in animals.9, 109 nevertheless, partial supportive modes can still cause vidd in the setting of overassist,8 so mode per se is not protective . On the contrary, in septic rabbits, controlled mechanical ventilation may protect from vidd61 and the use of modes that allow spontaneous breathing is contradictory to the documented benefit of neuromuscular blockers in the early course of severe acute respiratory distress syndrome (ards).120 the preferred ventilation mode, the optimal level of support (unloading), or the rate of support reduction for patients is currently unknown.39 spontaneous breathing is well maintained with socalled effort adapted modes 121 like neurally adjusted ventilatory assist (nava) or adaptive support ventilation (asv). Asv seems to mitigate deleterious effects of mechanical ventilation on the diaphragm of piglets.10 nava delivers assist in unison with the patient's inspiratory neural effort.93 as the support level is titrated against the patient's respiratory demand, patients are protected against overassist.122, 123 this approach was successfully used in various clinical icu settings97, 122, 124, 125 but most importantly in patients with critical illness myoneuropathy.97 currently, no medical treatment for vidd is available.126 antioxidants show benefits in animal models40, 71, 72, 127 and in a randomized trial in critically ill surgical patients.128 however, human data remain sparse . Targeting of proteolytic (especially proteasome) pathways in rodent models may further elucidate the pathophysiology and theoretically open novel therapeutic avenues . Inhibition of lysosomal proteases and calpain with leupeptin129 or the proteasome using bortezomib130 may be of interest as data indicate potential to completely or partially prevent vidd . However, results could not be reproduced with epoxomicin.131 overall, effects may be time dependent.129, 130, 131, 132 moreover, r548, a jak / stat inhibitor, maintained normal diaphragmatic contractility,133 and a recent foxo (forkhead boxo) animal knockout model suggests new potential therapeutic targets.134 while the effects of corticosteroids on vidd seem dose dependent conflicting,135, 136 propofol was accused as a causative agent in animal models.137 neuromuscular blockers do not to have additive effects on diaphragmatic dysfunction.136 moreover, moderate hypercapnia exerts protective effects on diaphragmatic muscular strength,138, 139 which fits to the overall concept of lung protective ventilation.140 in addition, the calcium sensitizer levosimendan may improve diaphragmatic neuromechanical efficiency in healthy volunteers, while the fast skeletal troponin activator ck2066260 improved diaphragmatic fibre strength ex vivo in an experimental human trial.24 further studies in critically ill patients with respiratory failure are warranted.141 in conclusion, prolonged periods of complete diaphragmatic rest should be avoided and diaphragmatic contractions preserved whenever possible . The clinical hallmark is respiratory muscle weakness which contributes to weaning failure and thus implies a significant health care burden . Major pathophysiological changes are disuse atrophy and microstructural changes including decreased protein synthesis, increased proteolysis, and oxidative stress, possibly linked to mitochondrial dysfunction . For the clinician, bedside ultrasound evaluation and assessment of the electrical diaphragmatic activity are promising tools for the diagnosis and monitoring of respiratory muscle dysfunction / vidd . Effort adapted ventilation modes may offer advantages . Whether this may lead to improved clinical outcomes needs to be established . The department of intensive care medicine has / had research contracts with orion corporation, abbott nutrition international, b. braun medical ag, csem sa, edwards lifesciences services gmbh, kenta biotech ltd, maquet critical care ab, omnicare clinical research ag and research and development / consulting contracts with edwards lifesciences sa, maquet critical care ab, and nestl . The money was paid into a departmental fund; the authors received no personal financial gain . Unrestricted educational grants from the following organizations for organizing a quarterly postgraduate educational symposium (berner forum for intensive care) were provided by: fresenius kabi; gsk; msd; lilly; baxter; astellas; astrazeneca; b.braun; csl behring; maquet; novartis; covidien; nycomed; pierre fabre pharma (robapharma); pfizer, orion pharma . All authors certify compliance with the ethical guidelines for authorship and publishing established by the journal of cachexia, sarcopenia, and muscle.142
The online version of this article (doi:10.1007/s40121 - 015 - 0094 - 6) contains supplementary material, which is available to authorized users . Extended spectrum -lactamases (esbl) are enzymes capable of hydrolyzing penicillins, broad - spectrum cephalosporins, and monobactams . These infections increase cost of care if inappropriate therapy, defined as non - susceptibility of pathogen to therapy by laboratory criteria, is administered . At present, the clinical and laboratory standards institute (clsi) and european committee on antimicrobial susceptibility testing (eucast) do not recommend routine screening for esbls or subsequent phenotypic confirmation testing that had been advocated previously [3, 4]. While this approach has been supported in the clinical practice setting by the introduction of reduced breakpoints for many beta - lactams, it may be of value, particularly when evaluating novel therapies, to understand the potency of various agents against esbl producers . However, if one utilizes esbl classification with aztreonam (atm), ceftriaxone (cro), or ceftazidime (caz) minimum inhibitory concentrations (mic) as a simple screen, without phenotypic confirmation via the clavulanate test, this approach may include other resistant genotypes (i.e., carbapenemase producers) that may not accurately predict the clinical utility of therapeutic agents under consideration . Phenotypic screening tests to detect resistance mechanisms such as carbapenemase production or ampc expression are available and can be used . While molecular testing would yield the most definite results, it is also cost prohibitive and unavailable to most clinical microbiological laboratories . In this study, we tested the recently us food and drug administration (fda)-approved compound, ceftolozane / tazobactam (c / t), which has demonstrated in vitro activity against some esbl organisms . The purpose of this study was to compare the impact of mic - based screening versus confirmatory testing of esbls on the susceptibility profile of c / t versus selected antimicrobials . In this study, 44 us hospitals collected non - duplicate, non - urine escherichia coli (n = 1306) and klebsiella pneumoniae (n = 1205) over the period of june 2013 and october 2014 . Participating sites identified the organisms using their established method and then transferred each isolate to trypticase soy agar slants for shipping . Isolates were transferred onto trypticase soy agar plates containing 5% sheep blood at the central processing laboratory (center for anti - infective research and development, hartford hospital, hartford hospital, hartford, ct, usa) for mic determination using broth microdilution as described by clsi . Mics were determined for c / t, cefepime (fep), cro, caz, ciprofloxacin (cip), atm, ertapenem (etp), piperacillin / tazobactam (tzp), meropenem (mem), and tobramycin (tob) against this collection of 2511 contemporary clinical enterobacteriaceae isolates . C / t was provided by cubist pharmaceuticals, while all others antibiotics were purchased from sigma - aldrich (st . E. coli 25922 and pseudomonas aeruginosa 27853 were utilized as quality control strains as recommended by clsi . Clsi states that the initial mic - based esbl screening for e. coli and k. pneumoniae should be performed by evaluating the mic profile of one of the following drugs: cefpodoxime 4 g / ml, caz 1 g / ml, atm 1 g / ml, or cefotaxime 1 g / ml . It is noted that the use of more than one of these agents will improve detection of esbls . We defined a positive esbl screen as having an mic of 1 g / ml to 2 of the following: atm, cro, or caz . Subsequent clsi defined, phenotypic, esbl confirmation studies were undertaken using caz and cefotaxime with and without clavulanate . Probable carbapenemase - producing organisms were excluded based on etp (1 g / ml) or mem (2 g / ml) susceptibility profiles . While molecular - based confirmation of these enzymes would have been definitive, methodologies readily available to the clinical microbiology laboratory were utilized . Recently, c / t has been approved in the us and susceptibility interpretative criteria have now been provided by the fda of 2 g / ml for the enterobacteriaceae . This surveillance program was reviewed by the institutional review board (irb) at the coordinating center, hartford hospital . Since all samples were collected as part of routine medical care and no interventions were undertaken as a result of this testing, informed consent was deemed unnecessary by the irb . Four - hundred and forty - two (18%) isolates [e. coli (n = 231), k. pneumoniae (n = 211)] screened positive for esbl production based on mic - based criteria . Of these, 274 (62%) isolates [e. coli (n = 146), k. pneumoniae (n = 128)] were phenotypically confirmed positive for esbl production . With further exclusion of the 28 (10%) isolates [e. coli (n = 13), k. pneumoniae (n = 14)] that demonstrated carbapenem non - susceptible, a left shift in the mic distribution (i.e., enhanced potency) against phenotypically confirmed esbl was noted for c / t, etp, tzp, and mem (table 1). Figure 1 highlights these screening- and confirmation - based observations for c / t and tzp . Improvements in% s between 8% and 17% were noted for confirmed versus screened - positive esbl isolates for c / t, etp, tzp, and mem . (table 1) overall,% s of the other -lactams, tob and cip, was poor against phenotypically confirmed esbls . Comparing the mic90 values for all esbl mic - based screen - positive versus phenotypically confirmed positive, we observed a reduction of twofold for c / t, sixfold for etp, and eightfold for mem (table 2).table 1percent susceptibility of all agents against mic - based screen - positive (screen +) and phenotypically confirmed (confirmed +) esbl escherichia coli and klebsiella pneumoniae isolatespercent susceptibility for antimicrobialsc / tfepcrocazcipatmetptzpmemtoball screen + (n = 442)64211018231674537945 confirmed + (n = 274)7582914691619342 confirmed +, carbapenem ns removed (n = 246)829291371006710042 e. coli screen + (n = 231)79291324272386699054 confirmed + (n = 146)889310101096759847 confirmed +, carbapenem ns removed (n = 139)91941110101007810047 k. pneumoniae screen + (n = 211)471251118962366534 confirmed + (n = 128)6171718282468835 confirmed +, carbapenem ns removed (n = 107)717171621005210036 atm aztreonam, caz ceftazidime, cip ciprofloxacin, cro ceftriaxone, c / t ceftolozane / tazobactam, etp ertapenem, esbl extended spectrum -lactamase, fep cefepime, mem meropenem, mic minimum inhibitory concentration, ns non - susceptible, tob tobramycin, tzp piperacillin / tazobactamfig . 1the mic distribution of c / t and tzp for the mic - based screen - positive isolates (screen +), phenotypically confirmed positive (confirmed +) and confirmed + with carbapenem ns isolates removed (confirmed +, no carbapenem ns) of all enterobacteriaceae . C / t ceftolozane / tazobactam, mic minimum inhibitory concentration, ns non - susceptible, tzp piperacillin / tazobactamtable 2mic90 (g / ml) of all agents against mic - based screen - positive (screen +) and phenotypically confirmed (confirmed +) escherichia coli and klebsiella pneumoniae isolatesmic90 (g / ml) for antimicrobialsc / tfepcrocazcipatmetptzpmemtoball screen + (n = 442)12812812812832128325123264 confirmed + (n = 274)32128128128321280.55120.12564 confirmed +, carbapenem ns removed (n = 246)212812812832640.125640.06464 atm aztreonam, caz ceftazidime, cip ciprofloxacin, cro ceftriaxone, c / t ceftolozane / tazobactam, etp ertapenem, esbl extended spectrum -lactamase, fep cefepime, mem meropenem, mic minimum inhibitory concentration, ns non - susceptible, tob tobramycin, tzp piperacillin / tazobactam percent susceptibility of all agents against mic - based screen - positive (screen +) and phenotypically confirmed (confirmed +) esbl escherichia coli and klebsiella pneumoniae isolates atm aztreonam, caz ceftazidime, cip ciprofloxacin, cro ceftriaxone, c / t ceftolozane / tazobactam, etp ertapenem, esbl extended spectrum -lactamase, fep cefepime, mem meropenem, mic minimum inhibitory concentration, ns non - susceptible, tob tobramycin, tzp piperacillin / tazobactam the mic distribution of c / t and tzp for the mic - based screen - positive isolates (screen +), phenotypically confirmed positive (confirmed +) and confirmed + with carbapenem ns isolates removed (confirmed +, no carbapenem ns) of all enterobacteriaceae . C / t ceftolozane / tazobactam, mic minimum inhibitory concentration, ns non - susceptible, tzp piperacillin / tazobactam mic90 (g / ml) of all agents against mic - based screen - positive (screen +) and phenotypically confirmed (confirmed +) escherichia coli and klebsiella pneumoniae isolates atm aztreonam, caz ceftazidime, cip ciprofloxacin, cro ceftriaxone, c / t ceftolozane / tazobactam, etp ertapenem, esbl extended spectrum -lactamase, fep cefepime, mem meropenem, mic minimum inhibitory concentration, ns non - susceptible, tob tobramycin, tzp piperacillin / tazobactam after confirmatory esbl testing using the clavulanate test, 38% of strains identified as potential esbl producers using a simple mic screen were excluded . For agents with potential activity against esbls such as c / t, tzp, etp, and mem, reduced susceptibility the reason for this discordance is that carbapenemase producers, ampc, and other types of resistance would not be differentiated by mic - based screen testing alone . As such, although mic screening selects for resistant organisms, inclusion of these other genotypes may falsely under - predict the potency of several antibiotics against esbl producers and if such a method is used during published surveillance studies, it may limit applicability of these data to geographic areas not plagued with carbapenemase producers . For this reason, in scenarios, such as the clinical laboratory, when molecular testing is not available, clavulanate - based phenotypic confirmatory testing is recommended to better evaluate empiric therapy options against esbl . David p. nicolau is on the speaker bureau for cubist and has received grants from cubist . This surveillance program was reviewed by the institutional review board (irb) at the coordinating center, hartford hospital . Since all samples were collected as part of routine medical care and no interventions were undertaken as a result of this testing, informed consent was deemed unnecessary by the irb . This article is distributed under the terms of the creative commons attribution - noncommercial 4.0 international license (http://creativecommons.org/licenses/by-nc/4.0/), which permits any noncommercial use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the creative commons license, and indicate if changes were made.
Rising pharmaceutical prices have been the subject of intense research and debate over the last several years as drug therapies play an ever - increasing role in our treatment of illness and disease . A significant proportion of expenditures incurred by medicaid, the insurer of over 40 million medically and categorically needy persons, is devoted to paying for prescription drugs . Recently passed legislation, namely the medicare prescription drug, improvement and modernization act of 2003 (mma), will likely have a considerable impact on medicaid expenditures, as medicare will become the primary drug insurer for approximately 6 million beneficiaries eligible for both the medicare and medicaid programs (known as dually eligible beneficiaries). The trends and analyses contained within include information on dually eligible beneficiaries, as well as all classes of medicaid beneficiaries . Medicaid, the nation's largest program that provides health care to the poor and near - poor, was created in 1965 . It is jointly funded by the federal government, along with state governments, and is intended to assist people who meet certain eligibility criteria . Each state has its own guidelines that determine what medical services it covers, however, there are certain services that must be covered in order for states to receive federal funds . One service that is not required of states is coverage for outpatient prescription drugs, yet every state has elected to provide such coverage for at least some of their beneficiaries . Thinly - stretched state budgets, along with spiraling drug costs, have compelled states to implement certain cost - control mechanisms as they strive to balance the provision of quality health care with the need for fiscal restraint . Among the list of cost - control measures currently employed by many states are monthly utilization limits, automatic generic substitution, creation of maximum allowable cost (mac) programs, and prior authorization requirements (national pharmaceutical council, 2002). Spending trends and the impacts some of these provisions have on state budgets and on medicaid beneficiaries are scrutinized in this issue of the health care financing review . The contribution from baugh, pine, blackwell, and ciborowski is an examination of medicaid prescription drug costs throughout the decade of the 1990s . During that 10-year period, spending for drugs was no exception as it increased from $4.4 billion in 1990 to $20 billion in 2000 . On a per recipient basis, spending soared from $256 in 1990 to $975 in 2000 . Among the disabled find that increases in drug spending averaged better than 20 percent for the period studied . In fact, this group had the highest medicaid drug payments of any of the other eligibility groups including the aged, children, and adults . In 2000, drug spending for the disabled topped $11.6 billion, which represented 58 percent of medicaid's drug payments . They also report that the number of medicaid recipients increased by just under 2 percent per year throughout the 1990s . The largest group of medication recipients was children (8.3 million), more than double the number of adults receiving medicaid drugs (4 million). With medicaid drug bills ascending at double - digit rates, cost control policies take on added importance . This issue features an analysis of potential cost containment strategies as they relate to generic drugs . The objective of abramson, harrington, missmar, li, and mendelson was to characterize the mac lists of five states and compare them to each other, as well as to drug prices that are regulated by the federal upper limits (ful) program . Both the mac and the ful programs contribute to savings garnered by medicaid by encouraging pharmacies to dispense generic medications rather than brand - name drugs . They also influence prices by limiting the reimbursements medicaid can make for generic drugs . Comparing mac programs with one another, the researchers conclude there is a relatively high degree of variation in the breadth, depth, and price aggressiveness across these programs . Discussions with medicaid officials indicate the reasons for these differences are the tedious nature of mac list creation and administration, and state mac programs frequently have trouble acquiring reliable drug pricing data . From an administration perspective, the investigators recommend that states focus their mac list efforts on those drugs with the highest sales volume . Moreover, states should ensure that their mac lists contain as many forms and strengths for covered drugs as possible to reduce price variability within drug name . Finally, states should collaborate with each other on their mac list operations . To address the data acquisition problem, abramson et al . Additionally, states should gather pricing information from alternative sources, as well as implement formal policies that require price disclosure . In comparing mac to ful lists, the authors also report that state mac lists typically contain more drugs and assign lower prices to those medications . This finding is attributable to mac programs usually having a greater degree of latitude on issues of quantity and price . For instance, fuls drugs account for almost two - thirds of total nationwide generic drug sales . Also, some state mac lists include medications also found on fuls lists, but ful prices are considerably higher . Removing barriers to drug treatment is likely to have certain access effects such as increasing the number of patients on a particular drug, or changing the characteristics of the treated population . To examine this, and other issues related to drug access, mccombs, mulani, and gibson analyzed the various impacts of offering open access to second - generation antipsy chotics in the california medicaid program (medi - cal). They found a substantial increase in the likelihood that a patient in the medi - cal program would start antipsychotic drug therapy without having a mental health condition diagnosed in the previous 6 months when there was open access to such therapy (38.4 percent closed access; 51.7 percent open access). Moreover, the authors found a sizable increase in the number of episodes initiated per month once open access was granted to the second - generation antipsychotics . For patients restarting therapy (with the newly approved medications), this effect appeared to be temporary as the rate of restart episodes eventually fell below the levels observed prior to open access . Similar results were observed for patients who switched to the newer medicines without a break from their usual medicine, and for those who augmented their original therapy with the newer drugs . With respect to changing characteristics of the treatment population, mccombs et al . Note that, although the average age in the open access period was slightly higher compared to the mean age for the closed access period (44.8 years closed; 45.1 years open access), the proportion of patients with an episode of therapy under age 25 nearly doubled from 7.7 to 12.4 percent . Similarly, although less dramatic, was an increase in the proportion of beneficiaries age 65 or over (15.8 percent closed; 18.4 percent open). The authors conclude that while the average monthly cost of treating patients taking antipsychotics increased across all service types and the persistence on the initial drug use declined under open access, reductions in the future use of nursing home care and psychiatric hospitalizations helped to offset these higher costs . Questions related to drug prices and their availability are pondered worldwide (danzon and furukawa, 2003). This issue features an article by duckett on australia's pharmaceutical benefit scheme (pbs), one branch of that country's universal health insurance arrangement . Growing from a list of 139 life saving and disease preventing drugs in 1939, duckett reports that in 2003, the pbs covered over 600 generic products marketed as 2,602 different brands . Frequently, gaining access to the drugs on the list requires contact with the administering agency of the pbs and the health insurance commission . There are also times when the medical practitioner is required to certify the presence of certain indications to justify the need for a medication . Duckett states when a pharmaceutical is listed on the pbs under several different brand names, pharmacists are permitted to dispense generically identical forms of the drug . As a rule, the pbs will only pay for the least expensive version of that drug while the consumer must pay any additional costs associated with obtaining a specific branded drug . His commentary indicates expenditures on pharmaceuticals have been growing faster than australia's overall economy in recent years . Moreover, prescription drug expenditures are the fastest growing segment of that country's health care bill, averaging 15 to 20 percent growth per year . In general, the australian blueprint for providing pharmaceuticals performs well with respect to several criteria including equity and efficiency . From an equity perspective, the pbs helps to minimize financial barriers to drugs through low copays for australians age 65 or over . He cites a study that showed just 2 percent of australia's aged population reported not filling a prescription due to cost . From an efficiency point of view, the pbs is successful due, in part, to its requirement that all new drugs face a rigorous cost - effectiveness test prior to being listed on the pbs . Some argue that by passing this test, any added expenditures to the program are likely offset by savings in other sectors of health care or by increases in productivity . The medicaid highlights by tepper and lied provide a somewhat different perspective on drug spending increases . Tepper and lied illustrate that ffs drug expenditures, as a percent of ffs total expenditures, climbed to over 11 percent in 2001 . That compares to a rate of about 6 percent in 1985 . At the state level in 2001, california led all others with approximately $3.1 billion in total drug spending, followed by new york with $2.9 billion . New jersey led all states in prescriptions per beneficiary with 33, despite being ninth in the nation on total drug spending ($0.64 billion). Medicaid, the nation's largest program that provides health care to the poor and near - poor, was created in 1965 . It is jointly funded by the federal government, along with state governments, and is intended to assist people who meet certain eligibility criteria . Each state has its own guidelines that determine what medical services it covers, however, there are certain services that must be covered in order for states to receive federal funds . One service that is not required of states is coverage for outpatient prescription drugs, yet every state has elected to provide such coverage for at least some of their beneficiaries . Thinly - stretched state budgets, along with spiraling drug costs, have compelled states to implement certain cost - control mechanisms as they strive to balance the provision of quality health care with the need for fiscal restraint . Among the list of cost - control measures currently employed by many states are monthly utilization limits, automatic generic substitution, creation of maximum allowable cost (mac) programs, and prior authorization requirements (national pharmaceutical council, 2002). Spending trends and the impacts some of these provisions have on state budgets and on medicaid beneficiaries are scrutinized in this issue of the health care financing review . The contribution from baugh, pine, blackwell, and ciborowski is an examination of medicaid prescription drug costs throughout the decade of the 1990s . During that 10-year period, spending for drugs was no exception as it increased from $4.4 billion in 1990 to $20 billion in 2000 . On a per recipient basis, spending soared from $256 in 1990 to $975 in 2000 . Among the disabled find that increases in drug spending averaged better than 20 percent for the period studied . In fact, this group had the highest medicaid drug payments of any of the other eligibility groups including the aged, children, and adults . In 2000, drug spending for the disabled topped $11.6 billion, which represented 58 percent of medicaid's drug payments . They also report that the number of medicaid recipients increased by just under 2 percent per year throughout the 1990s . The largest group of medication recipients was children (8.3 million), more than double the number of adults receiving medicaid drugs (4 million). With medicaid drug bills ascending at double - digit rates, cost control policies take on added importance . This issue features an analysis of potential cost containment strategies as they relate to generic drugs . The objective of abramson, harrington, missmar, li, and mendelson was to characterize the mac lists of five states and compare them to each other, as well as to drug prices that are regulated by the federal upper limits (ful) program . Both the mac and the ful programs contribute to savings garnered by medicaid by encouraging pharmacies to dispense generic medications rather than brand - name drugs . They also influence prices by limiting the reimbursements medicaid can make for generic drugs . Comparing mac programs with one another, the researchers conclude there is a relatively high degree of variation in the breadth, depth, and price aggressiveness across these programs . Discussions with medicaid officials indicate the reasons for these differences are the tedious nature of mac list creation and administration, and state mac programs frequently have trouble acquiring reliable drug pricing data . From an administration perspective, the investigators recommend that states focus their mac list efforts on those drugs with the highest sales volume . Moreover, states should ensure that their mac lists contain as many forms and strengths for covered drugs as possible to reduce price variability within drug name . Finally, states should collaborate with each other on their mac list operations . To address the data acquisition problem, abramson et al . Additionally, states should gather pricing information from alternative sources, as well as implement formal policies that require price disclosure . In comparing mac to ful lists, the authors also report that state mac lists typically contain more drugs and assign lower prices to those medications . This finding is attributable to mac programs usually having a greater degree of latitude on issues of quantity and price . For instance, fuls drugs account for almost two - thirds of total nationwide generic drug sales . Also, some state mac lists include medications also found on fuls lists, but ful prices are considerably higher . Removing barriers to drug treatment is likely to have certain access effects such as increasing the number of patients on a particular drug, or changing the characteristics of the treated population . To examine this, and other issues related to drug access, mccombs, mulani, and gibson analyzed the various impacts of offering open access to second - generation antipsy chotics in the california medicaid program (medi - cal). They found a substantial increase in the likelihood that a patient in the medi - cal program would start antipsychotic drug therapy without having a mental health condition diagnosed in the previous 6 months when there was open access to such therapy (38.4 percent closed access; 51.7 percent open access). Moreover, the authors found a sizable increase in the number of episodes initiated per month once open access was granted to the second - generation antipsychotics . For patients restarting therapy (with the newly approved medications), this effect appeared to be temporary as the rate of restart episodes eventually fell below the levels observed prior to open access . Similar results were observed for patients who switched to the newer medicines without a break from their usual medicine, and for those who augmented their original therapy with the newer drugs . With respect to changing characteristics of the treatment population, mccombs et al . Note that, although the average age in the open access period was slightly higher compared to the mean age for the closed access period (44.8 years closed; 45.1 years open access), the proportion of patients with an episode of therapy under age 25 nearly doubled from 7.7 to 12.4 percent . Similarly, although less dramatic, was an increase in the proportion of beneficiaries age 65 or over (15.8 percent closed; 18.4 percent open). The authors conclude that while the average monthly cost of treating patients taking antipsychotics increased across all service types and the persistence on the initial drug use declined under open access, reductions in the future use of nursing home care and psychiatric hospitalizations helped to offset these higher costs . Questions related to drug prices and their availability are pondered worldwide (danzon and furukawa, 2003). This issue features an article by duckett on australia's pharmaceutical benefit scheme (pbs), one branch of that country's universal health insurance arrangement . Growing from a list of 139 life saving and disease preventing drugs in 1939, duckett reports that in 2003, the pbs covered over 600 generic products marketed as 2,602 different brands . Frequently, gaining access to the drugs on the list requires contact with the administering agency of the pbs and the health insurance commission . There are also times when the medical practitioner is required to certify the presence of certain indications to justify the need for a medication . Duckett states when a pharmaceutical is listed on the pbs under several different brand names, pharmacists are permitted to dispense generically identical forms of the drug . As a rule, the pbs will only pay for the least expensive version of that drug while the consumer must pay any additional costs associated with obtaining a specific branded drug . His commentary indicates expenditures on pharmaceuticals have been growing faster than australia's overall economy in recent years . Moreover, prescription drug expenditures are the fastest growing segment of that country's health care bill, averaging 15 to 20 percent growth per year . In general, the australian blueprint for providing pharmaceuticals performs well with respect to several criteria including equity and efficiency . From an equity perspective, the pbs helps to minimize financial barriers to drugs through low copays for australians age 65 or over . He cites a study that showed just 2 percent of australia's aged population reported not filling a prescription due to cost . From an efficiency point of view, the pbs is successful due, in part, to its requirement that all new drugs face a rigorous cost - effectiveness test prior to being listed on the pbs . Some argue that by passing this test, any added expenditures to the program are likely offset by savings in other sectors of health care or by increases in productivity . The medicaid highlights by tepper and lied provide a somewhat different perspective on drug spending increases . Tepper and lied illustrate that ffs drug expenditures, as a percent of ffs total expenditures, climbed to over 11 percent in 2001 . That compares to a rate of about 6 percent in 1985 . At the state level in 2001, california led all others with approximately $3.1 billion in total drug spending, followed by new york with $2.9 billion . New jersey led all states in prescriptions per beneficiary with 33, despite being ninth in the nation on total drug spending ($0.64 billion).
The prevalence of papillary thyroid microcarcinoma is increasing in recent years, and a large proportion of these patients are young women . Therefore, minimally invasive endoscopic thyroidectomy with central neck dissection (cnd) emerged and showed well - accepted results with improved cosmetic outcome, accelerated healing, and comforting the patients . Robotic thyroidectomy via a transaxillary approach was first described by kang et al . In 2009 . Thereafter, various operative approaches, including axillary - breast - anterior chest approach, transoral periosteal approach, and retroauricular approach, were introduced by many surgeons . The aim of this prospective randomized study was to evaluate the safety and effectiveness of robotic total thyroidectomy with cnd via bilateral axillo - breast approach (baba), compared to the conventional low - collar incision method for the treatment of papillary thyroid microcarcinoma . The study was approved by the research ethics committee of jinan military general hospital of people's liberation army (pla). The prospective randomized clinical study involved 100 patients with papillary thyroid microcarcinoma (17 males and 83 females), with an average age of 41.2 years (range: 2163 years), from march 2014 to january 2015, in jinan military general hospital of pla . Before grouping, we described the operative methods and relative complications between robotic thyroidectomy and open thyroidectomy to the patients . Then, the patients were randomly given a number and assigned to the corresponding group: 50 patients received robotic total thyroidectomy with cnd via baba (using da vinci si surgical system, intuitive surgical inc ., sunnyvale, ca, usa), and 50 were operated with conventional low - collar incision approach . The inclusion criteria for this prospective randomized study were as follows: (1) intrathyroidal papillary carcinoma smaller than 10 mm in diameter; and (2) no evidence of central or lateral lymph node metastasis . All patients were evaluated preoperatively via sonography, fine - needle aspiration cytology, thyroid function, and/or computed tomography (ct). The exclusion criteria included a history of thyroiditis, graves disease, previous neck or thyroid surgeries, irradiation history, obesity (body mass index> 30 kg / m), enlarged thyroid gland detected by ultrasound examination (one thyroid lobe volume> 40 ml), and a possible extrathyroidal extension or abutment on the thyroid capsule, especially when occurrence was near the tracheoesophageal groove detected by preoperative sonography or ct . We analyzed the medical records and evaluated surgical outcomes including the total operative time, estimated intraoperative blood loss, numbers of lymph node removed, visual analog scale (vas), postoperative hospital stay time, complications, and numerical scoring system (nss) in all patients . The cosmetic results were recorded using a scoring system ranging from 1 to 4 levels (1: extremely, 2: fairly, 3: normal, and 4: not at all), and nss was used for patients self - assessment in the outpatient clinic at 1 month and 3 months after the operation to grade the cosmetic outcome between 0 and 10 . All patients were followed up in outpatient clinic at 1, 3, and 6 months, and then every 6 months to detect thyroid function and thyroglobulin (tg) level . Robotic total thyroidectomy under general endotracheal anesthesia, patients were placed on the operating table in the supine position with the neck extended slightly and both arms slightly abducted to allow insertion of the axillary port . After draping, we draw the instrument arm trajectory lines and working area on patient's chest and neck for reference [figure 1]. Using a 23-gauge spinal needle, we injected diluted epinephrine solutions (1:200,000, 100 ml) and ropivacaine hydrochloride (100 mg) into the subcutaneous areas of both breasts and axilla, as well as the subplatysmal layer of the neck for easy dissection and less bleeding . Drawing instrument arm trajectory lines and working area . Baba included 5 mm or 8 mm axillary crease incisions and two circumareolar incisions about 8 mm on the left side and 12 mm on the right side, respectively . The 12-mm circumareolar incision was used as a camera port and can be moved to the left breast if the surgeon prefers . Blunt dissection was carefully performed with a vascular tunneler from the incision superiorly to the suprasternal fossa [figure 1, area a] in trajectory lines and laterally to the medial border of the sternocleidomastoid muscle . Once the working space was found, the robotic column was placed parallel to the camera port and trocars were inserted into tunnels . Place the camera port first (e port) using a disposable 12 mm trocar . Place the instrument arm 2 or 3 port in the trocars of the axilla followed by docking the robot cart [figure 2]. Following above procedures, the 30 endoscope was placed in the circumareolar area on the side of the lesion through a 12 mm trocar . Then, the harmonic scalpel was placed on the circumareolar area on the contralateral side through an 8 mm trocar . Furthermore, a 5 mm maryland dissector was placed in the axillary area (same side as the lesion) and 5 mm prograsp forceps (intuitive inc ., sunnyvale, ca, usa) were placed in the axillary area on the contralateral side, as shown in figure 2 . The working space was extended superiorly, laterally, and inferiorly to the level of the thyroid cartilage, the medial border of each sternocleidomastoid muscle, and the anterior chest, respectively [figure 1, areas a and b]. After the sternocleidomastoid muscle and the strap muscle were identified, we cut through the linea alba cervicalis incision with a harmonic scalpel between the strap muscles from the suprasternal notch to thyroid cartilage . The general principle of surgical procedures for robotic total thyroidectomy was the same as conventional open thyroidectomy . We should identify the tracheal wall, expose it as a midline landmark, and dissect the lower pole of thyroid from the adipose tissue or trachea . During this procedure, we should avoid injuries to the tracheal wall . With delicate blunt dissection, both thyroid lobes were exposed and examined for pathological changes, and with a strictly median orientation, the primary isthmus was removed using the harmonic scalpel . The upper pole of the thyroid was drawn downward, and superior thyroid vessels were identified and individually divided close to the thyroid capsule to avoid injuring the external branch of the superior laryngeal nerve . During dissection, the superior parathyroid gland should be identified and left intact in situ [figures 3 and 4]. Identifying and protecting parathyroid glands and the recurrent laryngeal nerve . Robotic total thyroidectomy with central neck dissection showing the course of the ipsilateral (right) recurrent laryngeal nerve and the superior parathyroid gland . The thyroid was retracted medially, and the middle thyroid vein was identified and coagulated off carefully . Careful dissection was performed to avoid the inferior thyroid artery and the recurrent laryngeal nerve (rln). We can easily identify and preserve the rlns under magnified 1015 times surgical view [figures 3 and 4]. After the inferior thyroid vein and thyrothymic ligament had been recognized, the thyroid was retracted upward and medially . Finally, proper application of the harmonic scalpel can successfully remove the thyroid gland from the trachea . Circular resection is recognized as a safe and highly effective method in robotic thyroidectomy via baba for bilateral thyroid lesions . Particular attention should be paid to ensure sufficient distance of the heated harmonic scalpel from the rln and the visible parathyroids . We dissected the level 4 lymph nodes (delphian / prelaryngeal, pretracheal, and paratracheal lymph nodes) using the robotic system . During ipsilateral or bilateral cnd, the lymph nodes of the central compartment were carefully dissected to avoid injuries to the rln and parathyroids . Application of carbon nanoparticles suspensions injection can stain the lymph nodes of the center compartment while leaving the parathyroid glands unstained (because of the negative development of parathyroid glands to carbon nanoparticles, chongqing lummy pharmaceuticals, china), thus helping to identify and protect parathyroid glands and the rln [figures 3 and 4], as well as facilitating the identification and clearance of lymph nodes . The resected specimen was extracted through enlarging the axillary skin incision by a specimen pouch . Vacuum - assisted draining system should be placed in the operative area though the axilla tunnel . The wounds were closed with 5 - 0 absorbable monofilament sutures using an atraumatic needle, followed by placement of steri - strips . Conventional open approach patients in the conventional open approach group underwent conventional open total thyroidectomy with central lymph node dissection in the supine position with neck extension under general endotracheal anesthesia . The lower layer of the platysma was cut through, and subplatysmal flap dissection was performed from the sternal notch to the level of the thyroid cartilage superiorly . The linea alba cervicalis of the strap muscles was divided vertically, and the thyroid gland was exposed . Dissection of the thyroid and central lymph nodes was performed with harmonic focus (ethicon endo - surgery inc .,, every effort was made to identify and preserve all parathyroid glands and the rlns with the negative development of parathyroid glands by carbon nanoparticles [figure 5]. Parathyroid glands were transplanted in the sternocleidomastoid muscle once we found that the blood supply to the glands was compromised . Then, the surgical field was douched by 500 ml sterile distilled water (42c). The wounds were closed by an atraumatic needle with 5 - 0 absorbable sutures . Identifying and exposing recurrent laryngeal nerve, the negative development of parathyroid glands by carbon nanoparticles . Patients with no complications were usually discharged on day 3 to day 5 after the surgery . Descriptive statistics of quantitative variables, mean standard deviation (sd) and range were used to describe central tendency and dispersion . Fisher's exact test was used if the assumptions of chi - square test were violated . Independent samples t - test was used to compare the level of quantitative variables between the two groups . The study was approved by the research ethics committee of jinan military general hospital of people's liberation army (pla). The prospective randomized clinical study involved 100 patients with papillary thyroid microcarcinoma (17 males and 83 females), with an average age of 41.2 years (range: 2163 years), from march 2014 to january 2015, in jinan military general hospital of pla . Before grouping, we described the operative methods and relative complications between robotic thyroidectomy and open thyroidectomy to the patients . Then, the patients were randomly given a number and assigned to the corresponding group: 50 patients received robotic total thyroidectomy with cnd via baba (using da vinci si surgical system, intuitive surgical inc ., sunnyvale, ca, usa), and 50 were operated with conventional low - collar incision approach . The inclusion criteria for this prospective randomized study were as follows: (1) intrathyroidal papillary carcinoma smaller than 10 mm in diameter; and (2) no evidence of central or lateral lymph node metastasis . All patients were evaluated preoperatively via sonography, fine - needle aspiration cytology, thyroid function, and/or computed tomography (ct). The exclusion criteria included a history of thyroiditis, graves disease, previous neck or thyroid surgeries, irradiation history, obesity (body mass index> 30 kg / m), enlarged thyroid gland detected by ultrasound examination (one thyroid lobe volume> 40 ml), and a possible extrathyroidal extension or abutment on the thyroid capsule, especially when occurrence was near the tracheoesophageal groove detected by preoperative sonography or ct . We analyzed the medical records and evaluated surgical outcomes including the total operative time, estimated intraoperative blood loss, numbers of lymph node removed, visual analog scale (vas), postoperative hospital stay time, complications, and numerical scoring system (nss) in all patients . The cosmetic results were recorded using a scoring system ranging from 1 to 4 levels (1: extremely, 2: fairly, 3: normal, and 4: not at all), and nss was used for patients self - assessment in the outpatient clinic at 1 month and 3 months after the operation to grade the cosmetic outcome between 0 and 10 . All patients were followed up in outpatient clinic at 1, 3, and 6 months, and then every 6 months to detect thyroid function and thyroglobulin (tg) level . Robotic total thyroidectomy under general endotracheal anesthesia, patients were placed on the operating table in the supine position with the neck extended slightly and both arms slightly abducted to allow insertion of the axillary port . After draping, we draw the instrument arm trajectory lines and working area on patient's chest and neck for reference [figure 1]. Using a 23-gauge spinal needle, we injected diluted epinephrine solutions (1:200,000, 100 ml) and ropivacaine hydrochloride (100 mg) into the subcutaneous areas of both breasts and axilla, as well as the subplatysmal layer of the neck for easy dissection and less bleeding . Drawing instrument arm trajectory lines and working area . Baba included 5 mm or 8 mm axillary crease incisions and two circumareolar incisions about 8 mm on the left side and 12 mm on the right side, respectively . The 12-mm circumareolar incision was used as a camera port and can be moved to the left breast if the surgeon prefers . Blunt dissection was carefully performed with a vascular tunneler from the incision superiorly to the suprasternal fossa [figure 1, area a] in trajectory lines and laterally to the medial border of the sternocleidomastoid muscle . Once the working space was found, the robotic column was placed parallel to the camera port and trocars were inserted into tunnels . Place the camera port first (e port) using a disposable 12 mm trocar . Place the instrument arm 2 or 3 port in the trocars of the axilla followed by docking the robot cart [figure 2]. Following above procedures, the 30 endoscope was placed in the circumareolar area on the side of the lesion through a 12 mm trocar . Then, the harmonic scalpel was placed on the circumareolar area on the contralateral side through an 8 mm trocar . Furthermore, a 5 mm maryland dissector was placed in the axillary area (same side as the lesion) and 5 mm prograsp forceps (intuitive inc ., sunnyvale, ca, usa) were placed in the axillary area on the contralateral side, as shown in figure 2 . The working space was extended superiorly, laterally, and inferiorly to the level of the thyroid cartilage, the medial border of each sternocleidomastoid muscle, and the anterior chest, respectively [figure 1, areas a and b]. After the sternocleidomastoid muscle and the strap muscle were identified, we cut through the linea alba cervicalis incision with a harmonic scalpel between the strap muscles from the suprasternal notch to thyroid cartilage . The general principle of surgical procedures for robotic total thyroidectomy was the same as conventional open thyroidectomy . We should identify the tracheal wall, expose it as a midline landmark, and dissect the lower pole of thyroid from the adipose tissue or trachea . During this procedure both thyroid lobes were exposed and examined for pathological changes, and with a strictly median orientation, the primary isthmus was removed using the harmonic scalpel . The upper pole of the thyroid was drawn downward, and superior thyroid vessels were identified and individually divided close to the thyroid capsule to avoid injuring the external branch of the superior laryngeal nerve . During dissection, the superior parathyroid gland should be identified and left intact in situ [figures 3 and 4]. Identifying and protecting parathyroid glands and the recurrent laryngeal nerve . Robotic total thyroidectomy with central neck dissection showing the course of the ipsilateral (right) recurrent laryngeal nerve and the superior parathyroid gland . The thyroid was retracted medially, and the middle thyroid vein was identified and coagulated off carefully . Careful dissection was performed to avoid the inferior thyroid artery and the recurrent laryngeal nerve (rln). We can easily identify and preserve the rlns under magnified 1015 times surgical view [figures 3 and 4]. After the inferior thyroid vein and thyrothymic ligament had been recognized, the thyroid was retracted upward and medially . Finally, proper application of the harmonic scalpel can successfully remove the thyroid gland from the trachea . Circular resection is recognized as a safe and highly effective method in robotic thyroidectomy via baba for bilateral thyroid lesions . Particular attention should be paid to ensure sufficient distance of the heated harmonic scalpel from the rln and the visible parathyroids . We dissected the level 4 lymph nodes (delphian / prelaryngeal, pretracheal, and paratracheal lymph nodes) using the robotic system . During ipsilateral or bilateral cnd, the lymph nodes of the central compartment were carefully dissected to avoid injuries to the rln and parathyroids . Application of carbon nanoparticles suspensions injection can stain the lymph nodes of the center compartment while leaving the parathyroid glands unstained (because of the negative development of parathyroid glands to carbon nanoparticles, chongqing lummy pharmaceuticals, china), thus helping to identify and protect parathyroid glands and the rln [figures 3 and 4], as well as facilitating the identification and clearance of lymph nodes . The resected specimen was extracted through enlarging the axillary skin incision by a specimen pouch . Vacuum - assisted draining system should be placed in the operative area though the axilla tunnel . The wounds were closed with 5 - 0 absorbable monofilament sutures using an atraumatic needle, followed by placement of steri - strips . Conventional open approach patients in the conventional open approach group underwent conventional open total thyroidectomy with central lymph node dissection in the supine position with neck extension under general endotracheal anesthesia . The lower layer of the platysma was cut through, and subplatysmal flap dissection was performed from the sternal notch to the level of the thyroid cartilage superiorly . The linea alba cervicalis of the strap muscles was divided vertically, and the thyroid gland was exposed . Dissection of the thyroid and central lymph nodes was performed with harmonic focus (ethicon endo - surgery inc .,, every effort was made to identify and preserve all parathyroid glands and the rlns with the negative development of parathyroid glands by carbon nanoparticles [figure 5]. Parathyroid glands were transplanted in the sternocleidomastoid muscle once we found that the blood supply to the glands was compromised . Then, the surgical field was douched by 500 ml sterile distilled water (42c). The wounds were closed by an atraumatic needle with 5 - 0 absorbable sutures . Identifying and exposing recurrent laryngeal nerve, the negative development of parathyroid glands by carbon nanoparticles . Patients with no complications were usually discharged on day 3 to day 5 after the surgery . For descriptive statistics of quantitative variables, mean standard deviation (sd) and range were used to describe central tendency and dispersion . For analysis of the differences in proportions, fisher's exact test was used if the assumptions of chi - square test were violated . Independent samples t - test was used to compare the level of quantitative variables between the two groups . The clinicopathologic characteristics of 100 patients with papillary thyroid microcarcinoma are summarized in table 1 . The robotic total thyroidectomy with cnd permanent pathological diagnosis showed that all 100 patients had papillary thyroid microcarcinoma . In the robotic group, mean time of creating workspace was 16.0 5.3 min, docking stage time was 10.5 3.2 min, and console stage time was 92.0 11.5 min . The mean total operative time of the robotic group was longer than that of conventional open approach group (118.8 16.5 min vs. 90.7 10.3 min, p <0.05). This study found the statistically significant differences between the two groups in terms of the vass for pain assessment (2.1 1.0 vs. 3.8 1.2, p <0.05) and nss (8.9 0.8 vs. 4.8 1.7, p <0.05). The differences of the two groups in the estimated intraoperative blood loss, postoperative hospital stay time, numbers of lymph node removed, primary tumor sizes, total drain volumes, mean draining days, and number of patients whose tumors showed multicentricity or multifocality were not statistically significant (p> 0.05). The complications in both groups, such as hypoparathyroidism (temporary), rln paralysis (temporary), seroma, skin burn, flap necrosis, hematoma, wound infection, chyle leakage, and postoperative tg levels, were not statistically different (all p> 0.05). Among the 100 patients, 35 (35%) had subclinical central lymph node metastasis . The more patients (19 patients) in robotic group had lymph node metastasis (vs. 16 patients in conventional open approach group), but this did not influence the final results in the staging (according to the american joint committee on cancer staging, 7 edition) of both groups (p> 0.05). Clinical data and postoperative outcomes between robotic and conventional open approach groups the data were shown as mean standard deviation unless otherwise indicated . Bmi: body mass index; cnd: central neck dissection; rln: recurrent laryngeal nerve; vas: visual analog scale; nss: numerical score system; tg: thyroglobulin . Postoperative cosmetic result was extremely satisfying in robotic group except one patient's suprasternal fossa became shallow with minimal numbness and tingling [figure 6]. One patient in the robotic group experienced transient vocal cord palsy, but this resolved within 2 months without any treatment . After the hoarseness had disappeared, we observed normal movement of the vocal cords by laryngoscope examination . Neither iatrogenic implantation nor metastasis occurred in punctured porous channel or chest wall was found by inspection or ultrasound during follow - up . Scar cosmesis 1 month after robotic total thyroidectomy with central neck dissection, which could be covered by bra . The conventional surgical approach is to extend the incision into a large transverse incision to complete the required neck dissection . Because the anterior neck is prominent and constantly exposed part of the body, an unsightly neck scars furthermore, a majority of patients with papillary thyroid microcarcinoma are young women, who prefer no scar in the neck . Therefore, surgeons have made every effort to resolve the scar problem . In 1997, the feasibility of robotic thyroidectomy with the transaxillary approach has been demonstrated by kang et al . In 2009, which further improved the cosmesis . The obvious advantage of the extracervical approach is the absence of visible scar in the neck or upper chest . According to anatomical access, novel approaches such as postauricular and transoral approaches have been reported . To the best of our knowledge, some modified approaches have been reported in literature, which could be grouped into small cervical incisions, either the natural body orifice or the surface of the body; the distant approaches such as the chest wall, periareolar, axillary, postauricular, and transoral; and combined approaches . All of the approaches have their own merits and drawbacks, such as the transaxillary approach is limited in arriving at the lymph nodes of the contralateral central compartment . However, many reports have described the feasibility of thyroidectomy with robotic system for benign thyroid lesions . Some studies had reported that total thyroidectomy with prophylactic central lymph node dissection is necessary in patients with papillary thyroid microcarcinoma . For any new emerging technique, while tailoring the surgical approach to the patients concerns and desires is important, adhering to fundamental oncosurgical principles should always be a priority . The subclinical central lymph node metastasis rate was 35%, and the rate of multicentricity or multifocality in papillary thyroid microcarcinoma was 15% . This comparative study suggested that there were no significant differences regarding the oncological and the technical safeties between two groups . Robotic procedure uniquely provided a three - dimensional vision, the flexible robotic instruments with seven degree of freedom, and precise simulation - based technology . These enabled surgeons to access deep, narrow spaces for a complete cnd and to identify and preserve the rln and parathyroid glands (parathyroid gland autotransplantation: 10 in robotic group versus 16 in conventional open approach group, p> 0.05). However, robotic procedure is more invasive, more expensive, and more time - consuming than conventional open thyroidectomy . However, the formation of visual thinking facilitated by robots can make up for the lack of tactile feedback . Robotic surgery could provide surgeons with the best opportunities to transform the thinking mode . In the future, we intend to compare the long - term follow - up results and oncologic safety in a large sample size . According to the results of the study the baba permits a full and symmetrical surgical view of important anatomic (structures such as the superior and inferior thyroidal vessels, the rlns, the parathyroid glands, and the trachea), bilateral thyroid exploration, more workspace for instrument use, and the removal of larger nodules . In terms of clinical safety and effectiveness, robotic total thyroidectomy with cnd was similar to open surgery . However, some young women would refuse to receive this surgery since the baba would involve their breast . Further studies are needed to explore other better approaches to minimize this disadvantage . In conclusion, robotic total thyroidectomy with cnd via baba had same safety and effect as conventional open procedure for patients with papillary thyroid microcarcinoma, but its cosmetic effect was more satisfactory . This study was supported by the grants from the third batch special foundation of china postdoctoral science foundation (no . 201003759), and the president funding of jinan military general hospital of pla (no . This study was supported by the grants from the third batch special foundation of china postdoctoral science foundation (no . 201003759), and the president funding of jinan military general hospital of pla (no.
Aortic stenosis is a common heart disorder that affects nearly 5% of persons older than 75 years . Many patients with severe aortic stenosis are not surgical candidates for surgical valve replacement owing to associated multiple medical problems and comorbidities . Recently, transaortic valve implantation (tavi) showed a promise as an alternative option to conventional open heart surgery [3 - 8]. Multislice detector computed tomography (mdct) is playing an increasingly important role in patient s screening protocol before tavi, provides detailed anatomic assessment of aortic valve morphology and calcification, aortic root measurement, aortic annulus size, comprehensive assessment of the aorta and suitability of iliofemoral access, and determines appropriate coaxial angles to optimize the valve implantation procedure [9, 10]. Mdct images are acquired through the chest, abdomen, and pelvis with a large imaging field of view, and potential incidental non - cardiac findings (incfs) can be detected . The prior unknown abnormalities that are identified on mdct without previous suspicion of disease and unrelated to the indications of the examination are referred to as incidental findings . The incfs may affect the patient s life expectancy following tavi and overall usefulness of the procedure . Currently, there are limited published data with inconstant results on the prevalence of the incfs; most published data are small patient s cohort with variable definition and classification of incfs . The goal of this study was to investigate the prevalence of incfs in pre - tavi protocol patients who underwent mdct as routine investigational workup before tavi procedure . Sixty - seven consecutive patients with severe symptomatic aortic stenosis referred to our institution for pre - tavi assessment and were retrospectively evaluated between january 2010 and march 2015 . The local ethics committee approved the retrospective evaluation of mdct data of all patients . As a result of the retrospective nature of this study, waiver of informed consent mean age was 73 8 years, 35 (52%) patients were male, and 32 (48%) patients were female . Patients with common contraindication to cta with intravenous contrast such as acute or chronic renal failure or prior contrast reaction were excluded . Patients underwent retrospective ecg gated ct with 64 detectors (hd 750 discovery; general electrics, milwaukee, wi), and the ct parameters were as follows: collimation 64 0.625 mm, section acquisition 0.625, tube voltage 120 kv, tube current based on body mass index, rotation time 330 ms, pitch 0.25 - 0.35 depending on heart rate, scan direction craniocaudal, and iterative reconstruction with 40% adaptive statistical iterative reconstruction (asir). Contrast materials injection protocol was total dose of material from 90 to 120 ml of iodixanol 320 (ge, health care, princeton, nj). The injection rate was 5 ml / s followed by 50 ml of normal saline . Ct images were reconstructed at slice thickness of 0.75 mm every 0.5 mm for coronaries, mediastinal and abdominal images were reconstructed at 2.0 mm thickness every 1 mm, and images analysis by multiplanar reformation was performed with dedicated software (cardio q x - press, general electrics). Images processing datasets included manually measured vessels diameters (aortic root dimensions, aorta, iliac and common femoral arteries), the distance of the coronary ostia to the aortic annulus, and the presence and extent of calcification and touristy . Incfs were diagnosed where an abnormality was found without previous clinical suspicion or known prior disease and were classified into three groups according to the anatomical location . The musculoskeletal groups encompassed the bone, joints, muscles and abdominal and chest wall as well as breasts . Furthermore, on the basis of clinical relevance, non - cardiac findings were classified into three groups . Group 1 findings were clinically significant and needed immediate attention and further evaluation; group 2 findings were clinically significant and needed further follow - up by more imaging with other imaging modalities such as magnetic resonance imaging or ultrasound; group 3 were incidental findings with no further follow - up or recommendations . Sixty - seven consecutive patients with severe symptomatic aortic stenosis referred to our institution for pre - tavi assessment and were retrospectively evaluated between january 2010 and march 2015 . The local ethics committee approved the retrospective evaluation of mdct data of all patients . As a result of the retrospective nature of this study, waiver of informed consent mean age was 73 8 years, 35 (52%) patients were male, and 32 (48%) patients were female . Patients with common contraindication to cta with intravenous contrast such as acute or chronic renal failure or prior contrast reaction were excluded . Patients underwent retrospective ecg gated ct with 64 detectors (hd 750 discovery; general electrics, milwaukee, wi), and the ct parameters were as follows: collimation 64 0.625 mm, section acquisition 0.625, tube voltage 120 kv, tube current based on body mass index, rotation time 330 ms, pitch 0.25 - 0.35 depending on heart rate, scan direction craniocaudal, and iterative reconstruction with 40% adaptive statistical iterative reconstruction (asir). Contrast materials injection protocol was total dose of material from 90 to 120 ml of iodixanol 320 (ge, health care, princeton, nj). The injection rate was 5 ml / s followed by 50 ml of normal saline . Ct images were reconstructed at slice thickness of 0.75 mm every 0.5 mm for coronaries, mediastinal and abdominal images were reconstructed at 2.0 mm thickness every 1 mm, and images analysis by multiplanar reformation was performed with dedicated software (cardio q x - press, general electrics). Images processing datasets included manually measured vessels diameters (aortic root dimensions, aorta, iliac and common femoral arteries), the distance of the coronary ostia to the aortic annulus, and the presence and extent of calcification and touristy . Incfs were diagnosed where an abnormality was found without previous clinical suspicion or known prior disease and were classified into three groups according to the anatomical location . The musculoskeletal groups encompassed the bone, joints, muscles and abdominal and chest wall as well as breasts . Furthermore, on the basis of clinical relevance, non - cardiac findings were classified into three groups . Group 1 findings were clinically significant and needed immediate attention and further evaluation; group 2 findings were clinically significant and needed further follow - up by more imaging with other imaging modalities such as magnetic resonance imaging or ultrasound; group 3 were incidental findings with no further follow - up or recommendations . The mean age of the included 67 patients was 73 8 years, and 35 male and 32 female (table 1) showed patient characteristics . Forty - six patients (69%) had chest findings (table 2), 57 patients (85%) had abdominal findings (table 3) and 22 patients (33%) had musculoskeletal findings (table 4). The total number of the incfs in the 67 patients was 248, and number and percentage based on anatomical location were as follows: 95 (38%) chest findings, 129 (52%) abdominal and pelvic findings, and 24 (10%) musculoskeletal findings . Of these 248 findings, 23 (9%), 62 (25%), and 153 (66%) findings belong to the first category, the second category, and the third category, respectively (fig . 1). Number and distribution of 248 non - cardiovascular incidental findings in 67 patients by system and categories . Three patients had significant, non - incidental, non - cardiovascular findings including gastric cancer, liver cirrhosis with partly ablated hepatoma and left hip prosthesis loosening, and one patient had malignant course of anomalous left coronary artery . Distribution of incfs in category 1 (findings that required treatment and immediate attention) showed most patients (83%) belong to chest findings, 13% belong to abdominal findings, and 4% belong to musculoskeletal findings . Distribution of incfs in category 2 showed abdominal findings represent 58%, 12 patients (18%) had liver findings that require further evaluation including solid lesions, one capsular retraction and one liver cirrhosis . Twenty - six patients (39%) had simple renal cysts belonging to category 3, while only one patient had cyst with possible internal enhancement requiring further evaluation classified as category 2 . Fifteen patients (22%) had heterogeneous prostate enlargement with (seven patients) and without calcifications, chest findings (37%), and musculoskeletal (5%). Twelve patients (18%) had thyroid findings including thyroid enlargement (three), nodules (six) and calcifications, and only 3% had musculoskeletal . Distribution of incfs in category 3 showed chest findings represent 53% of the total chest incfs, abdominal findings represent 90% of total abdominal incfs, and musculoskeletal findings represent 20% of total musculoskeletal incfs . Examples of category 3 incfs include: mosaic lung attenuation, diverticulosis, gallstones, right middle lobe / lingular sub - segmental atelectasis, simple renal cysts, fatty liver, and several small to borderline sized mediastinal lymph nodes . The main findings in this study are that all patients who underwent pre - tavi mdct workup had incfs . This study is one of few studies that report the prevalence and detailed nature of the non - cardiovascular findings in such specific population . This is the only study, to the best of our knowledge, that addresses the incidental findings based on anatomical location such as chest, abdominal and musculoskeletal findings . This system - based approach will be of great assist to interpreting physician to look for and report these findings more precisely in a systematic method . A more conservative approach in the classification of incidental findings was followed; particularly in category 1, there are findings that we think that need to be managed more promptly, such as anterior abdominal wall fat stranding and early abscess formation, and pulmonary edema and presence of lung infection, e.g., tree in bud appearance and patchy ground glass opacity . Early identification and prompt management of such abnormality may have a major impact in patients management and long - term outcome . The most common incf belongs to abdominal category; a similar study showed that 99% of 200 patients had 456 of non - incfs, and most patients had multiple findings that fell into more than one group . Incidental abdominal and pelvic findings contributed to 129 (52%) of the total findings (table 3); interestingly, few patients belong to category 1, such as moderate to severe ascites, but majority of findings in category 2 that need further follow - up, and group 3 findings that incidental and further workup or recommendation was required . As per table 3, there are multiple findings in category 2 including liver lesions, adrenal nodules, pancreatic and renal lesion, and presence of these incidental findings obviously, as per standard practice, initiates more workup . Chest incfs compose 95 (38%) of total findings, findings that need urgent management are more common than abdominal findings, and these findings include lung consolidation, atelectasis, pulmonary edema, pericardial effusion, and evidence of ongoing lung infection such as ground glass opacities and tree in bud . Prompt identification and management of these incidental findings is of a great clinical value to prevent further clinical deterioration and to avoid short- and long - term complication . Only lung nodules, hilar and mediastinal abnormalities, and thyroid nodules in category 2 need further follow - up; on the other hand, there are multiple incidental chest findings that do not need any further workup (table 2). Musculoskeletal findings are the smallest group, only 24 (10%), only one finding, anterior abdominal wall thickening, foci of air, and fat stranding that we thought it needs immediate attention . Findings required follow - up such as vertebral collapse with possible cord compression and non - aggressive bone lesions . However, the majorities of msk findings are incidental and require no further workup such as non - complicated abdominal wall hernia, lipoma, mild scoliosis, and calcified nodes in the axillary and pelvic muscle (table 4). The first category findings are likely related to patient s severe aortic stenosis . This includes moderate amount of ascites in three patients, moderate to large amount pleural effusion in six patients, moderate to large amount pericardial effusion in three patients and pulmonary edema in three patients, and anterior abdominal wall infection in msk findings . The second category findings are more interesting as they might require further evaluation or correlation with additional radiological or non - radiological tests . Lung nodule is a common incidental finding with the new generation chest ct examinations and has been reported in up to 51% of smokers aged 50 years or older . Fleischner s society recommendations for follow - up and management of lung nodules smaller than 8 mm detected incidentally at non - screening ct in persons 35 years of age or older depend on the nodule size (average of length and width) and individual s risk factor of lung cancer like smoking . Twelve patients (18%) had thyroid findings including thyroid enlargement, nodules, and calcifications . Incidental thyroid nodules (itns) are common . Further evaluation of itn including neck ultrasound and possibly fna should take into consideration nodule size, imaging features and the patient s life expectancy, based primarily on age and comorbidities . Only one patient had a cyst with possible internal enhancement requiring further evaluation classified as category bosniak two . Incidental renal findings are common and mostly cystic in nature and classified as bosniak category 1 . Twelve patients (18%) had liver findings that require further evaluation including solid lesions, one capsular retraction and one liver cirrhosis . Low (age <40 years) and average (age> 40 years) risk individuals have no known 1ry malignancies with a propensity to metastasize to the liver, cirrhosis, or other hepatic risk factors . Further evaluation is not required for such patients with sharply marginated solitary or multiple masses with low - attenuation (20 hu). Lesion size, density features, stability as compared to prior if available, presence or absence of 1ry malignancy are important factors that lead to the proper management . Presence of macroscopic fat with or without calcifications is diagnostic of benign adrenal myelolipoma . Homogenous adrenal nodule with hu less than 10 in non - enhanced ct scan is most likely fat rich adenoma . However, tavi dedicated ct scans are aortic angiography which might interfere with this role . Adrenal lesion of 1 - 4 cm size and benign features (low density, homogeneous, smooth margins) with no prior imaging or history of 1ry malignancy could be followed up after 1 year with ct or mri . The third category findings are more common, and typically these finding are incidental and require no further workup . For abdominal findings such as diverticulosis, gallstones, and fatty liver, common chest incidental findings such as granuloma scatter small cyst, and small pleural effusion and small calcified mediastinal lymph nodes, particularly have no major clinical relevance . Lindsay et al in a recent study demonstrated that incidental findings in pre - tavi ct are common, and do not independently identify patients with poor outcomes after tavi procedure . Detection of incidental findings on ct should not necessarily influence or delay the decision to perform tavi . In a total number of 260 patients incfs were observed in 204 patients (70.6%), eight (3.9%) malignances were detected, and the authors concluded a high prevalence of non - cardiac findings on routine pre - tavi ct examination . In another study by gufler et al, in a total of 131 patients, 31% patients had significant non - cardiac findings, 19% chest and 12% abdominal findings . Five lesions were considered potentially malignant, two renal cell carcinomas, one hepatoma, one mediastinal lymphoma, and one metastatic bladder cancer . The study found that prevalence of malignant incidental finding in pre - tav ct is 3.8% . However, this prevalence is not high in tavi patients with average age of 81.6 years compared to general population . A 2-year survival after tavi procedure was investigated by stachon et al, and the presence of potentially malignant findings (pmf) versus no pmf did not significantly influence decision or time to treatment . Several findings which are highly suspicious for malignancy were less likely associated with invasive treatment; the authors concluded that the frequently occurring radiological pmf did not influence 2-year survival after tavi procedure . This is a single - center study with relatively small number of patients, which might have some invalid representations of the population; however, compared to other similar studies, our patients comparable to others, typical tavi patients are elderly with multiple associated commodities . One of the other important limitations is no follow - up of most of the patients, the real significance and impact of non - cardiac findings are unknown, but all our patients underwent successful tavi procedure and discharged home without any in - hospital complications, and most of the patients as per radiological findings will follow with their primary care physicians . Incfs are common in routine pre - tavi ct presumably due to advanced age of patient population . These findings have variable clinical significance and some of them might require acute treatment while other might require additional evaluation and follow - up . Most incfs do not influence the decision to perform tavi procedure, and do not affect post - procedure outcome . It is imperative to report pre - tavi ct; beside cardiac and vascular findings, such incidental findings must be reported in a systemic method in a system - based approach to avoid missing important findings . This is a single - center study with relatively small number of patients, which might have some invalid representations of the population; however, compared to other similar studies, our patients comparable to others, typical tavi patients are elderly with multiple associated commodities . One of the other important limitations is no follow - up of most of the patients, the real significance and impact of non - cardiac findings are unknown, but all our patients underwent successful tavi procedure and discharged home without any in - hospital complications, and most of the patients as per radiological findings will follow with their primary care physicians . Incfs are common in routine pre - tavi ct presumably due to advanced age of patient population . These findings have variable clinical significance and some of them might require acute treatment while other might require additional evaluation and follow - up . Most incfs do not influence the decision to perform tavi procedure, and do not affect post - procedure outcome . It is imperative to report pre - tavi ct; beside cardiac and vascular findings, such incidental findings must be reported in a systemic method in a system - based approach to avoid missing important findings.
Avaliar o emprego da tcnica de perfuso pulmonar ex vivo (ppev) clinicamente com a finalidade de transplante . Estudo prospectivo envolvendo o recondicionamento de pulmes limtrofes, definidos por critrios especficos, tais como relao pao2/fio2 <300 mmhg, com um sistema de ppev . Entre fevereiro de 2013 e fevereiro de 2014, os pulmes de cinco doadores foram submetidos ppev por at 4 h. durante a ppev, a mecnica pulmonar foi avaliada continuamente . Amostras do perfusato foram colhidas a cada hora, assim como foi realizada a avaliao funcional dos rgos . A mdia de pao2 dos pulmes captados foi de 262,9 119,7 mmhg, sendo que, ao final da terceira hora de perfuso, essa foi de 357,0 108,5 mmhg . A capacidade de oxigenao dos pulmes apresentou discreta melhora durante a ppev nas primeiras 3 h (246,1 35,1; 257,9 48,9; e 288,8 120,5 mmhg, respectivamente), sem diferenas significativas entre os momentos (p = 0,508). As mdias de complacncia esttica foram de, respectivamente, 63.0 18,7; 75,6 25,4; e 70,4 28,0 mmhg aps 1, 2 e 3 h, com melhora significativa entre a hora 1 e 2 (p = 0,029), mas no entre a hora 2 e 3 (p = 0,059). A resistncia vascular pulmonar permaneceu estvel durante a ppev, sem diferenas entre os momentos (p = 0,284). Os pulmes avaliados permaneceram em condies fisiolgicas de preservao; no entanto, o protocolo no foi efetivo para promover a melhora na funo pulmonar, inviabilizando o transplante . Ex vivo lung perfusion (evlp) is a novel strategy for reconditioning lungs, with a view to increasing the number of viable organs for transplantation and to reducing the mortality of patients awaiting a transplant . 1 3 the principle of the technique is to replicate the physiological environment that provides the substrates needed to maintain cell metabolism . Initially, evlp was described by steen et al . 3 for the evaluation of non - heart - beating donor lungs, and, subsequently, it was modified for application to the reconditioning of extended - criteria donor lungs by the toronto lung transplant group, in canada . 4 lung reconditioning with subsequent transplantation has been employed by various transplant groups, with positive outcomes . 2 reported on a series of 20 reconditioned extended - criteria donor lungs in which the pao2/fio2 ratio increased significantly at the end of the reconditioning process (2 - 4 h). Those authors found that the rate of primary graft dysfunction 72 h after transplantation was comparable for both recipients of evlp lungs and recipients of conventionally selected lungs, and that no adverse events were directly attributable to evlp . In other centers, such as those in vienna (austria), paris (france), and turin (italy), the outcomes were similar to those obtained by the toronto group . 5 despite the positive outcomes, in the united kingdom, a study showed that, of 13 reconditioned lungs, only 6 were transplanted, which represents a utilization rate of 46% . 6 the present article reports on the use of evlp clinically to prepare donor lungs for transplantation in brazil . The study was approved by the brazilian national health council - comisso nacional de tica em pesquisa (conep, brazilian national research ethics committee; registration no . 16026; process no . We used human brain - dead donor lungs that were rejected for conventional transplantation because their pao2/fio2 ratio was <300 mmhg . Between february of 2013 and february of 2014, there were five lung recoveries for the lung reconditioning protocol . Immediately before recovery, bronchoscopic inspection with bal was performed . The bal procedure involved instillation of 40 ml of sterile saline (0.9% sodium chloride) into the segments of the basal pyramid of the right lower lobe, followed by aspiration until approximately 50% of the instilled amount was recovered and stored in a collection vial . The lungs were perfused with perfadex (vitrolife, gteborg, sweden) as the preservation solution, stored at 4c in a special bag, and sent to the heart institute surgery center of the university of so paulo school of medicine hospital das clnicas, in the city of so paulo, brazil, to undergo the procedure . The pulmonary artery and pulmonary veins were sutured onto special plastic cannulas, and the lung block was placed in a perfusion chamber (xvivo perfusion, gteborg, sweden). The perfusion system consists of cannulas, a membrane deoxygenator, a heat exchanger, a leukocyte filter, and a centrifugal pump (maquet, toronto, canada). The system is primed with 1.5 l of steen solution (vitrolife), 500 mg methylprednisolone, 500 mg/500 mg imipenem / cilastatin sodium, and heparin (3,000 iu). The established flow corresponded to 40% of the cardiac output during ventilation at physiological levels: a tidal volume (vt) of 6 ml / kg; a respiratory rate of 7 breaths / min; a positive end - expiratory pressure of 5 cmh2o; and an fio2 of 21% . For flow increase, as well as for reheating and ventilation 7 perfusion time was set at 4 h, and ventilatory and pulmonary gas exchange parameters were evaluated at the end of each hour . At the end of hour 3, perfusion was interrupted in the lungs with no prospects of improvement in lung function or respiratory mechanics with a view to transplantation . At evaluation, the ventilator was set at a vt of 10 ml / kg of body weight, a respiratory rate of 10 breaths / min, a positive end - expiratory pressure of 5 cm cmh2o, and an fio2 of 100% . A sample of the perfusate was collected from the outlet of the pulmonary artery and pulmonary vein for blood gas analysis, which measured paco2, mixed venous carbon dioxide tension, pao2, mixed venous oxygen tension, lactate levels, and glucose levels . Ventilatory parameters measured included peak pressure, plateau pressure, mean airway pressure, static compliance, and vt . The samples were fixed in 10% formalin for 24 h, embedded in paraffin, sectioned on a microtome into 5 m thick slice, and stained with h&e . The presence / absence of pathological changes was investigated under light microscopy by a pathologist . Quantitative data are expressed as means and standard deviations . For each variable, the assumption of normal distribution was tested with the shapiro - wilk test . For comparison of means, we used repeated measures anova and the paired student's t - test . The probability of a type i () error was set at 0.05 for all inferential analyses . Descriptive and inferential statistics were calculated using the spss statistics software package, version 21 (ibm corporation, armonk, ny, usa). For comparison of means, we used repeated measures anova and the paired student's t - test . The probability of a type i () error was set at 0.05 for all inferential analyses . Descriptive and inferential statistics were calculated using the spss statistics software package, version 21 (ibm corporation, armonk, ny, usa). The lungs of five donors were submitted to evlp at the heart institute surgery center . However, no transplants were performed, because none of the lungs met the functional criteria for transplantation . Four male donors and one female donor were used, and the mean length intubation was 5.6 days . All lungs were obtained from brain - dead donors, because the current legislation in brazil does not allow the use of non - heart - beating donors . The most common cause of death was traumatic brain injury, followed by subarachnoid hemorrhage . The reason the donor lungs were rejected for conventional transplantation was poor blood gas values (pao2/fio2 ratio = 100%). In three cases, discard was associated with radiological changes, and in two cases in which the pao2/fio2 ratio was satisfactory, the lungs were discarded either because of indefinite histology for brain neoplasm (this being the cause of brain death) or because the poor clinical condition of the recipient precluded transplantation (table 1). Table 1.characteristics of the selected donors . Gender age, years cause of death mv, days pao2, mmhg donor m18tbi3264,01f50sah7283,02m19tbi362,53m41tbi6334,64m29sah9370,65mv: mechanical ventilation; m: male; f: female; tbi: traumatic brain injury; and sah: subarachnoid hemorrhage . Arterial blood gas sampling with a positive end - expiratory pressure of 5 cmh20 and an fio2 of 100% . Mv: mechanical ventilation; m: male; f: female; tbi: traumatic brain injury; and sah: subarachnoid hemorrhage . Arterial blood gas sampling with a positive end - expiratory pressure of 5 cmh20 and an fio2 of 100% . The mean oxygenation capacity (po2) measured during evlp showed little variation, with no statistically significant differences (figure 1). After 1, 2, and 3 h of evlp, respectively, po2 values were 246.1 35.7 mmhg, 257.9 48.9 mmhg, and 288.8 120.5 mmhg (figure 1). A comparison of the po2 values measured at recovery with those observed at the end of evlp showed that, although the final mean po2 was higher, there were no statistically significant differences (figure 1). The mean pao2 of the recovered lungs was 262.9 119.7 mmhg at baseline, compared with 357.0 108.5 mmhg after 3 h of evlp . There were no statistically significant differences among the three time points evaluated (1 h, 2 h, and 3 h after the initiation of ex vivo perfusion). The mean static compliance after 1, 2, and 3 h, respectively, was 63.0 18.7 mmhg, 75.6 25.4 mmhg, and 70.4 28.0 mmhg, with a significant difference in results between hour 1 and hour 2 (p = 0.029).in most of the evlp procedures, the perfusion time was 180 minutes; in only one case was the perfusion time 240 minutes . A comparative evaluation of the lung biopsy samples collected before and after evlp showed that, in three cases, there was worsening of edema and formation of visible thrombi . The results of bal examination revealed colonization by virulent bacteria with a high potential to cause pulmonary infection in three cases . The evlp technique has been disseminated worldwide, with positive outcomes at various centers . In 2013, research groups from toronto, vienna, and paris presented their clinical results with evlp at the international society of heart and lung transplantation meeting . 5 the results showed that the reconditioned lung utilization rate was high and that the rates of primary graft dysfunction within the first 72 h were low . In the usa, the technique has been validated in a multicenter clinical trial and approved by the food and drug administration . 8 despite the clearly demonstrated advances, we found discrepancies among the centers regarding the evlp lung utilization rate for transplantation . In the present study, we report the use of evlp at our center . We performed five lung perfusions; however, evlp was unable to improve lung function because, in general, the recovered lungs showed pronounced physiological changes upon arrival at our surgery center . Several factors may be related to the limited number of lung recoveries for evlp in brazil . In our country, organs are obtained only from brain - dead donors . This limitation leads to there being a small supply of lungs for evlp, in comparison with the mean rates of other centers where lungs from non - heart - beating donors are used . Reported that 44% of 50 non - heart - beating donor lungs subjected to evlp were used for transplantation in toronto . 1 the use of lungs from non - heart - beating donors is well established in the literature, and related data have been compiled into the international society of heart and lung transplantation registry, showing that the use of such lungs results in an increase in the number of transplants . In addition, a multicenter study reported that the outcomes of transplantation of lungs from non - heart - beating donors were similar to those of transplantation of lungs from brain - dead donors, suggesting that transplant teams should make continuous efforts to increase the number of transplants using non - heart - beating donors . 9 in our sample, poor blood gas values alone were not the primary cause of donor rejection . The most common reason for donor rejection in 2013 was infection (in 35%), followed by morbidity history (in 16.7%), including smoking and use of inhaled drugs, and blood gas changes (in 15.6%). The evlp lung utilization rate for transplantation varies among the transplant centers that use the evlp technique . The research groups in lund (sweden) and in toronto are the most successful, with utilization rates of 67% and 87%, respectively . However, the newcastle group, in the united kingdom, reported a utilization rate of 46% and attributed it to complications related to the quality of the recovered lungs . Like the newcastle group, we extended the criteria for donor lung recovery, including those with blood gas changes associated with edema and contusion . 6 certainly the limited number of perfusions performed by our group and the inclusion of donor lungs with poor blood gas values associated with other changes had a significant impact on our utilization rate . The use of extended - criteria donor lungs resulted in a greater likelihood of dysfunctions, such as infection, alveolar infiltrate, excess secretions, and pulmonary embolism, compromising the evlp reconditioning process . The main radiological findings were pulmonary infiltrate (contusion or congestion) and infection, confirmed by h&e histology and bal culture at the end of evlp . The comorbidities detected in three lungs by radiological imaging and histopathological analysis before evlp may have negatively impacted reconditioning, given that the functional activity of those lungs did not progress satisfactorily . In addition, histological analysis of the lung tissue at the end of perfusion showed increased edema, thrombus formation, and other vascular changes in these cases . In the fourth case, in which there were mild radiological and histopathological changes, evlp occurred satisfactorily, increasing lung functional indices up to values close to the acceptable threshold for transplantation . Regarding growth in bal cultures, we detected colonization by virulent bacteria, with a high potential to cause pneumonia in recipients with suppurative disease, in three donors . At our center, previous studies reported high rates of bal culture positivity in lung donors . A retrospective study reported that, of 49 lungs recovered for conventional transplantation, 31 had positive cultures, mainly for staphylococcus aureus and pseudomonas aeruginosa . 10 our database provides evidence that the current situation remains unchanged, given that, in 2013, 26 of the 27 lungs recovered for conventional transplantation had positive cultures . This underscores the importance of investigation of the specific characteristics of the donors at each center so that possible remedial action can be taken before the use of evlp . Postoperative complications related to the development of pneumonia can be prevented by combining evlp and specific prophylactic antibiotic regimens . In one of the cases reported here, we found, on the basis of histopathological results, that the donor had bronchiectasis . In another case, both bronchiectasis and microthrombi had been missed on imaging . In the two cases, although evlp was not satisfactory enough for the reconditioning process, it was important in the diagnosis of the comorbidities . We deem it necessary to use additional imaging modalities, such as ct, as well as early fiberoptic bronchoscopy to minimize the number of pathologically compromised donor lungs . In a case report, machuca et al . Reported the use of evlp for the treatment of lungs from a donor with pulmonary thromboembolism, with the addition of a thrombolytic agent to the perfusate, which resulted in improvement in the lungs and subsequent transplantation . 11 in an experimental study conducted by our team, we found a significant improvement in arterial blood gas values after 1 h of evlp . 12 13 however, we highlight some methodological differences relative to the present study, such as the use of an fio2 of 100% throughout perfusion and the use of an open left atrium . In recent years, evlp has proven to be very important in the rehabilitation of extended - criteria donor lungs, increasing the number of transplants performed . Often, the severity of the comorbidities inherent in the donor lung has a major impact on the achievement of positive outcomes from the use of the evlp technique . Among the various organ procurement centers, there are differences with regard to donor care protocols, that is, there is no consensus regarding thyroid hormone replacement, use of desmopressin (an antidiuretic hormone analog that has mild vasopressor activity), or use of a feeding tube . During the development of our protocol, we found that donor lungs in brazil have a higher rate of complications after brain death than do those available for reconditioning at other centers . The here reported non - utilization of evlp donor lungs for transplantation suggests the need for investigation of the specific characteristics of the donors at our center . Such information may be highly relevant to making adjustments to the reconditioning technique so as to meet specific needs more fully, thus increasing the likelihood of success with evlp . Therefore, we conclude that, although the evlp technique used at our center contributed to maintaining the lungs under physiological conditions, it did not effectively improve lung function, thus precluding the use of the lungs for transplantation.

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