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A 9-month - old female patient was brought to a health institute with complaints of coughing persisting for 2 weeks . Her hematologic tests revealed the presence of hyperleucocytosis (107.000/mm), and consequently she was referred to us with the initial diagnosis of leukemia . Her history revealed the presence of vomiting, and diarrhea in addition to her complaints of coughing within the first week . She had nt been vaccinated . Her oropharynx was hyperemic, and crepitant rales were heard bilaterally over basal lobes of her lungs . Some of her laboratory values were as follows: hemoglobin: 10.6 gr / dl, wbc: 102.900/mm, platelets: 797.000/mm, normal hepatic, and renal function test results; uric acid: 2.5 mg / dl, and ldh: 386 u / l . Results of peripheral smear analysis were as follows: neutrophils: 26%, lymphocyte: 72%, monocyte: 2%, absence of atypical cells, and blast cells . Dimensions of mediastinum were within normal limits, and infiltration in the right paracardiac region was seen . Polymerase chain reaction (pcr) performed with throat swab, and respiratory tract panel realized for adenovirus infection yielded positive results . Pcr test could not detect any evidence of pertussis infection . Respiratory tract infection caused by an adenovirus, and hyperleucocytosis day of her hospitalization wbc was 66.680/mm . During follow - up of the patient, her respiratory symptoms, and complaints of coughing regressed at the end of the first week of her hospitalization . On 7 . Whole blood cell counts, and results of her peripheral smear are shown in table 1 . Complaints of the patient regressed, and her clinical problems resolved during her follow - up, and consequently she was discharged with prescription of a vacination plan . Whole blood cell count, and peripheral smear results of the patient x wbc: whole blood cell . Classical whooping cough infection courses with paroxysmal fits of coughing, vomiting after coughing, deep inspirium, and intermittent symptoms of coughing lasting longer than 28 days up to 3 months . Adenovirus infections most frequently present with manifestations of respiratory tract infections, and gastroenteritis . In our patient coughing episodes lasted for 3 weeks, and in the first week diarrhea, and vomiting accompanied complaints of coughing . In the pediatric age group acute leukemias are the most frequent causes of hyperleukocytosis . Apart from leukemias, infections, drugs, bleeding, hypersensitivity reactions, splenectomy, and solid tumors induce leukemoid reaction leading to development of hyperleukocytosis . However hyperleukocytosis secondary to absolute lymphocytosis is seen in pertussis, tuberculosis, and viral infections . Among viral infections, whole blood cell count of our patient demonstrated hyperleukocytos, anemia, and reactive thrombocytosis . Apart from bordetella pertussis infection the manifestations of pertussis - like coughing can be encountered during the course of infections caused by chlamydophila pneumoniae, chlamydiae trochomatis, mycoplasma pneumoniae, adenoviruses, and bordetella bronchiseptica . In a study on 149 patients who presented with clinical manifestations of pertussis, the most frequently detected infectious agents, apart from bordetella pertussis were adenoviruses, parainfluenza, mycoplasma pneumoniae, and rsv . In this study, adenoviruses, and mycoplasma pneumoniae were detected in 3, and 1.3% of the cases, respectively . In patients presenting with clinical manifestations of pertussis where bordetella pertussis cannot be isolated or cannot be confirmed serologically, investigation for infectious agents as mycoplasmae, chlamydiae, and adenoviruses has been recommended . Gold standard for the diagnosis of pertussis is detection of bacterial growth on culture which has a 100% specificity . Its sensitivity changes depending on many factors as stage of the disease, initiation of antimicrobial treatment, sampling method, quality of the material used for sampling, conditions of delivery of the sample to the laboratory, and experience of the laboratory, and ranges between 12, and 60 percent . The world health organization recommends combined use of antibiograms, and pcr technique for the diagnosis of pertussis . Sensitivity, and specificity of pcr method have been indicated as 7099%, and 86100%, respectively . In our patient bacterial growth pcr respiratory tract panel revealed presence of adenovirus, and absence of mycoplasma pneumoniae, and rsv . Although the patient presented to our clinic with pertussis - like coughing, complaints of vomiting, and diarrhea at the beginning of the disease, shorter duration of the coughing when compared with whoopig cough, and in consideration of laboratory test results, presumptive diagnosis of pertussis - like syndrome secondary to adenovirus infection, and hyperleucocytosis were made . Since all her necessary vaccinations were not performed, and diagnostic laboratory methods were not 100% sensitive, bordatella infection could not be ruled out conclusively . In conclusion, in patients presenting with clinical manifestations of pertussis, and hyperleucocytosis, if especially etiologic agent cannot be cultivated, then adenovirus infections should be considered in the differential diagnosis, and relevant investigations should be carried out.
In this paper, three researchers with different primary research interests (ocular toxicology ftf, dry mouth jjs, and dry eye wdm) examine several hypotheses concerning medications and their possible roles in causing ded . Hypothesis - driven studies are especially difficult for dry eye since diagnosis of this disease relies in part on subjective testing . In the future, perhaps testing such as osmolarity, standardized meibomian gland tests [2, 3], fourier - domain optical coherence tomography, and so forth will be incorporated into clinical practice and will provide a greater degree of repeatability and predictability than is currently available . Until these become available, the studies cited here are heavily dependent on subjective assessments, case reports, and summaries of expert committees or organizations . The data presented here may be speculative and lacking quantification, but may be conceptually useful until a more evidence - based understanding of dry eye is achieved . This paper is also an attempt to give the clinician some guidelines to assist in their clinical decision making . The primary secretory glands of the eye are the main and accessory lacrimal glands, the meibomian glands, and other accessory glands . For the mouth the primary secretory glands are the parotid (serous secretion) and submandibular (mixed mucus and serous) with sublingual (mucus) and minor salivary glands (mucus). Both the mouth and eyes are supplied by acinar exocrine glands that produce proteins, electrolytes, and enzymes in an aqueous medium . Mucus secretions for the eye are primarily provided by the epithelial goblet cells of the conjunctiva and primarily the submandibular and 600 plus salivary glands of the mouth . Both the mouth and external eye are lined by stratified squamous epithelium [5, 6]. Pharmacologically both systems react in a very similar manner, with cholinergic and anticholinergic agents probably giving the most drying effects both for the eye and mouth (table 1). Of the top 100 best - selling systemic drugs in the united states in 2009, the physician's desk reference mentioned the possibility of 22 of these drugs causing dry eye and dry mouth and another 34 causing only dry mouth . Therefore, 56% of these drugs carry a possible sicca effect . Dry eye and dry mouth are primarily diagnosed by subjective complaints and are so recorded by pharmaceutical companies in phase ii and iii studies . For dry mouth, the complaints are generally straightforward; subjects relate that they have a dry mouth or need increased fluids when they eat and often complain of difficulty in speech articulation . On the other hand, dry eye may start with tearing and have complaints of burning, itching, foreign body sensation (sandy), mucus discharge, photophobia, and blurred vision and occasionally may start with tearing . . Currently it is defined as a multifactorial disease of the tears and ocular surface that results in symptoms of discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface . It is accompanied by increased osmolarity of the tear film and inflammation of the ocular surface . While those interested in clinical toxicology are aware that some drugs cause dry mouth, dry eye, and decreased sweating, to date there is no incidence data for drugs causing dry eye and/or dry mouth . We suggest that the drugs which have the most sicca effects probably affect both the eyes and mouth . As better tests become available, this hypothesis could be evaluated on a drug by drug basis . We recommend that doctors ask their dry eye patients if any of their drugs caused a dry mouth as this may indicate that it also may be causing or aggravating their dry eye . This drug might then be substituted by another or given at a time when its peak drying effect would occur during sleep . The most common definition of polypharmacy is the use of five or more prescription drugs . This is a problem mostly affecting the older population and continues to increase with age . The national ambulatory medical survey for person 60 years and older showed 12.4% used 2 prescription drugs daily, 27.3% used 3 - 4, and 36.7% used 5 or more . This does not include the average of 2 or more over - the - counter medications (i.e., vitamins, aspirin, and decongestants) and then another 0.5 or more herbal preparations per day . Polypharmacy may be a problem because many medications interact with each other in ways that are difficult to predict and become increasingly complex as additional medications are added . There may also be a threshold effect for a particular mechanism or relating to a particular combination of drugs which then becomes clinically evident, whereas any single drug would not breach this threshold . The side effects of drugs, generally, have been reported to be 3 times greater in the older population [7, 23] which suffers most from dry eye . It is also rare that we understand most drug interactions involving more than 2 medications . A study by schein et al . Evaluating 2,481 individuals ages 6584 years, found a strong correlation between the addition of each new class of drug known to have a sicca effect and an associated increased prevalence of dry eye and dry mouth . The adjusted odds ratio was 1.8 (0.74.0) for one drug, 2.9 (1.26.9) for three drugs, and 7.0 (2.718.0) for five drugs, confirming the marked effect of adding known systemic drugs in causing dry eye and dry mouth . The importance of over - the - counter medications in polypharmacy as it pertains to the eye should not be overlooked . Both observational and incidence - based epidemiologic studies showed as little medication as a daily aspirin and/or multiple daily vitamins can cause dry eye . Possibly a multiple vitamin pill may be considered a polypharmacy in a single medication . It is probable, that in many cases, more ocular drying occurs with advancing or multiple diseases, especially with such conditions as collagen vascular disease, regardless of medications . This may make dry eye causation difficult to determine as to disease, polypharmacy, or both . Based on the above, we suggest that oral polypharmacy should itself be considered as a significant factor . Polypharmacy as a cause of dry eye has the potential to be even more important than with dry mouth since the risk of ocular sicca will be amplified by exposure to both topical and systemic medications . There are few population - based observational studies examining systemic medications causing dry eye . This data has been variable and conflicting in part due to differing definitions and design . The beaver dam population - based, age - adjusted, cumulative 10-year dry eye study examined 3,722 individuals . This represents the only - incidence based study tabulating dry eye that occurred only after drug exposure and thus more likely to be a causative agent . In a previous prevalence study . A recent cross - sectional survey on over 26,000 males over the age of 50 showed an increased prevalence of dry eye when treated with medications for depression and prostatic disease . This epidemiologic study showed antiandrogens as a cause of dry eye since the conjunctiva has 1 receptors (table 2). There is no population - based study regarding dry eye for any individual prescription drug, only for classes of drugs . Therefore, guidelines for any single medication are based on softer data as we are left with the literature, which is incomplete, and with postmarketing surveillance databases (fda, who, and nr), which remain inadequate . Incomplete data and the difficulty of comparing data make it challenging to draw conclusions with regarding causality . The who classification system, causality assessment of suspected adverse reactions, has definitions which include certain, probable / likely, possible, unlikely, unconditional / unclassified, and unassessable / unclassified categories . This was developed for all of medicine and is the standard for the national registry of drug - induced ocular side effects as a guide to determine causality . The system relies on a clinical event, laboratory findings, or an identifiable pattern not explained by a concurrent disease, drug, or chemical and has a scientifically plausible mechanism for causation . Dechallenge (the response to withdrawal of the drug) and rechallenge (the response to the drug being reintroduced) data is weighted heavily . Based on this system, we have listed in table 3 our assessment of which systemic drugs may cause or aggravate dry eye . Systemic drugs may cause dry eye secondary to decreased tear production, alteration of nerve input including reflex secretion and decreased corneal sensation or a direct inflammatory effect on secretory glands . Some may cause increased evaporation by changes in tear film composition, ocular surface abnormalities, number and quality of blinking, changes in mucus producing cells, and inflammatory changes in various ocular tissues . Of the 9 systemic drugs known to be secreted in the tears, rarely are drugs or its metabolites looked for in tears; however this may be an underappreciated as a cause or coincidental with ocular surface disease and ded . The role of systemic medication in ded is underappreciated, but available data to support this is still lacking since separating disease - induced ded from drug - induced ded is difficult . Evaluation of topical ocular medications dry eye is more complicated than that with systemic drugs . Much of the data comes from trials primarily in young patients in phase 2 and 3 studies required for fda approval . Dry eye side effects in these trials may be reported as various symptoms of dry eye without the patients saying that their eyes actually feel dry and therefore are not reported as such . There is a seldom objective sicca testing and therefore the term dry eye is not always mentioned in the physician's desk reference . Evaporative dry eye is primarily of a set of symptoms associated with meibomian gland dysfunction leading to increased evaporation and osmolarity, in conjunction with ocular surface inflammation and possibly reduced lacrimal output . Many of the mechanisms whereby topical medications may cause dry eye differ little from systemic medications . However, the potential additional effects on evaporation from topical medication should increase the total incidence of dry eye . Complications from topical medications may result from their typically higher drug concentrations, greater frequency of application, preservative effects, higher incidence of drug - induced blepharitis, and drug - induced ocular surface inflammation [1013]. All of these, either individually or combined, may result in tear film changes leading to increased evaporation and dry eye . Using the who causality classification system, we present our assessment of preservative - free topical ocular drugs which probably cause or aggravate dry eye (table 4). Of the top 200 best selling drugs in 2009, surprisingly 7 are eye drop medications . These are latanoprost, cyclosporine, bimatoprost, moxifloxacin, travoprost, brimonidine, and olopatadine . Other than cyclosporine, all have shown probable ocular drying effects, although olopatadine (an antihistamine) may have marginal effects . The greatest potential for a topical ocular medication to have a drying effect may be with long - term therapy, polypharmacy, tear film, or corneal surface changes and especially drug - induced blepharitis . This potential is present with glaucoma therapy which has 40% of the 14 + billion dollar prescription topical ocular medication market . The importance of the number and frequency of each additional application or medication in causing a significant increase in ocular surface disease and dry eye are well documented [25, 26]. Inflammation plays a significant and central role in the pathogenesis of dry eye and keratoconjunctivitis sicca . However, the role of inflammation secondary to medication in causing dry eye is complicated and not fully understood . It is not clear whether these reactions produce allergic, toxic, or inflammatory side effects . Many mechanisms are possible since medications may interfere with goblet cells, meibomian glands, ocular surfaces of the conjunctiva and cornea, and poorly understood tear film properties . As pointed out by baudouin et al . [28, 29], the complexity of inflammation occurring on the ocular surface may be in part due to medications . Recently a report of probable glaucoma medication-(with preservative) induced meibomian gland dysfunction was associated with a 50% incidence of dry eye; however, the patient had the signs of dry eye but did not have the symptoms . This is not surprising since there may be a poor correlation between objective and subjective signs of dry eye . With use of each additional known sicca - causing systemic class of drugs, ocular sicca complaints were also increased . The same is true for each additional topical ocular medication . What is not known is whether oral plus topical ocular dry - eye - producing agents are additive since the study has not been done . There is evidence that topical ocular drugs, including topical preservatives, may cause ocular surface inflammation and trigger dry eye and these effects are likely to be additive to the sicca effects of systemic drugs . Many commercial chronic topical ocular medications can cause changes in the ocular tear film and/or blepharitis (table 4). There is disagreement as to whether or not long - term bak can cause dry eye . Some feel that changes described from long - term bak use can be normal aging changes or not due to the preservative and they ask for better designed, long - term studies . There is no doubt that topical preservatives can cause ocular surface toxicity, which may take a number of forms, one of which includes ded . There have been randomized, controlled multicenter, double - masked, prospective parallel group studies for 6 months with 605 patients comparing travoprost 0.0015 and 0.004%, bimatoprost, and timolol in 379 patients upto 1 year and latanoprost in 829 patients up to 2 years . Travoprost contains an ionic buffering system as a preservative, bimatoprost contains bak 0.05 mg / ml, latanoprost 0.02% bak, and timolol 0.01% bak . None of these studies showed significant anterior segment pathology which could cause dry eye . Baudouin et al ., recently reviewed their extensive experience and the literature from over the past 20 years, which showed that topical ocular mediations containing bak caused tear film instability, ocular surface changes, conjunctival inflammation, epithelial apoptosis, and subconjunctival fibrosis . There are 170 papers (medline) that pertain to bak effects on the eye, amplifying the experience of baudouin and colleagues . A study of 9,658 patients, using objective and subjective criteria, found significantly more signs and symptoms of ocular surface disease in patients taking glaucoma medications with preservatives (95+% using bak) than those using preservative - free formulations . Dry eye sensation was reported as twice as common, foreign body sensation three times, and stinging and burning two and a half times as common in the preservative group as compared to the preservative - free group . The incidence of anterior and posterior blepharitis was about 3 times more common with glaucoma medications with preservatives . All of the above findings were highly significant and had p <0.0001 values . Rossi et al ., using the definition of dry eye as the presence of superficial punctuate keratitis and decreased fluorescein break - up time, showed a higher incidence of dry eye in patients taking topical ocular medication containing bak than controls (p <0.01). Dry eye was found in 40% of those taking 2 - 3 drops per day compared to 11% once a day and 5% for no eye drops . Using the ocular surface disease index (osdi) for measuring the symptoms of dry eye, show a 59% prevalence rate of dry eye in at least one eye in patients on topical ocular glaucoma medications with bak compared to their overall prevalence rate of 27% in non - glaucoma patients . In glaucoma patients on topical ocular medications, there was an abnormal schirmer's result in 61% of cases, lissamine green staining in 22%, decreased tear film break - up times in 78% and decreased tears in 65% . Each additional bak containing eye drop was associated with 2 times higher odds of showing abnormal results on lissamine green staining test (p <0.034). Using the osdi for measuring dry eye symptoms, showed that scores significantly worsen when comparing patients on a single topical ocular glaucoma medication as to those on 2 medications (p <0.007) or 3 medications (p <0.0001). In these 3 studies (fechtner, leung, and rossi), patients were not stratified as to length of time on the drug [25, 26, 33]. Discovered marked differences between topical ocular medications with and without preservatives while the study by leung et al . Showed the impact of a single drop containing bak which may speak for more of drying effect of bak compared to the chronic exposure to the drug itself [33, 38]. Since most commercial medications contain bak this masks preservative versus drug influence as to causation of dry eye . There are, however, numerous reports placing heavier emphasis on bak [25, 26, 33, 37, 38]. Studied 124 patients and found that long - term (over 3 years) combination topical ocular glaucoma therapy was a significant risk factor for trabeculectomy surgery failure . Failure was associated with statistically significant more inflammatory cells in the superficial and deep substantia propria . Preoperative subclinical inflammation induced by previous exposure to long - term topical ocular glaucoma medication was felt to be the cause of this inflammation and higher surgical failure rate . Punctal stenosis is not uncommon from chronic exposure to topical ocular medication [40, 41]. In the nr, there are numerous cases of unilateral punctual stenosis occurring only on the side of the patient's monocular glaucoma . Schwab et al . Showed long - term glaucoma medication with preservatives caused conjunctival foreshortening with shrinkage including conjunctival scarring . This was confirmed by thorne et al . In a series of 145 cases in which 97.4% of these patients had antiglaucoma medication . The standard of proof demanded by the fda and international dry eye workshop implicating bak and dry eye has not been met . In our opinion it is in large part because the long - term studies that are necessary are extremely difficult and expensive . The marketplace has acted to some degree already, with 1015% of artificial tears being sold are preservative free, not including non - bak preservatives . We suggest, that in addition, this action may interact synergistically with the effects of polypharmacy or with a previously compromised ocular surface . Since so many classes of drugs cause both a dry mouth and dry eye (table 1) and since polypharmacy is the number one cause of dry mouth and possibly important in dry eye, the effects of polypharmacy must be ruled out before diagnosing primary sjgren's syndrome . In a dry eye and mouth study which showed that the addition of each class of drugs increased the prevalence of dry eye or dry mouth, the authors also found that if a female took 7 prescription drugs with known sicca effects on the mouth or eyes, there was also an increased prevalence of vaginal dryness . In evaluating patients for primary sjgren's syndrome any changes in medication, especially during the previous 3 months, should be considered as potentially causing or worsening dry mouth or dry eye . Labeling a patient as having primary sjgren's without unequivocal serologic and tissue studies (minor salivary gland biopsy) may add significant emotional stress resulting from the perceived implications of an autoimmune disease diagnosis . The role of medications in causing or aggravating ded is complex and this paper suffers from oversimplification and incomplete data . The role of systemic and topical ocular medications in causing dry eye is probably underappreciated . There is a need to determine if the drying effects of systemic and topical ocular medications are additive or synergistic.oral polypharmacy needs to be studied as a cause of dry eye . The total number of drugs should include not only prescription drugs, but topical ocular medications, over - the - counter medications, and herbals as well.clinicians should remember that if a medication causes a dry mouth, it may also cause or aggravate dry eye.it is probable that the duration of topical ocular therapy is relevant as a cause of dry eye . Topical bak may be the primary factor in causing ded and ocular surface disease in a given patient.it is important to question the diagnosis of primary sjgren's syndrome without adequate laboratory confirmation if the patient has a history of polypharmacy or if onset of dry eye or mouth occurred within 3 months of a change in medication . Available on the national registry of drug - induced ocular side effects website, http://www.eyedrugregistry.com/, arebibliography of a drug's side effect profile listed in tables 3 and 4;list of drugs causing dry mouth;causes of polypharmacy . The role of systemic and topical ocular medications in causing dry eye is probably underappreciated . There is a need to determine if the drying effects of systemic and topical ocular medications are additive or synergistic . The total number of drugs should include not only prescription drugs, but topical ocular medications, over - the - counter medications, and herbals as well . Clinicians should remember that if a medication causes a dry mouth, it may also cause or aggravate dry eye . It is probable that the duration of topical ocular therapy is relevant as a cause of dry eye . Topical bak may be the primary factor in causing ded and ocular surface disease in a given patient . It is important to question the diagnosis of primary sjgren's syndrome without adequate laboratory confirmation if the patient has a history of polypharmacy or if onset of dry eye or mouth occurred within 3 months of a change in medication . Available on the national registry of drug - induced ocular side effects website, http://www.eyedrugregistry.com/, are bibliography of a drug's side effect profile listed in tables 3 and 4;list of drugs causing dry mouth;causes of polypharmacy . Bibliography of a drug's side effect profile listed in tables 3 and 4; list of drugs causing dry mouth; causes of polypharmacy.
Currently, glaucoma is defined as a disturbance of the structural or functional integrity of the optic nerve that causes characteristic atrophic changes in the optic nerve and retinal nerve fiber layer (rnfl) associated with visual field changes with or without increase in intraocular pressure (iop).1 as this injury is largely irreversible, early detection of glaucomatous damage is visually important . Examination of optic nerve head and rnfl is necessary in both diagnosis and follow - up of glaucoma.2 changes in the optic disc and rnfl often precede the appearance of visual field defect with standard automated perimetry (sap). Unfortunately, rnfl defect can be difficult to identify during clinical examination as this is a subjective method needing clear optical media, highly efficient red free filter, and advanced defect.3,4 therefore, objective investigations of measuring these structures facilitate ophthalmologists to reach to definite diagnosis . Optical coherence tomography (oct) is a noninvasive imaging modality that uses low - coherence light to obtain a high - resolution cross - section of anterior and posterior segments of the eye and quantitative assessments of different layers.5 short wave automated perimetry (swap) is more sensitive to early glaucoma than standard perimetry.6 with swap, a large goldmann size v blue target is projected against a bright yellow background . The background reduces the sensitivities of the green and red cones, thus isolating the short wavelength - sensitive blue cones and their associated small, bistratified retinal ganglion cells.6 the purposes of the study are to assess the role and diagnostic ability of oct and swap to distinguish between normal, glaucoma suspects, and surely diagnosed glaucomatous eye . This consecutive prospective study was conducted on patients attending the outpatient s clinic of menoufia university hospital and banha teaching hospital by the same operator during the period from january 2013 to october 2015 . A total of 70 subjects (140 eyes) were included in the study and divided into three groups: group a, 10 healthy volunteers (20 eyes); group b, 30 patients (60 eyes) with glaucoma suspect; and group c, 30 patients (60 eyes) with already diagnosed glaucomatous eyes . The selection criteria were as follows: best - corrected visual acuity at least 6/12, with spherical refractive error between + 2d and 2d and astigmatism the exclusion criteria were as follows: patients with any type of retinal pathology, history of retinal or refractive laser procedures, history of retinal surgery, patients with secondary glaucoma, history of diabetes, significant media opacity (as cataract), or pachymetry> 480 m or <540 m were excluded . Patients with unreliable visual fields defined as false - negative> 33%, false - positive> 33%, and fixation errors> 20% were also excluded . All patients had a full ophthalmic examination including visual acuity, refraction, goldman applanation tonometry, gonioscopy, pachymetry, dilated fundus examination, standard automated visual field examination, swap using humphrey field analyzer (carl zeiss meditec, inc), and finally nerve fiber layer thickness measurement by oct (carl zeiss meditec, inc). All of them had iop less than 21 mmhg, with absence of glaucomatous optic neuropathy (gon) with normal visual field indices . Glaucoma suspects either had (normal tension, normal visual fields with glaucomatous optic neuropathy) or (normal visual fields, absent glaucomatous optic neuropathy and high iop higher than 21 mmhg). Glaucomatous eyes show gon associated with characteristic visual field changes in the corresponding hemi field . Glaucomatous eyes were subdivided according to the deviation of sap (hodapp s classification)7 into three subgroups . Early was defined by visual field loss with an md <6 db, moderate glaucoma with md between 6 and 12 db, and severe glaucoma with md worse than 12 db . The oct cirrus (carl zeiss meditec, inc, dublin, ca, usa) employs a low - coherence interferometer to assess peripapillary tissue thickness . A good quality image was defined as an image with signal - to - noise ratio less than 50 db . By using inbuilt rnfl thickness average analysis, mean rnfl thickness can be measured.8 a functional disc map was made by dividing the optic nerve head into 12 equal radial sectors, each one of them for 30. three different parameters were performed for rnfl thickness: the average thickness of rnfl for the entire circumference of the optic disc.rnfl thickness in each sector (clock hours).the thickness at each quadrant (90), namely, superior (46135), nasal (136225), inferior (226315), and temporal (31645). The thickness at each quadrant (90), namely, superior (46135), nasal (136225), inferior (226315), and temporal (31645). Swap is a modification of sap using humphrey field analyzer 745i (zeiss humphrey systems, dublin, ca, usa). It utilizes a 440 nm 1.8 target at 200 milliseconds duration on a 100 candelas / m yellow background to selectively test the short wave length - sensitive cones and their connection . First field done to any patient was discarded, whether white / white (w / w) or swap . Testing was done in more than one session to prevent the effects of patient s fatigue . No more than two fields were done on one single visit, and no more than one swap testing was done per day . All visual fields that were used for analysis satisfied the following reliability criteria: fixation losses <25% and false - positive and false - negative responses <20% . For each reliable field, mean deviation (md) and pattern standard deviation (psd) results were collected, tabulated, and statistically analyzed using the ibm personal computer and statistical package for the social sciences (spss, chicago, il, usa) version 16 . In the statistical comparison between the different groups, the significance of difference was tested using one of the following tests: student s t - test and mann whitney test: used to compare mean of two groups of quantitative data of parametric and nonparametric, respectively.analysis of variance test (f value): used to compare mean of more than two groups of quantitative data . Intergroup comparison of categorical data was performed by using chi - square test (-value) and fisher s exact test (fet).correlation coefficient: to find relationships between variables . A p - value <0.05 was considered statistically significant (s), while a value> 0.05 was statistically insignificant . A p - value <0.01 was considered highly significant in all analyses . Student s t - test and mann whitney test: used to compare mean of two groups of quantitative data of parametric and nonparametric, respectively . Analysis of variance test (f value): used to compare mean of more than two groups of quantitative data . Intergroup comparison of categorical data was performed by using chi - square test (-value) and fisher s exact test (fet). A p - value <0.05 was considered statistically significant (s), while a value> 0.05 was statistically insignificant . The selection criteria were as follows: best - corrected visual acuity at least 6/12, with spherical refractive error between + 2d and 2d and astigmatism the exclusion criteria were as follows: patients with any type of retinal pathology, history of retinal or refractive laser procedures, history of retinal surgery, patients with secondary glaucoma, history of diabetes, significant media opacity (as cataract), or pachymetry> 480 m or <540 m were excluded . Patients with unreliable visual fields defined as false - negative> 33%, false - positive> 33%, and fixation errors> 20% were also excluded . All patients had a full ophthalmic examination including visual acuity, refraction, goldman applanation tonometry, gonioscopy, pachymetry, dilated fundus examination, standard automated visual field examination, swap using humphrey field analyzer (carl zeiss meditec, inc), and finally nerve fiber layer thickness measurement by oct (carl zeiss meditec, inc). All of them had iop less than 21 mmhg, with absence of glaucomatous optic neuropathy (gon) with normal visual field indices . Glaucoma suspects either had (normal tension, normal visual fields with glaucomatous optic neuropathy) or (normal visual fields, absent glaucomatous optic neuropathy and high iop higher than 21 mmhg). Glaucomatous eyes show gon associated with characteristic visual field changes in the corresponding hemi field . Glaucomatous eyes were subdivided according to the deviation of sap (hodapp s classification)7 into three subgroups . Early was defined by visual field loss with an md <6 db, moderate glaucoma with md between 6 and 12 db, and severe glaucoma with md worse than 12 db . The oct cirrus (carl zeiss meditec, inc, dublin, ca, usa) employs a low - coherence interferometer to assess peripapillary tissue thickness . A good quality image was defined as an image with signal - to - noise ratio less than 50 db . By using inbuilt rnfl thickness average analysis, mean rnfl thickness can be measured.8 a functional disc map was made by dividing the optic nerve head into 12 equal radial sectors, each one of them for 30. three different parameters were performed for rnfl thickness: the average thickness of rnfl for the entire circumference of the optic disc.rnfl thickness in each sector (clock hours).the thickness at each quadrant (90), namely, superior (46135), nasal (136225), inferior (226315), and temporal (31645). The thickness at each quadrant (90), namely, superior (46135), nasal (136225), inferior (226315), and temporal (31645). Swap is a modification of sap using humphrey field analyzer 745i (zeiss humphrey systems, dublin, ca, usa). It utilizes a 440 nm 1.8 target at 200 milliseconds duration on a 100 candelas / m yellow background to selectively test the short wave length - sensitive cones and their connection . First field done to any patient was discarded, whether white / white (w / w) or swap . Testing was done in more than one session to prevent the effects of patient s fatigue . No more than two fields were done on one single visit, and no more than one swap testing was done per day . All visual fields that were used for analysis satisfied the following reliability criteria: fixation losses <25% and false - positive and false - negative responses <20% . For each reliable field, mean deviation (md) and pattern standard deviation (psd) were recorded . Results were collected, tabulated, and statistically analyzed using the ibm personal computer and statistical package for the social sciences (spss, chicago, il, usa) version 16 . In the statistical comparison between the different groups, the significance of difference was tested using one of the following tests: student s t - test and mann whitney test: used to compare mean of two groups of quantitative data of parametric and nonparametric, respectively.analysis of variance test (f value): used to compare mean of more than two groups of quantitative data . Intergroup comparison of categorical data was performed by using chi - square test (-value) and fisher s exact test (fet).correlation coefficient: to find relationships between variables . A p - value <0.05 was considered statistically significant (s), while a value> 0.05 was statistically insignificant . Student s t - test and mann whitney test: used to compare mean of two groups of quantitative data of parametric and nonparametric, respectively . Analysis of variance test (f value): used to compare mean of more than two groups of quantitative data . Intergroup comparison of categorical data was performed by using chi - square test (-value) and fisher s exact test (fet). A p - value <0.05 was considered statistically significant (s), while a value> 0.05 was statistically insignificant . Regarding age and sex there were no statistically significant differences between the groups, but female sex was found to be a significant factor . Rnfl thickness values in all parameters (average and quadrants) measured by oct are listed in table 2 . Rnfl thickness in the nasal and temporal quadrants was significantly thinner than in superior and inferior quadrants, demonstrating the so - called double - hump pattern . Md and psd of sap and swap are listed in table 3 . In the glaucoma suspect group, rnfl thickness measured by oct was outside normal limits in at least 1 hour in 7 eyes . Twenty - one eyes (35%) were following is nt rule while the other 39 eyes were against (65%). Forty - three eyes of 60 (71.7%) had iop equal to or higher than 21 mmhg at least in two different times with no gon . Twenty - one eyes of 43 (35%) had glaucomatous field changes in swap (generalized depressed, paracenteral scotomas, arcuate scotomas, nasal step, temporal wedge, and altitudinal defect). These 17 eyes (28.3%) had glaucomatous field changes in swap (generalized depressed, paracenteral scotomas, arcuate scotomas, nasal step, temporal wedge, and altitudinal defect). Thirty - eight eyes (63.3%) in this group had glaucomatous field changes in swap (generalized depressed, paracenteral scotomas, arcuate scotomas, nasal step, temporal wedge, and altitudinal defect). In glaucoma group, rnfl thickness measured by oct was outside normal limits in at least 1 hour in 9 eyes . Forty - three eyes (71.7%) did not comply with the is nt rule, while 17 eyes (28.3%) did . All the 60 eyes had glaucomatous field changes in sap (generalized depressed, paracenteral scotomas, arcuate scotomas, nasal step, temporal wedge, and altitudinal defect). Fifty - two eyes of 60 (86.7%) had corresponding oct findings (rnfl thickness defect), while 8 eyes (13.3%) did not have this corresponding oct findings . All of the 52 eyes showed similar but more advanced and denser glaucomatous field changes in swap correlated to these finding in sap . Average rnfl thickness showed a stronger correlation (highly statistically significant p=0.001) with swap md (0.387) than that was found between average rnfl thickness and psd (0.111) of swap (p=0.009). Inferior quadrant thickness showed a highly significant statistically correlation with md (0.563) and psd (0.185; p=0.001). Superior quadrant thickness showed a highly statistically significant correlation with swap (md 0.19; p=0.001), while there was no significant correlation with psd (p=0.094). Table 4 shows that the strongest correlation in this group was found between rnfl inferior quadrant thickness and swap md (0.563), which was found to be highly statistically significant p=0.001 . Average rnfl thickness showed a stronger correlation (highly statistically significant p=0.001) with swap md (0.171) than that was found between average rnfl thickness and psd (0.145) of swap (p=0.007). Inferior and superior quadrants thickness showed a highly statistically significant correlation with md (0.252 and 0.173; p=0.001) and psd (0.416 and 0.134; p=0.001 and 0.004). Nasal quadrant thickness showed a highly statistically significant correlation with swap (md 0.113; p=0.003). Table 5 shows that the strongest correlation in this group was found between rnfl inferior quadrant thickness and swap psd (0.416), and this was p=0.001, highly statistically significant . Average rnfl thickness showed a stronger correlation (highly statistically significant p=0.001) with swap md (0.387) than that was found between average rnfl thickness and psd (0.111) of swap (p=0.009). Inferior quadrant thickness showed a highly significant statistically correlation with md (0.563) and psd (0.185; p=0.001). Superior quadrant thickness showed a highly statistically significant correlation with swap (md 0.19; p=0.001), while there was no significant correlation with psd (p=0.094). Table 4 shows that the strongest correlation in this group was found between rnfl inferior quadrant thickness and swap md (0.563), which was found to be highly statistically significant p=0.001 . Average rnfl thickness showed a stronger correlation (highly statistically significant p=0.001) with swap md (0.171) than that was found between average rnfl thickness and psd (0.145) of swap (p=0.007). Inferior and superior quadrants thickness showed a highly statistically significant correlation with md (0.252 and 0.173; p=0.001) and psd (0.416 and 0.134; p=0.001 and 0.004). Nasal quadrant thickness showed a highly statistically significant correlation with swap (md 0.113; p=0.003). Table 5 shows that the strongest correlation in this group was found between rnfl inferior quadrant thickness and swap psd (0.416), and this was p=0.001, highly statistically significant . Primary open - angle glaucoma is currently incurable, but glaucoma subspecialists agree that early diagnosis and treatment are essential for controlling the disease and reducing vision loss . The diagnosis of glaucoma requires a multifaceted analysis of patients history and clinical findings, including assessments of visual function and imaging studies.9 oct is considered as an objective investigative tool that provides quantitative information about rnfl thickness . In addition, there are no effects of refractive error or corneal birefringence on oct information.10 in our study, there was significant difference in rnfl thickness among control group, glaucoma, and glaucoma suspected . Previous studies have shown that rnfl thickness measured by oct is a good tool for differentiating glaucomatous from normal eyes.11 chen et al12 showed that average rnfl thickness was the best parameter for differentiating glaucoma from normal eyes . Sibota and sony11 found that average rnfl thickness followed by inferior rnfl thickness had the highest power to discriminate between glaucomatous and normal eyes.11 in recent studies, chen & huang showed that inferior rnfl was the best parameter for differentiation.12 results in our study showed that oct and short - wavelength perimetry were well correlated . There was a statistically significant correlation between md, psd, and both average rnfl thickness (0.622 and 0.334; p=0.001 and 0.009) and inferior quadrant among the glaucoma group (0.75 and 0.431; p=0.001). However, superior rnfl thickness significantly correlated only with md (0.436; p=0.001). We also found that among the glaucoma suspect group there was a statistically significant correlation between md, psd as visual field indices, and inferior quadrant (0.502 and 0.645; p=0.001), followed by average rnfl thickness (0.413 and 0.381; p=0.001 and 0.003), and finally with superior quadrant (0.417 and 0.366; p=0.001 and 0.004). However, nasal rnfl thickness significantly correlated only with md (0.377; p=0.003). Vishva and glen13 showed that structure function relationship was enhanced with rnfl thickness as the correlation r between rnfl thickness and visual field indices ranged from 5% nasal to 25% inferotemporal and was relatively stronger than other neuroretinal rim measurement and was not significantly different from that with bruch s membrane opening - based horizontal rim width . In addition, sibota et al11 found a correlation between visual field parameters (md and psd) and the average rnfl thickness . There was a significant positive correlation with md and a significant negative correlation with psd . The same holds good for yalvac and altunsay,14 who found a significant correlation between global indices of visual field md, psd, and rnfl thickness . He also showed a less significant correlation between md and rnfl thickness . In this study, we found that md measured by swap is lower than that measured by sap, and psd measured by swap is higher than that measured by sap . We also found that 38 eyes (63.3%) in the glaucoma suspect group that had glaucomatous field changes in swap (generalized depressed, paracentral scotomas, arcuate scotomas, nasal step, temporal wedge, and altitudinal defect) were free by sap . Later, five eyes showed glaucoma based on the results of a repeatable visual field defect by swap; of these, four were classified as high risk and one as medium risk . Both rnfl thickness measured by oct and swap indices are good discrimination tools between glaucomatous, glaucoma suspect, and normal eyes . There is superior ability of swap over sap in detecting glaucomatous changes in glaucoma suspect group.
Leprosy patients with less than five skin lesions are grouped into paucibacillary (pb) group for treatment purposes without taking into account their size and extent of lesions or the number of nerves involved; the clinical classification of leprosy is not considered relevant . They are uniformly treated with pb multidrug therapy for a fixed duration of 6 months as per who recommendations . This group potentially consists of patients with diverse clinical, bacteriological, and histopathological features . This categorization of leprosy based on the number of skin lesions for treatment purposes is arbitrary and is purely for the convenience of field workers . In 1998, the who expert committee on leprosy proposed mdt - pb for leprosy patients with 1 - 5 skin lesions but did not mention any scientific basis for such a recommendation except citing the nonreliability of skin smear services, which were considered relevant till then . As per clinical classification for control programs of 1982 by who, bacterial index <2 + on ridley scale was termed pb and that with bacterial index> 2 + was termed multibacillary (mb). This was changed in 1988, in which bacterial index of zero was considered as pb and the ones with bacterial index of 1 + or above as mb . There has been a substantial decrease in the numbers of leprosy patients after the implementation of mdt for leprosy . In the national leprosy elimination programme in india, the proportion of mb cases was seen to increase in states heading toward elimination . However, this increase was marginal as pb cases still account for more than 60% of the leprosy cases in india in the year 2004 . Study of this pb group for variations and patterns is not only important in the present context, but also relevant in devising future strategies in leprosy control programs . In this study, we analyzed the clinical, bacteriological, and histopathological features of this group of patients based on detailed clinical examination, skin smear findings, and skin histopathology . Thirty - one patients of leprosy with 15 skin lesions attending the department of dermatology, jjmmc, davangere, karnataka, over a period of 2 years were included in this study . The diagnosis was based on the presence of hypopigmented hypoesthetic skin lesions and cutaneous and/or peripheral nerve thickening . The sections were stained with hematoxylin and eosin (h&e) and modified fite stain, and studied by pathologist . The type and character of granuloma and the presence of acid fast bacilli (afb) were noted . The bacillary index of granuloma (big) was calculated in the tissue biopsy specimen . Of the 31 patients, 19 (61.2%) had single skin lesions [figure 1], 7 (22.5%) had two, 4 (12.9%) had three, 1 (3.22%) had four skin lesions . Their clinical classification was tuberculoid leprosy (tt) in 1 patient and borderline tuberculoid (bt) in 30 patients . Slit skin smear (sss) for afb was negative in all patients . Well - defined raised erythematous lesion the histopathological findings of h and e stained skin biopsies were as follows: tt (1), bt (23) [figures 25] il (6) patients . The big values on modified fite stain were 1 + in two biopsies which showed features of bt on histopathology [table 1]. Pan dermal granuloma with langhans giant cells hugging the epidermis, h and e; 4 initial sections lymphohistiocytic aggregate around nerve twigs, h and e; 40 fite faraco stain afb in nerve twig, h and e; 40 skin biopsies with histopathological features and big in the bt group, 23 out of 30 patients showed concordance, i.e., clinicopathological correlation was 76.6% . Clinicopathological correlation among 31 patients under study the percentage of positivity of afb was 0% in sss, and 6.45% in skin biopsies . This study reveals that patients with 15 skin lesions present with varied clinical and histopathological features which points to the nonhomogeneous nature of this group . Two patients with clinical features of bt leprosy had big of 1 + on skin biopsy, which highlights the importance of fite faraco stain on skin biopsies which reveals the presence of m. leprae bacilli better than sss, thus indicating the importance of histopathology and afb stain on skin biopsy to rule out mb type in patients clinically grouped as pb type . The significance of our observation is that patients with 15 skin lesions do not have disease of similar severity, and can reveal afb in few of them . It is known that clinical features in leprosy patients reflect only the gross morphology of the underlying pathological changes . Significant discrepancies have been reported between the bacteriological and immunological status of the nerve and skin compared to the clinical diagnosis. [46] these features should also be considered while establishing treatment programs for leprosy . It has been observed that a few bt patients harbor afb in their nerves for many years, even though they become clinically inactive following mdt . Bacterial clearing capacity within a lepromin - induced granuloma is not invariably present in all tuberculoid and indeterminate leprosy patients . This study thus makes it mandatory to study tissue biopsy findings in all leprosy patients which were not considered relevant for treatment purposes until now, and thus could be given a status in the categorization and assessment of severity of the disease . The significance of the finding of afb (1 + or more) and histopathology of mb type of leprosy in tissue biopsies, in patients grouped as pb leprosy should be resolved so that these patients could be given the drug therapy and duration of therapy they warrant.
Akabane virus (akav) is an arthropod - borne virus belonging to the genus orthobunyavirus in the family bunyaviridae . It causes epizootic and sporadic outbreaks of abortions, premature births, stillbirths, and congenital abnormalities characterized by arthrogryposis, hydranencephaly, or microanencephaly in cattle, sheep, and goats . Akav is widely distributed in the tropical and temperate zones in australia [3, 4], southeast asia, east asia [6, 7], the middle east, and africa . Akav infection of adult cattle causes a transient viraemia without obvious clinical signs, while infection of pregnant cattle often causes fetal damage resulting in abortion, stillbirth, or various congenital abnormalities . During 1972 to 1975, this disease had led to the birth of more than 42,000 abnormal calves in japan, causing significant economic loss in cattle industry [6, 11]. Subsequently, major outbreaks resulting in congenital defects in ruminants caused by akav were reported from israel, australia, taiwan, korea, and turkey [7, 10, 1215]. In sudan, akav infection was recognized based on serological evidence in sheep, goats, and cattle in various ecological zones of the country . The present study aimed at investigating the current situation of akav infection in sudan as a part of an ongoing research project on viral causes of reproductive problems of cattle in the country . The investigation area extends from the latitudes 11 78 to 19 61 north and longitudes 22 45 to 37 21 east . Sera were collected from dairy cattle in white nile, kassala, north kordofan, sennar, blue nile, north darfur, red sea, gadarf, khartoum, and gazira states . = 4pq / l, where n is the required number of individuals to be examined; p is a known or estimated prevalence; q = (1 p); l is the allowable error . Since location to location variation was expected, the sample size was increased and all collected sera (n = 361) were tested (table 1). Blood samples were collected from 361 dairy cattle in 10 states of sudan (table 1). Animal sampled included individuals with history of abortion and infertility from two states, namely, khartoum and gazira states . Animals of both sexes and various age groups (less than 6 months, less than 1 year, 1 - 2 years, 2 - 3 years, and more than 3 years); breeds (crossbred and indigenous) under various management systems (extensive, semi - intensive, and intensive) and in different seasons including dry (march june); rainy (july october), and winter (november february) were bled . Sera were extracted by centrifugation at 1,500 rpm for 10 minutes and kept at 20c until they were tested for presence of antibodies against akav using elisa . A total of 68 out of the 361 sera samples were collected from animals suffering from reproductive problems (infertility and abortion). Commercial competitive elisa antibodies kit (i d vet innovative diagnostics, france) was used to detect antibodies (anti - g1) against akav according to manufacturer's instructions . The serological results and other information gathered during this investigation such as locality, sex, season, management system, signs of reproductive problem, and breed (indigenous and crossbred) of the sampled animals were edited and analyzed statistically using statistical package (spss version 16). To identify the association of the risk factors with the specific seroprevalence, the chi - square (test) was used . The statistical significance level used was p 0.05 . The overall prevalence rate of akav antibodies found in dairy cattle samples was 29.4% (106/361) (table 1). The prevalence rate varied from highest (69.6%) (64/92) in khartoum state to lowest (3.3%) (1/30) in sennar state . Likewise the prevalence of akav antibodies was significantly (p <0.001) higher in crossbred animals (39.9%) than in indigenous breed animals (8.9%) and in females (33%) than in males (14.3%; p <0.001). The prevalence rates were significantly higher in intensively (37%) managed animals (p <0.001) (table 2). The seroprevalence was significantly high among 2 - 3 years old (45.3; p <0.001) and in the winter season (58.1%; p <0.001). The prevalence rates were also high in cases of infertility (76.2%) and abortion (48.9%) when compared to apparently healthy animals (22.9%) and were significantly associated with cases of infertility (p <0.03) (table 3). Prevalence of akav antibodies in domestic ruminants in different ecological zones of sudan indicated prevalence rates of up to 27% in sheep, 36% in goats, and 47% in cattle . The prevalence rates in cattle ranged between 10.6% and 47.8% in different areas . In the present study, prevalence rates of akav antibodies in dairy cattle varied greatly ranging from 3.3% in sennar state to 69.6% in khartoum state with an overall prevalence rate of 29.4% . This higher seroprevalence in khartoum state may be due to the fact that in this state 59 cattle sera out of 92 serum samples investigated were suffering from abortion (39 cases) and infertility (20 cases) (table 3). This result confirmed records of jun et al . Who discussed the associations between this virus and abortion suggesting that this virus may significantly increase abortion risk among cattle and sheep . . Stated that transmission of akav in sudan may not be an annual event with more sporadic occurrences, a fact that may lead to variations in prevalence rates from year to year and from one area to another . Thus, while these authors recorded a prevalence rate of 10.6% for sennar state, the prevalence recorded in the present study was 3.3% for the same area . However, both studies indicated wide geographic distribution of akav seropositivity in cattle in sudan indicating that the vector(s) of aka virus are widespread; unfortunately information regarding the vector(s) in the country is lacking . It is interesting, however, that, inspite of the observed high prevalence and wide distribution of akav infection in sudan, no evidence of outbreaks of congenital malformations, as has been reported in other parts of the world, has been recorded in dairy cattle in sudan . The present study also revealed that prevalence rates were highly associated with locality, breed, sex, season, age of animals, and management system . The prevalence rate was significantly higher in khartoum state (69.6%; p <0.001) than in other states . Animals sampled in khartoum state were from intensively managed crossbred dairy cattle that reported reproductive problems based on a questionnaire survey administered prior to collection of samples in khartoum and in gazira states . The prevalence rates were also significantly higher in crossbred animal (39.9%; p <0.001) than in indigenous ones (8.9%) and in females (33%; p <0.001) than in males which may reflect higher degree of attractiveness of these animals to insect vectors . The prevalence rates were also higher in intensive and semi - intensive management systems (37.0% and 36.1%, resp .) Than in extensive systems . The previous two systems are generally concentrated around urban areas, and crossbred animals usually constitute the majority of animals in these systems while extensive systems are usually practiced in the vast grass lands of eastern and western sudan and usually involve indigenous animals . In addition vector arthropods may not be as numerous in the extensive system as in the intensive and semi - intensive system where microenvironmental conditions suitable for vector breeding and survival may be available year - round . Seroprevalence rates were much higher in winter season (58.1%) than in dry (25.6%) or rainy (25.3%) season . Winter season in much of the sudan is usually very mild with an average temperature that is usually above 30c . When examining prevalence rates of akav antibodies in individuals with reproductive problems, the results revealed that these rates were significantly higher in cases of infertility (76.2%; p <0.03) than in abortion cases (48.9%) and were in both cases higher than in apparently healthy (22.9%) animals . However, the high prevalence rates observed in both infertility and abortion cases may indicate the importance of akav infection in the bovine reproductive syndrome in sudan warranting further investigations . The present study clearly indicated the widespread prevalence of akav infection in dairy cattle in sudan . Absence of clinically recognized symptoms of akav infections with noticeable outbreaks of abortion and foetal malformations may lead to underestimation of the importance of the disease in the sudan . This situation may, however, change and the disease can pose considerable threat with the increasing trend toward intensive raising of exotic breed dairy cattle to satisfy the rising local demand for milk and other dairy products . Hence, further countrywide epizootiological studies of akav infections in cattle, sheep, and goats are called for to provide information required for formulating well - designed control programs.
After the increasing use of laparoscopy, robotic surgery has provided a new era in minimally invasive surgery . Safety and clinical outcomes have been reported in various fields . The most common complications include ileus, deep vein thrombosis, and pulmonary embolism [1, 2]. However, postoperative acute renal failure (arf) is a rare complication in laparoscopic surgery and has not been reported in robotic surgery . Herein we report a case of postoperative acute renal failure in a patient who had undergone robot - assisted prostatectomy . A 63-year - old man presented with adenocarcinoma of prostate, clinical stage t1c, preoperative serum psa level 7.21 ng / ml, gleason score 3 + 2, and serum creatinine level 1.3 mg / dl and underwent a robot - assisted prostatectomy . The patient was placed in the trendelenburg position at an angle of 30 from the horizontal with the legs split . The operation time was 400 min and intraoperative blood loss was less than 100 ml . Throughout the operation the end - tidal carbon dioxide (co2) level was maintained between 37 and 38 mmhg . There was a mild increase in abdominal drainage levels to 500 ml per day . Bilateral percutaneous nephrostomy tubes were placed, and patent ureters without urine extravasation found in antegrade pyelography . The serum creatinine level rose to 3.8 mg / dl 12 h after operation . Pulmonary edema developed on the first postoperative day and the patient received hemodialysis and intensive care . Acute respiratory distress syndrome developed and consequently resulted in duodenal ulcer bleeding despite the use of antacid . Renal function recovered after comorbidity was controlled 2 months after surgery while serum creatinine level was 2.0 mg / dl . Serum creatinine level was 2.0 mg / dl and psa level was less than 0.01 ng / ml after one - year follow - up . Robotic surgery adds a new dimension to this field and provides three - dimensional visualization and allows advanced freedom of instrumentation motion . Currently there are four systems being used to treat patients and the numbers are increasing . The physiological effects of robotic surgery on the human body are similar to those of laparoscopy assisted surgery [1, 2]. Deterioration of renal function after laparoscopic surgery has been reported in various fields, including cholecystectomy, nephrectomy, and bariatric surgeries [47]. The common mechanisms of complications are related to: (1) increased intra - abdominal pressure (iap), (2) co2 insufflation and (3) rhabdomyolysis syndrome . To obtain adequate working space, maintaining pneumoperitoneum status the increased iap is associated with decreased urine output and deterioration of renal function in animal models and humans [69]. The major physiological effects of increased iap are direct renal parenchymal and vessel compression and decrease of renal arterial supply and venous drainage [8, 9]. This consequently stimulates the renin, aldosterone, and angiotensin systems, enhances renal vasoconstriction and decreases renal blood flow . Insufflation with co2 may depress myocardial contractility through blood absorption effects and increase systemic vascular resistance, which results in a decrease in cardiac output and activates the renin - angiotensin system . Both iap and insufflation with co2 increase plasma renin and angiotensin ii and subsequently cause reduction of renal blood flow and deterioration of renal function . In our case, oliguria was found 6 h postoperatively at a rate of 20 ml / h . Concluded that the predisposing risk factors included diabetes, hypertension, exaggerated surgical position, body shape character (muscular frame or morbid obesity), hypovolemia, operation time of more than 5 h, and preexisting renal dysfunction . In their study, they found the serum creatinine kinase (ck) level to be elevated to more than 5,000 u / l in all seven patients . Compared to our patient, the ck level was 1,831 there is no strong correlation between ck and arf from literature review, and the possibility of rhabdomyolysis in this patient should still be considered . In conclusion, we report the first case of acute renal failure after robot - assisted radical prostatectomy . The treatment outline included fluid balance, electrolyte correction, and prevention of secondary complication . Patients with prolonged surgery (> 5 h), morbid obesity, and pre - existing renal function impairment are at higher risk of the development of rhabdomyolysis . Minimizing the operation time, adequate patient padding, and intraoperative circulation maintenance are helpful in the prevention of postoperative acute renal failure.
Tophi can present as a first sign of hyperuricemia but literature has limited reports as far as flexor tendon contracture presenting as a first clinical sign of gout is concerned . We report a case of tophaceous gout with finger flexion contracture as its first sign . A 45 years old man presented in our out patient department with complaint of inability to extend middle finger of his left hand along with a mass growing over volar aspect of forearm for over few months . On physical examination a firm hard mass around 2 * 2 cm was found over volar aspect of forearm just proximal to flexor retinaculam . Clinical suspicion of tb tenosynovitis, was kept as a first diagnosis followed by soft tissue neoplasm and subsequently surgical exploration was performed . A whitish chalky infiltration of tendon of fds, synovial adhesion of other tendons along with median nerve embedded within hypertrophied synovium of flexor tendon noted . Patient underwent synovectomy and excision of tendon of fds to middle finger following histo - pathological evaluation a diagnosis of tophaceous gout was made with serum uric acid level being 8.6 mg / dl . A review of the literature reveals that gouty arthritis of the wrist is rare in isolation . Gout at the wrist as the initial appearance of the condition occurs between 0.8 to 2% of all gout cases . Gout occurs when serum uric acid levels are persistently higher than 6.8mg / dl . Since tophaceous gout can be seen scarcely in well controlled hyper - uricaemic patient thus making preoperative diagnosis of intratendinous tophi even more strenuous . Tophi are chalky, gritty accumulations of monosodium urate crystals that build up in soft tissue of an untreated gouty joint . The peak incidence of gout is between the ages of 3050 with the prevalence increasing with age . Tophi can present as a first sign of hyperuricemia but literature has limited reports as far as flexor tendon tophaceous infiltration presenting as a first clinical sign of gout is concerned [2 - 4]. We report a rare case in which intratendinous tophaceous gout within the flexor digitorum superficialis (fds) tendon could not be differentiated from tubercular tenosynovitis or neoplasm preoperatively more so as it was seen in a patient with no prior history suggestive of hyperuricemia . A 45 years old man presented in our out patient department with complaint of inability to extend middle finger of his left hand along with a mass growing over volar aspect of forearm for over 2 months [fig 1]. There was previous history of pain at wrist on finger movement which subsided gradually along with the decline in extension of middle finger . There was no associated history of fever, loss of weight or appetite, night sweats, malaise or fatigue . There was no history of trauma, pain in other joints of the body, morning stiffness of the back or hand joints, or continuous use of vibratory tools . Hard mass around 2 * 2 cm was found over volar aspect of forearm just proximal to flexor retinaculam . It was non tender, non compressible, mobile at right angle to anatomical axis of forearm . Movement at wrist was normal with restriction of movement of middle finger in sagittal plane . Laboratory findings showed all within normal limits except serum uric acid which was 8.6 mg / dl . Magnetic resonance imaging (mri) showed that the mass was within the fds which on axial t1 [fig 2] appeared as heterogenous isointense lesion while on coronal t2 stir [fig 3] showed heterogenous mixed hypointense hyperintense lesion . Clinical suspicion of tubercular tenosynovitis, soft tissue neoplasm and tophaceous gout was made and subsequently surgical exploration was performed . An incision along the flexor crease, extending further proximally while maintaining the longitudinal orientation taken . On longitudinal slit of thickened tendon sheath chalky white liquid emanated . On futher exploration there was whitish chalky infiltration of tendon of fds [fig 4], synovial adhesion of other tendons along with median nerve embedded within hypertrophied synovium of flexor tendons . Since the mass was noted well proximal to flexor retinaculam that probably could be the reason for flexor contracture and sparing of median nerve . Patient underwent synovectomy and tenotomy of fds to middle finger because of heavy intratendinous infiltration of the tophus along with destruction of the tendinous tissue . Histopathological evaluation of the chalky white substance showed fibroconnective tissue showing hyalinized areas with evidence of saponification surrounded by palisades of foreign body type giant cells and chronic inflammatory cells . The postoperative course was uneventful with no remarkable functional deficit from the removal of the superficialis endon to the middle finger [fig 6]. Gout at the wrist as the initial appearance of the condition occurs between 0.8 to 2% of all gout cases . Gout patients who are not treated have a 1930% chance of developing gout in the wrist during their lifetime . These nodules may not be recognized as tophi because the clinical diagnosis of gout in many instances is not straightforward . In the past reports, all intratendinous infiltrations of tophaceous gout occurred at the wrist and existed with carpal tunnel syndrome . We present an uncommon and unusual case of gout in the flexor tendon of forearm which occurred in isolation in a patient with no prior medical history of the recorded disease . Measurement of serum uric acid level in chronic tophaceous gout may or may not be conclusive of hyperuricemia as some patients with diabetes or even alcoholics can have normal to lower levels although fnac prior to definitive intervention would have helped us to achieve the diagnosis preoperatively but rather we had gone for definitive intervention but in no way over riding the role of fnac as the procedure of choice to achieve definitive diagnosis [8]. Surgical intervention like tenotomy or tenosynovectomy are required to debulk tophaceous deposits, improve smooth gliding of tendon and decompress nerves but primarily medical management to treat gout remains the gold standard . Short - term outcomes are consistently good but the risk of rupture [11] or recurrency remain if medical control is not achieved . A review of the literature reveals that gouty arthritis of the wrist is rare in isolation . Gout at the wrist as the initial appearance of the condition occurs between 0.8 to 2% of all gout cases . Gout occurs when serum uric acid levels are persistently higher than 6.8mg / dl . Since tophaceous gout can be seen scarcely in well controlled hyper - uricaemic patient thus making preoperative diagnosis of intratendinous tophi even more strenuous . In indian subcontinent tubercular tenosynovitis
They described a clinical entity in which central nervous system disease was associated with hyponatremia and paradoxically increased the renal sodium excretion . The patients also had a high urine osmolarity, high urine output, and a depleted extracellular volume . (2), the syndrome of inappropriate secretion of antidiuretic hormone (siadh) became the usual explanation for any hyponatremia associated with neurological and neurosurgical disorders . Recently, however, the cerebral salt wasting (csw) syndrome instead of siadh has been reported frequently in patients with acute brain disease (3 - 5). Csw syndrome is a disease that leads to hyponatremia resulting from the renal loss of sodium in the presence of disorders of the central nervous system (6) and is different in its pathogenesis from siadh showing dilutional hyponatremia due to an increase of antidiuretic hormone (adh). The treatment of csw syndrome is to supply sufficient water and sodium through a normal saline solution (7). We here report two cases of postoperative csw syndrome after a cranial vault remodeling surgery . A 21-month - old boy had an operation for syndactyly of both hands at 6 months after the birth, and then underwent cranial vault remodeling due to craniosynostosis with a chief complaint of trigonocephaly in the department of plastic surgery . The patient developed postoperative polyuria and hypotension, and his general condition was exacerbated after two postoperative days . The patient urinated 50 - 60 ml / hr (1,510 ml/24 hr) on the first postoperative day, and revealed 3 - 4 cmh2o of central venous pressure, 136 ml of blood loss through a suction drain, and 90/60 mmhg of blood pressure . After the third postoperative day, polyuria was developed with a change of urination to over 80 ml / hr (1,965 ml/24 hr), and the blood pressure and central venous pressure (cvp) decreased to 70/30 mmhg and to 1 - 3 cm h2o of cvp, respectively, which was associated with blood loss (52 ml) through the surgical drain . From the sixth postoperative day, the amount of the urine reduced to 70 ml / hr (1,730 ml/24 hr) and the blood pressure was maintained within the normal range at 100/50 mmhg because there was no more blood loss . Since then, the amount of urine was gradually decreased and finally stabilized at 30 - 40 ml / hr from the twelfth postoperative day . At admission to the hospital, the patient was 12.6 kg (50 - 75 percentile) in weight and 85 cm (50 - 75 percentile) in height . The temperature was measured at 36.5, the pulse was at 100/min, and the respiration was at 25/min . The patient had a clear consciousness, a little pale conjunctivae, and anicteric sclera . No cervical lymph node was palpated, and the thorax was symmetrically expanded without showing sunken thorax . Preoperatively microcytic anemia was observed with hemoglobin 9.7 g / dl, hematocrit 29.5%, and mcv 61.9 fl . Serum electrolyte levels were sodium (na) 138 meq / l, potassium (k) 4.5 meq / l, and chloride (cl) 107 meq / l, and urine specific gravity was 1.015 . Postoperative data of electrolytes and osmolarity of serum and urine are shown in table 1 . Preoperative levels of adh and atrial natriuretic peptide (anp) were 4 pg / ml and 19 pg / ml (<40 pg / ml), respectively . The anp and human brain natriuretic peptide (hbnp) were 89 pg / ml and 68.7 pg / ml (<100 pg / ml) on the first postoperative day, respectively . On the fifth and ninth postoperative day adh was 1.29 pg / ml and 3.68 pg / ml, anp 65 pg / ml and 51 pg / ml, and hbnp 13.8 pg / ml and 31.9 pg / ml, respectively . The infant was diagnosed as having csw syndrome that developed after cranial vault remodeling, and was supplied with water and sodium through intravenous normal saline administration from the early stage . The amount of urine was decreased from the sixth postoperative day, and the sodium level was stabilized . The infant currently shows normal in growth and development, weight gain, serum electrolytes, and urine osmolarity . A 4.3-yr - old boy came to the department of plastic surgery with chief complaints of down syndrome, syndactyly of the third and fourth toes on both feet, and trigonocephaly . Plastic surgeons performed cranial vault remodeling for craniosynostosis and managed the patient cooperatively with pediatricians for the postoperative polyuria and exacerbation of the general condition . The findings on the first and second postoperative day were as follows; daily urine amounts of 798 ml and 1,790 ml during 24 hr, cvp 5 - 8 cmh2o and 2 - 6 cmh2o, blood loss of 99 ml and 64 ml, and blood pressure of 100/60 mmhg and 96/60 mmhg, respectively . On the third postoperative day, the values were changed to 2,375 ml of 24-hr urine amount, 3 - 6 cmh2o of cvp, 9.5 ml of blood loss, and 90/50 mmhg of blood pressure . The 24-hr urine amount, cvp, and blood pressure were 2,360 ml, 1 - 3 cmh2o, and 90/50 mmhg, respectively, on the fourth postoperative day and were 1,750 ml, 5 - 7 cmh2o, and 100/60 mmhg on the fifth postoperative day . The findings on the eighth postoperative day were as follows; 24-hr urine amount of 463 ml, cvp of 6 - 7 cmh2o, blood loss of 45 ml, and blood pressure of 110 - 60 mmhg . Since then, the 24-hr urine amount was maintained at less than 700 ml, and the blood pressure was also stabilized at 100 - 110/50 - 65 mmhg . On the thirteenth postoperative day, there was no more blood loss, and the suction drain was removed . Growth percentiles at admission were 15 kg (0 - 25 percentile) in weight, 96 cm (3 - 10 percentile) in height, 45 cm (0 - 3 percentile) in head circumference . Vital signs were 37.7 of body temperature, 92 beats / min of pulse, 22 times / min of respirations, and 110/60 mmhg of blood pressure . While the patient had clear consciousness and good nutritional condition, the intelligence quotient and the physical quotient were comparatively retarded corresponding to about 15 months and 24 months, respectively . Preoperative laboratory findings included normocytic anemia with hemoglobin 9.5 mg / dl and mcv 83.5 fl . Meq / l, k 4.5 meq / l, cl 106 meq / l, urine - specific gravity of 1.015, pmosm of 293 mosm / kg, and umosm of 408 mosm / kg . The changes of electrolytes and osmolarity of serum and urine after surgery are shown in table 2 . Preoperative values of adh, anp, and hbnp were 3.25 pg / ml, 14 pg / ml, and hbnp 4.1 pg / ml, respectively . On the day of operation, the values were adh 2.3 pg / ml, anp 92 pg / ml, and hbnp 63 pg / ml . On the fifth postoperative day, they were adh 1.38 pg / ml, anp 36 pg / ml, and hbnp 6.4 pg / ml . The patient was diagnosed as having csw syndrome when the postoperative 24-hr urine amount was over 1,500 ml, and did not show hyponatremia after the water and sodium supply through normal saline . Since the eighth postoperative day, the patient was in good general condition with the urine output gradually decreasing and normal blood pressure . Then, the patient was discharged from the hospital on the seventeenth postoperative day without circulatory or neurologic sequelae . A 21-month - old boy had an operation for syndactyly of both hands at 6 months after the birth, and then underwent cranial vault remodeling due to craniosynostosis with a chief complaint of trigonocephaly in the department of plastic surgery . The patient developed postoperative polyuria and hypotension, and his general condition was exacerbated after two postoperative days . The patient urinated 50 - 60 ml / hr (1,510 ml/24 hr) on the first postoperative day, and revealed 3 - 4 cmh2o of central venous pressure, 136 ml of blood loss through a suction drain, and 90/60 mmhg of blood pressure . After the third postoperative day, polyuria was developed with a change of urination to over 80 ml / hr (1,965 ml/24 hr), and the blood pressure and central venous pressure (cvp) decreased to 70/30 mmhg and to 1 - 3 cm h2o of cvp, respectively, which was associated with blood loss (52 ml) through the surgical drain . From the sixth postoperative day, the amount of the urine reduced to 70 ml / hr (1,730 ml/24 hr) and the blood pressure was maintained within the normal range at 100/50 mmhg because there was no more blood loss . Since then, the amount of urine was gradually decreased and finally stabilized at 30 - 40 ml / hr from the twelfth postoperative day . At admission to the hospital, the patient was 12.6 kg (50 - 75 percentile) in weight and 85 cm (50 - 75 percentile) in height . The temperature was measured at 36.5, the pulse was at 100/min, and the respiration was at 25/min . The patient had a clear consciousness, a little pale conjunctivae, and anicteric sclera . No cervical lymph node was palpated, and the thorax was symmetrically expanded without showing sunken thorax . Preoperatively microcytic anemia was observed with hemoglobin 9.7 g / dl, hematocrit 29.5%, and mcv 61.9 fl . Serum electrolyte levels were sodium (na) 138 meq / l, potassium (k) 4.5 meq / l, and chloride (cl) 107 meq / l, and urine specific gravity was 1.015 . Postoperative data of electrolytes and osmolarity of serum and urine are shown in table 1 . Preoperative levels of adh and atrial natriuretic peptide (anp) were 4 pg / ml and 19 pg / ml (<40 pg / ml), respectively . The anp and human brain natriuretic peptide (hbnp) were 89 pg / ml and 68.7 pg / ml (<100 pg / ml) on the first postoperative day, respectively . On the fifth and ninth postoperative day adh was 1.29 pg / ml and 3.68 pg / ml, anp 65 pg / ml and 51 pg / ml, and hbnp 13.8 pg / ml and 31.9 pg / ml, respectively . The infant was diagnosed as having csw syndrome that developed after cranial vault remodeling, and was supplied with water and sodium through intravenous normal saline administration from the early stage . The amount of urine was decreased from the sixth postoperative day, and the sodium level was stabilized . The infant currently shows normal in growth and development, weight gain, serum electrolytes, and urine osmolarity . A 4.3-yr - old boy came to the department of plastic surgery with chief complaints of down syndrome, syndactyly of the third and fourth toes on both feet, and trigonocephaly . Plastic surgeons performed cranial vault remodeling for craniosynostosis and managed the patient cooperatively with pediatricians for the postoperative polyuria and exacerbation of the general condition . The findings on the first and second postoperative day were as follows; daily urine amounts of 798 ml and 1,790 ml during 24 hr, cvp 5 - 8 cmh2o and 2 - 6 cmh2o, blood loss of 99 ml and 64 ml, and blood pressure of 100/60 mmhg and 96/60 mmhg, respectively . On the third postoperative day, the values were changed to 2,375 ml of 24-hr urine amount, 3 - 6 cmh2o of cvp, 9.5 ml of blood loss, and 90/50 mmhg of blood pressure . The 24-hr urine amount, cvp, and blood pressure were 2,360 ml, 1 - 3 cmh2o, and 90/50 mmhg, respectively, on the fourth postoperative day and were 1,750 ml, 5 - 7 cmh2o, and 100/60 mmhg on the fifth postoperative day . The findings on the eighth postoperative day were as follows; 24-hr urine amount of 463 ml, cvp of 6 - 7 cmh2o, blood loss of 45 ml, and blood pressure of 110 - 60 mmhg . Since then, the 24-hr urine amount was maintained at less than 700 ml, and the blood pressure was also stabilized at 100 - 110/50 - 65 mmhg . On the thirteenth postoperative day, there was no more blood loss, and the suction drain was removed . Growth percentiles at admission were 15 kg (0 - 25 percentile) in weight, 96 cm (3 - 10 percentile) in height, 45 cm (0 - 3 percentile) in head circumference . Vital signs were 37.7 of body temperature, 92 beats / min of pulse, 22 times / min of respirations, and 110/60 mmhg of blood pressure . While the patient had clear consciousness and good nutritional condition, the intelligence quotient and the physical quotient were comparatively retarded corresponding to about 15 months and 24 months, respectively . Preoperative laboratory findings included normocytic anemia with hemoglobin 9.5 mg / dl and mcv 83.5 fl . Biochemical test was normal, and electrolyte levels showed na 140 meq / l, k 4.5 meq / l, cl 106 meq / l, urine - specific gravity of 1.015, pmosm of 293 mosm / kg, and umosm of 408 mosm / kg . The changes of electrolytes and osmolarity of serum and urine after surgery are shown in table 2 . Preoperative values of adh, anp, and hbnp were 3.25 pg / ml, 14 pg / ml, and hbnp 4.1 pg / ml, respectively . On the day of operation, the values were adh 2.3 pg / ml, anp 92 pg / ml, and hbnp 63 pg / ml . On the fifth postoperative day, they were adh 1.38 pg / ml, anp 36 pg / ml, and hbnp 6.4 pg / ml . The patient was diagnosed as having csw syndrome when the postoperative 24-hr urine amount was over 1,500 ml, and did not show hyponatremia after the water and sodium supply through normal saline . Since the eighth postoperative day, the patient was in good general condition with the urine output gradually decreasing and normal blood pressure . Then, the patient was discharged from the hospital on the seventeenth postoperative day without circulatory or neurologic sequelae . When hyponatremia develops in patients with cns lesions such as brain damage, brain hemorrhage, brain tumor, intracranial infections, and stroke, an appropriate treatment based on an accurate diagnosis is the most important issue . If a treatment inhibiting water supply is put on a child with csw syndrome, crucial hypotension and cerebral ischemia can be induced . Conversely, excess water and sodium supply into a patient with siadh can cause osmotic demyelinolysis (7). Csw syndrome is defined as a state of hyponatremia and extracellular water loss due to sodium loss into kidneys in the existence of a cns lesion, while siadh is a state of dilutional hyponatremia due to increased adh secretion (6). . We must be cautious in making diagnosis of the disease if the patient has an expanded extracelluar fluid (ecf) volumes and a condition causing a deficiency of a physiologic stimulator of renal sodium reabsorption (8). Our cases showed extracted ecf volumes and natriuresis without any other etiologic factors except brain surgery . Csw syndrome can be induced by the increased sodium excretion through the urine due to the secretion of anp and hbnp (9); however, in the existence of a brain lesion, the mechanism to increase the secretion of natriuretic peptide can hardly be known . Anp and bnp are secreted from the walls of atrium and ventricle, and the secretion is normally controlled by the heart volume and the pressure increase (6). The secretion of anp and bnp promotes the sodium excretion but inhibits the vasodilation and the renin - aldosterone axis (10). The treatment of csw syndrome is to maintain a positive sodium balance by supplying sufficient water and sodium (11). The water and sodium supply was generally performed through an intravenous injection of 0.9% normal saline solution or 3% salt solution . At this time, the correction hyponatremia of needs to be done to the levels to prevent the occurrence of central pontine myelinolysis . The rate of injection of saline solution should be at 1 - 2 mmol / l per hour and must be monitored not to exceed 25 meq / l per day . Occasionally, injection of a normal saline solution with loop diuretics like furosemide needs to be considered (7). The two cases of this report developed hypotension and natriuresis after a cranial vault remodeling surgery and manifested polyuria; however, any neurologic abnormalities such as reduced levels of consciousness were not observed in the cases . On the other hand, the first case showed an increase of the 24-hr urine amount up to 2,000 ml, a decrease of the central venous pressure down to 1 - 3 cmh2o, a continuous decrease of the blood pressure on the third postoperative day; and thereupon, the infusion volume of 0.9% normal saline was increased . The second case showed an increase of the 24-hr urine amount up to 2,375 ml at the third postoperative day, but owing to the maintenance of the injection of 0.9% normal saline, the decrease of blood pressure was not obvious as in the first case . Also, the two cases demonstrated normal ranges of adh both pre- and the postoperatively . Anp and hbnp were temporarily increased postoperatively, which can be observed in csw syndrome . Accordingly, considering that the patients had undergone cranial operations that could be accompanied by csw syndrone, we closely observed the central venous pressure and urine output immediately after the operations, and measured the urine and blood sodium values . And then, we could make a diagnosis of csw syndrome in the two cases . The experience of the drop in the blood pressure due to polyuria in the first case made it possible to prevent the drop of blood pressure in the second case by increasing the fluid volume from the early stage to prevent the water loss . Based on the experience, when hyponatremia after cns operation such as cranial surgery, the causal factor should be verified first via checking the urine sodium excretion amount, serum electrolyte levels, adh, and anp, while monitoring the changes of postoperative urine amount and central venous pressure, and then, an appropriate treatment should follow . In summary, we report our experience of two cases of children with csw syndrome after cranial surgery . The patients manifested natriuresis, polyuria, and hypotension, and showed improvement with the supply of water and sodium with 0.9% normal saline . Therefore, csw syndrome should always be considered when a child patient with a cns lesion manifests hyponatremia and polyuria.
Autoimmune pancreatitis has travelled a relatively short way from its first description by sarles et al . In 1961 to the formal suggestion of the very concept of autoimmune pancreatitis by yoshida et al . In 1995, and its subsequent inclusion in a new group of diseases with systemic involvement associated with an increase in serum igg4 levels, after the depiction by kamisawa et al . . Most descriptions have, up to now, been done on asian patients, even though there have been descriptions of patients in other geographical areas [4, 5]. In spite of the scant information available, mainly due to the few reported cases of the disease and its aforementioned short history, it is possible to assert that there are some differences in the clinical presentation of patients of different ethnicities . In conjunction with the scarcity of available information, there is the added problem that until recently, there is a lack of universally recognised criteria for the diagnosis of the disease . Asian, european, and north american consensus have so far been published [68] and most centers use distinct criteria, making difficult to establish comparison among patients in whom the diagnosis was made at different geographical areas . Recently, an international panel of experts met to develop the international consensus diagnostic criteria for autoimmune pancreatitis to favor the worldwide recognition of the illness . In this consensus, emphasis is placed on the advantages of classifying autoimmune pancreatitis into either of two types: lymphoplasmocytic sclerosing pancreatitis (lpsp) and idiopathic duct - centric pancreatitis (idcp), since it has been so far ascertained that there are significant variations in clinical presentation, depending on the histological characteristics of the pancreatitis under study . For this reason, it is important to obtain clinical descriptions of the disease, together with its histopathological characteristics, as this will allow us to lay the foundations for our understanding of the disease behaviour . The aim of this study is to identify and describe the general characteristics of patients diagnosed with autoimmune pancreatitis at a third - level hospital in mexico . From our hospital records, we identified 15 patients who met the histological criteria for autoimmune pancreatitis . A mexican mestizo is defined as an individual who was born in mexico and is a descendant from the native inhabitants of such region, and from individuals, mainly spaniards of white or african origin, or both, who came to america during the 16th century . The cut - off was 1868 mg / dl and 135 mg / dl, respectively . Immunohistochemical staining for igg4 was performed in all cases and was considered positive with> 10 igg4-positive plasma cells per high power field (hpf). Subtype classification as either lymphoplasmocytic sclerosing pancreatitis (lpsp) or idiopathic duct centric pancreatitis (idcp) was performed according to the 2011 international consensus diagnostic criteria for autoimmune pancreatitis . Available pretreatment computed tomography (ct) scans (n = 13) were reviewed by a single radiologist, while in 2 patients only written radiological reports were at hand . Pancreatic enlargement was defined as an increase in size of the gland in the absence of a discrete mass . Mass was defined as a lesion that had different density compared with surrounding pancreatic tissue . Biliary and pancreatic duct strictures were considered steroid - responsive if follow - up imaging showed normal biliary tree and pancreatic duct, respectively . Pancreatic mass / enlargement was considered steroid - responsive if abnormality resolved, with posttherapy imaging showing normal - appearing pancreas or pancreatic atrophy . Given the lack of immediate accessibility to serum igg4 measurements as a marker of disease, we remained without this response criterion . Patient's demographic and clinical characteristics at the outbreak of the disease are shown on table 1 . Patients were followed up at our institute for 34.9 months (range from 2 to 84 months). Throughout this period, four episodes of acute pancreatitis were reported and four patients were classified as having chronic pancreatitis . Moreover, weight loss was documented in 13 patients, with a mean loss of 13.3 6 kg . The median of total igg was 2400 mg / dl (range: 1060 mg / dl4740 mg / dl). The two patients with elevated levels of igg4 had values of 264 mg / dl and 591 mg / dl, with total igg of 1060 mg / dl and 1670 mg / dl, respectively, both within normal values of total igg . Additional sites of igg4 + plasma cell infiltration were histopathologically identified in 9 patients, some of them with more than one infiltration site: myofibroblastic tumour (n = 3), lymph node infiltration (n = 6), palate (n = 1), lacrimal gland (n = 1), salival gland (n = 1), gallbladder (n = 2), prostate (n = 1), orbit (n = 1), liver (n = 1), and kidney (n = 1). Nine patients (60%) underwent major abdominal surgery (biliodigestive derivation or whipple procedure). Radiological data before treatment were available for review in 13 patients, for the other 2 cases only written radiological reports were analyzed . Twelve patients had evidence of pancreatic enlargement as previously defined, 3 of this cases were interpreted as having a mass . The most common extrapancreatic involvement identified was abdominal lymphadenopathy in 5/15 cases, followed by interstitial lung involvement in 4/15 cases, and finally 3/15 patients had evidence of glandular involvement on head ct . Glandular organs, including pancreas, lachrymal glands, and minor and major salivary glands, showed predominantly acinar atrophy and a variable degree of interstitial and periductal inflammatory infiltrate with abundant plasma cells and fibrosis . Lymph nodes, including peripancreatic ones, had follicular hyperplasia and dilated sinuses with abundant plasma cells . All cases met criteria for lpsp (type 1). A total of 8 patients were treated with steroids . All of them received oral prednisone at doses of at least 20 mg / qd . All patients responded to treatment and, to this day, none has relapsed although this is not yet significant as the follow - up time from the beginning of treatment has been brief due to delay in diagnosis establishment . As was previously mentioned, two main histopathological variants of autoimmune pancreatitis are currently recognised, and they behave clinically in quite different manners . The recently established international consensus has suggested the use of this classification, but this is not, however, always feasible due to the need of a sufficiently large sample of tissue for a pathologist to study . In this work, we introduce the first series of cases of autoimmune pancreatitis in the mexican population, all of them confirmed by histopathological analysis . The review of these cases showed that all patients corresponded to the lpsp variant, which is the most frequently reported, even though our patients' mean age was much lower than that expected for this histological type (mean age of 61.6 versus 47.5 for out population). Nowadays, it is recognised that one of the most common symptoms is obstructive jaundice; however, we found it with a lower frequency than that previously reported, since in asian patients the frequency goes from 74 to 88% [12, 13], whereas only 53% of our patients had this symptom at the onset of the disease . It is worth remarking that we found a relatively high frequency of acute pancreatitis, which in a recent multicentre study was mainly associated to idcp . Abdominal pain was also reported with a higher frequency than expected according to previous reports . Extrapancreatic involvement has been mainly associated to lpsp with, in some cases, 2 to 4 organs affected . This is congruent with our findings, as we found 9/15 patients with multiorgan involvement, although organ involvement could probably be more common but pass unnoticed for not making a direct search of it . It is important to point out that, due to the clinical presentation of this disease, the differential diagnosis is established mainly with pancreas cancer . In this regard, even though pancreatic mass was identified in only 3 patients, pancreatic growth in the imagining study was found in 12 patients which, in conjunction with the weight loss in the majority, gave rise to a high rate of surgeries (biliodigestive derivation or whipple procedure). In order to avoid this, one of the main elements that hint at a diagnosis of autoimmune pancreatitis before the surgery alternative is the increase in serum igg4, which has been found to be high in 75% of patients in some series [16, 17]. In this concern, there are two important points in our series: firstly, igg4 measurement was requested in only 10/15 patients, which indicates the low suspicion levels prevalent with regard to the disease, something that we hope will change soon; secondly, that only 20% of our patients showed an increase in serum igg4 levels, which goes against what has been reported up to now . The search for cases was based on histopathological results, and perhaps if a systematic measurement of igg4 serum levels in patients suspected of harboring the disease had taken place, a greater prevalence of high igg4 would have been found . Be this as it may, only a systematic study of this pathology within our ethnic group can really answer whether these observations are truly an atypical manifestation . Recently published the clinical characteristics of patients with igg4-negative autoimmune pancreatitis, in which there were differences with respect to those patients that showed an increase, and these patients seem closer to the presentation of our cases . In conclusion, autoimmune pancreatitis is a disease that is currently undergoing a process of definition of its clinical and diagnostic characteristics . The first cases are being found within our population, and a greater body of evidence is still being needed in order to establish clearly its presentation mode . Notwithstanding this, this series results seem to indicate that, within the mexican population, patients with autoimmune pancreatitis with lpsp are affected from the disease at an earlier age, with a low percentage rate for high igg4 serum levels, and with the main symptoms being abdominal pain, pancreatitis, weight loss, and obstructive jaundice.
An 18-year - old male transferred to the emergency department presented loss of consciousness, cyanosis, and deteriorated vital signs . According to the rescue team transfer records, he called the control center himself because he was home alone . He complained of aggravating dyspnea after eating dinner and of sudden onset vomiting . During ambulance transfer the patient could not remember what happened after the arrival of the rescue team at his house and had no memory of his stay at the emergency room and intensive care unit (icu). Upon arrival to the emergency room, the patient presented with a cyanotic lip, stupor or semi - comatose mental state, and severe tachyarrhythmia . Endotracheal intubation was performed immediately . After drawing blood for an emergency laboratory study and portable chest x - ray, 1), a large bore needle (16 g) was inserted into the second intercostal space of the mid - clavicular line to decompress intrathoracic pressure . Immediately after insertion of the needle, percutaneously checked sao2 increased dramatically to higher than 95% . Closed bilateral thoracostomy followed, using a 24 fr chest tube . The patient was transferred to the icu for close monitoring of the patient's mental state and other parameters related to tension pneumothorax . The abga results upon arrival and after insertion of the chest tube were a ph of 7.272, pco2 of 56.1 mmhg, po2 of 78.6 mmhg, and sao2 78.6%, and a ph of 7.373, pco2 of 42.7 mmhg, po2 of 171.3 mmhg, and a sao2 of 99.3%, respectively . Within several hours after admission into the icu, the patient's mental state and vital signs were restored to within normal limits . Two hours after admission into the icu, endotrachial intubation was removed and spontaneous breathing was maintained with an adequate sao2 level . The possibility of hypoxic brain damage was evaluated by a neurosurgeon, who confirmed that there were no significant sequela . On the second day at the hospital, bilateral pneumothorax recurred during the application of continuous negative wall suction (-18 cmh2o). The operation was performed under general anesthesia with double - lumen endotracheal intubation . To avoid hemodynamic instability during the operation, the patient was positioned with the head elevated 30 degrees (semifowler's position) and with the legs slightly elevated, with both upper arms extended . During the operation, to improve the operation field view, the operation table was tilted to the contralateral side (fig . A large bullae on the left lung and single giant bulla on right upper lung were resected with an endo - gia linear stapling device . One long thoracic drain (28 fr) was inserted bilaterally into the thoracic cavity after mechanical abrasion of upper portion of the parietal pleura . The incidence of simultaneous bilateral primary spontaneous pneumothorax has been reported to be as low as 1.4% to 7.6% . Simultaneous bilateral primary spontaneous pneumothorax can result in a severely deteriorated condition, usually requiring intubation or resuscitation . The reported causes of bilateral pneumothorax include trauma, tumor, and iatrogenic causes [1 - 3]. Other more rare causes have been reported, including catamenial pneumothorax, sarcoidosis, pregnancy, and radiation . Simultaneous spontaneous bilateral tension pneumothorax is defined as when no tracheal shift occurs and when the degree of bilaterally lung collapse is similar in a chest x - ray . Patients with simultaneously developed bilateral tension pneumothorax may deteriorate rapidly, and immediate decompression is recommended . A vicious cycle may occur, leading to a deteriorated condition, with a chain reaction consisting of lowered venous return, lowered preload, lowered cardiac output and so on . The patient under study experienced a critically life threatened state, loss of consciousness, cyanosis, and tachyarrhythmia . The treatment methods include needle aspiration, percutaneous catheter drainage, and tube thoracostomy [1 - 4]. Indication for surgical management of pneumothorax include continuous air leakage after insertion of chest tube, recurring pneumothorax on the same side, simultaneous bilateral pneumothorax, pneumothorax developed after pneumonectomy, and having an occupational cause . At present, video - assisted thoracic surgery (vats) is the most frequently used form of surgery for treating pneumothorax . Kim et al . Reported a series of 66 patients that were randomized to treatment with either transaxillary minithoracotomy (tamt) or vats . First, a smaller incision was required when compared with tamt, which generally resulted in less pain . Second, the cosmetic effect is superior to that of tamt . Finally, most procedures are possible (mechanical pleurodesis, chemical pleurodesis, etc . ). Prospective studies of simultaneous bilateral spontaneous pneumothorax management are rare . Generally, cases of bilateral pneumothorax require definitive surgical therapy to reduce the risk of recurrence . This can be done either through vats or open thoracotomy, depending on the surgeon's preference . Our case presented the rare clinical situation of simultaneous bilateral tension pneumothorax, and a decision was made to treat with immediate large size needle decompression at the emergency room by a properly trained doctor . The procedure was a critical step which restored this patient from life - threatening tension pneumothorax.
Natural zeolites are vulcanic hydrated micro porous crystals with defined structures based on alo4 and sio4 tetrahedral building blocks connected through oxygen atoms and with an open structure which can accommodate a wide variety of positive ions . Due to their structure, zeolites exhibit versatile adsorptive, cation exchanger, dehydrating - rehydrating and catalytic properties . Some zeolites are already used in medicine as antidiarrheal, antibacterial, antifungal drugs and glucose absorbent . Moreover reactive oxygen species (ros) are highly reactive molecules which may be both important mediators of some physiological functions and also potential prooxidants . Imbalance between ros generation and antioxidant capacity induces a condition known as oxidative stress which may play a major role in the initiation and progression of numerous pathologies including cardiovascular dysfunction associated with vascular disease, hyperlipidemia, diabetes mellitus, hypertension and ischemia / reperfusion injury . The potential damage caused by an excess of ros is controlled by a series of antioxidant defence mechanisms and, among them, a key protective role is played by the antioxidant enzymes gluthatione (gsh) peroxidase, superoxide dismutase (sod) and gsh reductase . Presently, the antioxidant role attributed to zeolites, and specifically to clinoptilolite, is based on the ability to reduce lipid peroxidation, free radicals levels and also to increase total antioxidant status (tas) in serum . Due to their key protective role in preventing or slowing down oxidative damage, to this aim, in the present study we evaluated whether chabazite / phillipsite / analcime, tipical zeolites of the central - souther of italy, may affect the levels of gsh peroxidase, sod, gsh reductase and tas in a group of clinically healthy men, both non - smokers and smokers . Furthermore, since lipid peroxidation is a major indicator of oxidative stress, thiobarbituric acid reactive substances (tbars) were measured for screening and monitoring lipid peroxidation in our subjects . The fine powder of natural zeolites from central italy (near bolsena lake) was obtained by tribomechanical micronization and sterilization at 100c for 24 h (geomedical srl, viterbo, italy). The zeolitic - tuff used contained approximately 33.7% chabazite [caal2sio126(h2o)], 17.0% phillipsite [(k, na, ca)1 - 2(si, al)8o166(h2o)] and 6.1% analcime [naalsi2o6h2o]. Other components were: sanidine (18%), plagioclase (14.6%) and muscovite mica (10.6%). The total zeolitic content of the tuff was about 56.8% . The cation - exchange capacity and specific mass were 400 meq/100 g and 1.26 n particle size analysis showed a granulometry less then 1 m for 15% of the particles, 2 m for 20% of the particles, 5 m for 40% of the particles, 15 m for 80% of the particles and 100 m for 100% of the particles . In this study, 25 clinically healthy male (40 3 years; age mean standard deviation (sd)) were enrolled . Subjects gave informed consent to the examination protocol, conducted in accordance with the declaration of helsinki and approved by the institutional committee . These subjects were following a typical mediterranean diet and leading a non - sedentary life style . Of these, 12 were non - smokers and 13 were smokers (more then 20 cigarettes / day). Subjects received for 4 weeks, twice a day, 1/2 h before breakfast and dinner, 3 g of chabazite / phillipsite / analcime as powder suspension dissolved in water . Edta - anti - coagulated blood samples were taken before (t0) and at the end of the 4 weeks (t1). Plasma samples were obtained after blood centrifugation at 1800 g for 15 min at room temperature and stored at 20c until assay . Gsh peroxidase, sod, gsh reductase and tas were measured or on whole blood or on plasma samples by specific kits from randox laboratories ltd . (crumlin, belfast, uk) on the semi - automated analyzer for colorimetric and turbidimetric assays rx monza (randox laboratories ltd .) Following manufacture s instructions . U / l of whole blood for gsh peroxidase, 164240 u / ml of whole blood for sod, 3377 u / l of plasma for gsh reductase, 1.31.7 mmol / l of plasma for tas . Reported maximum intra- and inter - assay coefficients of variations were 3.79% and 4.90% for gsh peroxidase, 3.58% and 7.07% for sod, 2.58% and 4.32% for gsh reductase, 3.08% and 3.75% for tas, respectively . Tbars test was performed using the method described by richard et al . Briefly, after the reaction of thiobarbituric acid with malondialdehyde, the product was extracted in butanol and was measured spectrofluorometrically (excitation: 532 nm, emission 553 nm, slit 10 mm). Box - and - whisker diagrams were used to represent data . The smallest observation (sample minimum), lower quartile (25), median (50), upper quartile (75), and largest observation (sample maximum) are shown . Comparisons between groups were performed using student s two - tailed t test and a p value<0.05 was considered significant . The fine powder of natural zeolites from central italy (near bolsena lake) was obtained by tribomechanical micronization and sterilization at 100c for 24 h (geomedical srl, viterbo, italy). The zeolitic - tuff used contained approximately 33.7% chabazite [caal2sio126(h2o)], 17.0% phillipsite [(k, na, ca)1 - 2(si, al)8o166(h2o)] and 6.1% analcime [naalsi2o6h2o]. Other components were: sanidine (18%), plagioclase (14.6%) and muscovite mica (10.6%). The total zeolitic content of the tuff was about 56.8% . The cation - exchange capacity and specific mass were 400 meq/100 g and 1.26 n particle size analysis showed a granulometry less then 1 m for 15% of the particles, 2 m for 20% of the particles, 5 m for 40% of the particles, 15 m for 80% of the particles and 100 m for 100% of the particles . In this study, 25 clinically healthy male (40 3 years; age mean standard deviation (sd)) were enrolled . Subjects gave informed consent to the examination protocol, conducted in accordance with the declaration of helsinki and approved by the institutional committee . These subjects were following a typical mediterranean diet and leading a non - sedentary life style . Of these, 12 were non - smokers and 13 were smokers (more then 20 cigarettes / day). Subjects received for 4 weeks, twice a day, 1/2 h before breakfast and dinner, 3 g of chabazite / phillipsite / analcime as powder suspension dissolved in water . Edta - anti - coagulated blood samples were taken before (t0) and at the end of the 4 weeks (t1). Plasma samples were obtained after blood centrifugation at 1800 g for 15 min at room temperature and stored at 20c until assay . Gsh peroxidase, sod, gsh reductase and tas were measured or on whole blood or on plasma samples by specific kits from randox laboratories ltd . (crumlin, belfast, uk) on the semi - automated analyzer for colorimetric and turbidimetric assays rx monza (randox laboratories ltd .) Following manufacture s instructions . U / l of whole blood for gsh peroxidase, 164240 u / ml of whole blood for sod, 3377 u / l of plasma for gsh reductase, 1.31.7 mmol / l of plasma for tas . Reported maximum intra- and inter - assay coefficients of variations were 3.79% and 4.90% for gsh peroxidase, 3.58% and 7.07% for sod, 2.58% and 4.32% for gsh reductase, 3.08% and 3.75% for tas, respectively . Tbars test was performed using the method described by richard et al . Briefly, after the reaction of thiobarbituric acid with malondialdehyde, the product was extracted in butanol and was measured spectrofluorometrically (excitation: 532 nm, emission 553 nm, slit 10 mm). Box - and - whisker diagrams were used to represent data . The smallest observation (sample minimum), lower quartile (25), median (50), upper quartile (75), and largest observation (sample maximum) are shown . Comparisons between groups were performed using student s two - tailed t test and a p value<0.05 was considered significant . Gsh peroxidase, sod, gsh reductase and tas levels were measured before (t0) and after 4-weeks zeolites intake (t1). At t0, no statistically significant differences were observed for gsh peroxidase, sod, gsh reductase and tas levels between non - smokers and smokers subjects, although gsh peroxidase and gsh reductase mean values appeared more elevated in the non - smoking group . Among the analyzed parameters, tas levels resulted higher than the normal values in both groups, whereas all the other parameters were within the normal reference ranges . At t1, gsh peroxidase, gsh reductase and sod resulted increased compared to basal levels, both considering all subjects as joint and after subdivision in the two sub - groups, non - smokers and smokers (table 1 and fig . 1). Differently from the other parameters, a statistically significant reduction in tas levels was observed at t1 . Anyway, tas levels remained higher than the reference values in both groups (table 1 and fig . 1). As previously observed at t0, also at t1 gsh peroxidase, sod and tas levels were similar in both groups . Differently from t0, instead, a major increased in gsh reductase levels was observed in non - smoking compared to smoking subjects (94.98 20.17 u / ml tbars were measured for screening and monitoring lipid peroxidation which is considered as a major indicator of oxidative stress . As shown in table 1 and fig . 2, at t0 plasma tbars levels are significantly lower in non - smokers than in smokers subjects (2.1 0.3 nmol / ml sd vs 3.3 0.2 nmol / ml sd; p<0.05). At t1, tbars resulted decreased compared to t0, both considering all subjects as joint (t0: 2.7 0.6 nmol / ml sd; t1: 1.6 0.3 nmol / ml sd; p<0.001) and after subdivision in the two sub - groups, non - smokers (1.5 0.3 nmol / ml sd; p<0.05) and smokers (1.6 0.3 nmol / ml sd; p<0.01). In the present study we observed that 4-weeks chabazite / phillipsite / analcime intake in clinically healthy subjects, both non - smokers and smokers, increased the levels of gsh peroxidase, sod and gsh reductase, three antioxidant enzymes able to remove ros, and decreased tbars, a major indicator of lipid peroxidation utilized for monitoring oxidative stress, thus preventing or slowing down oxidative damage . Anthropologists have always maintained that our ancestor ingested, to cure themselves, different varieties of rocks taken from the surrounding environment . This diet behavior has profound biological bases, bonded to organic need to take the mineral elements necessary for life . Since today developed societies are exposed to a wide variety of exogenous and endogenous stress factors that may influence the generation of ros and antioxidant activity, the use of integrators and particular kind of food, like zeolites, may help to eliminate free radicals and toxic substances from the body . In addition to the previously suggested antioxidant properties of some zeolites, in the present study we observed that the intake of chabazite / phillipsite / analcime, three zeolites widespread in many volcanoclastic depositis in the central - southern of italy, gave a significant protection against oxidative stress by increasing the levels of important antioxidant enzymes and reducing oxidative stress . These zeolites have been used in our study for the following reasons: they are the most abundant zeolites of italian soil, they have an overall cation - exchange capacity of 400 meq/100 g and, finally, they have not been previously studied as medicinal food relative to their antioxidant potential . The exchange capacity, which indicates the ability to release beneficial elements while capturing and binding others, has been indicated as an important requirement for the therapeutic application of zeolites . For this reason, due to a cation - exchange capacity of 400 meq/100 g, which is greater than that of other zeolites (about 200 meq for the most used and studied clinoptilolite), chabazite / phillipsite / analcime may have the requirements to be more effective then other zeolites . Previous studies which evaluated the effect of smoking on the levels of antioxidant enzymes reported different conclusions, since both significant and not significant differences in the activities of these antioxidant enzymatic systems have been described . Although at t0 we observed that gsh peroxidase and gsh reductase mean values were lower in the smoking group, however we did not observe any statistically significant difference . After chabazite / phillipsite / analcime consume, increased levels of all the three antioxidant enzymes were observed in both groups . These data thus may indicate that chabazite / phillipsite / analcime are able to increase physiological mechanisms against oxidative stress in healthy subjects exposed to different risk factors of oxidative stress, such as smoking, and may be considered as protective agents against oxidative stress . The decrement of tas levels at t1 seems to be in contrast with previous studies which indicated that zeolites intake may help to increase tas . One possible explanation is that our subjects are healthy and daily following a typical mediterranean diet rich in natural antioxidants which contribute to increase tas to levels higher than the normal range . Thus, it is possible that after ingestion, chabazite / phillipsite / analcime remove not only toxins and biological waste but also absorb other molecules . Although chabazite / phillipsite / analcime reduced tas, however, it is to be noted that it remained higher than normal values . In addition, the observation that tbars were reduced after zeolites intake suggests that zeolites do not aggravate oxidative stress . On the contrary a general improvement was observed in both groups (non - smoking and smoking). Future studies considering other categories of subjects following different diets and also patients affected by pathologies characterized by increased oxidative stress, will be necessary to better clarify the effect of chabazite / phillipsite / analcime on tas . At now, the mechanisms mediating zeolites antioxidant effects are not fully understood and since orally - administered zeolites are not absorbed into the blood it is possible that their in vivo effect may be due to an indirect interaction with biochemical systems, such as toxin and biological waste removal from the gut, activation of the immune system via the mucosal associated intestinal lymphoid tissue and also by increasing the bioavailability of mineral elements, which are important co - factors for the enzymes . In conclusion, our results strongly indicated that chabazite / phillipsite / analcime may help to counteract the effects of oxidative stress by increasing the levels of antioxidant enzymes and reducing oxidative stress in apparently healthy subjects exposed to different risk factors of oxidative stress . Future studies will be necessary to better clarify which mechanisms mediate the observed zeolites effects and will be helpful to better identify some other potential applications of such compounds also in fields like cardiovascular prevention and atherosclerosis.
They may occur at any point and age along the line of the repaired cleft . Reported recurrence range, after surgical correction of cleft palate, varies between a wide range of 10% and 30% depending on the technique used and age at the time of primary repair . Small fistulas may be asymptomatic but patients commonly complain of regurgitation of liquids into the nose, and food may become impacted with resultant malodor . Palatal fistulas can be variedly located in reconstructed palate starting from alveolar margin to anterior third, middle third, or posterior one - third of palate . The probable cause for occurrence of anterior palatal fistulas (apfs) is anatomically shorter lesser segment, wide palatal cleft defect with thin palatal shelves, improper reflection of subperiosteal flap, and improper closure . This study is a retrospective record review of patients diagnosed with apf in 3.5 years (which reported in our unit between january 2010 and july 2013). A total number of 91 patients with anterior palate fistula were operated in this duration . There were forty cases of unilateral apf which were treated with one or another technique surgically depending on various factors influencing the choice of surgical technique . Patients were categorized in the study depending on the procedure followed for the apf closure . Analysis of preoperative, immediate postoperative, and 1-month follow - up pictures was done to check for the site of occurrence, type of cleft, evaluation of adjacent tissue, size of fistula, postoperative healing, and closure with various techniques to compare and evaluate the results and outcome along with complication if any . Out of the forty patients included in the study, primary palate closure of ten patients was done in our unit (bhagawan mahaveer jain hospital, bengaluru, india), whereas thirty were treated elsewhere for repair of cleft palate primarily . In the study, unilateral apfs were evaluated depending on their site of occurrence, type of cleft, evaluation of adjacent tissue, and the size of fistulae . Depending on the site of occurrence, apf were categorized into three locations: alveolar fistulae, alveolar fistulae extending to hard palate, and fistulae in hard palate [figure 1]. (c) fistula in hard palate all the palatal fistulas were evaluated for the adjacent tissue in which the quality of tissue was assessed if it is a normal anatomical tissue or secondary mucolized tissue postpalatoplasty . (b) normal anatomical tissue techniques adopted for closure of apf were anterior palate redo with bardach's principle [figure 3], closure with rotation of island flap [figure 4], closure with crevicular flap, closure with buccal advancement flap, and closure with tongue flap and vomer flap . (a) unrepaired anterior palatal fistula . As shown in table 1, forty cases were split for surgical correction into various options depending on their size, site, and quality of the tissue . Most of the cases were operated with a bardach's redo for fistula closure (n = 16) (40%) and crevicular flap technique (n = 13) (32.5%). Our overall success (satisfactory results) was 77.5% as observed in 31 out of 40 cases with individual success rates for bardach's and crevicular being 75% and 77%, respectively . There was reduction in size of fistula in three cases (7.5%) and a remnant pinpoint hole in four cases (10%) among all the operated cases . We observed just two breakdowns of repair in forty cases . During evaluation of all the three cases managed with vomer flap, results were considered satisfactory in case of the absence of any symptomatic or asymptomatic fistula, no particular complaints by patient or nasal regurgitation . Indicating the split up of 40 cases into various surgical techniques adapted for apf closure and comparison of result even today, cleft palate and its associated surgical repair are rated among the greatest challenges in reconstructive surgery . A successful treatment outcome expected after repair of cleft palate includes achieving normal speech without increasing maxillofacial growth disturbances . Factors predisposing to development of postoperative dehiscence or fistula include width of the palatal cleft, amount of deficiency of palatal segment, misplacement, and distortion of the cleft segment . Other extrinsic variables considered for the fistula formation are the timing of repair, sex of the patient, surgical procedures, and the operating surgeon . Early dehiscence and fistulas are primarily caused by errors in technique such as inadequate mobilization, closure under tension, injury at reintubation, poor handling of the tissue, failure to achieve a layered closure, and postoperative bleeding and infection . Denny and amm proposed closure of palatal fistula with anterior palate redo with bardach's principle . They documented the technique of total palatal elevation to be a safe and reproducible technique for closure of hard palate fistulas using local palatal flaps . The authors were of the opinion that this technique possesses added advantage of allowing direct visualization of all the elements essential for a successful repair . Our results held resemblance to this concept since we were able to achieve a successful treatment outcome in 75% of the cases . Henderson discussed the use of advancement island flap for the closure of apf and named it as the tadpole flap . Although lesser number of defects were repaired in the current case series with island flap, but almost all of them showed satisfactory healing outcome . Carstens in his study put forward the sequential cleft management with sliding sulcus technique and alveolar extension palatoplasty . He stated that conventional methods of cleft lip repair deprive the anterior (buccolingual) alveolar mucoperiosteum of blood supply from the facial - internal maxillary arcade . After 6 months, while performing palatoplasty, the lingual incisions permanently isolate the lingual mucoperiosteum from vascular supply which is the greater palatine artery, thereby transforming the osteogenic alveolar mucoperiosteum from a richly supplied boundary zone between the two angiosomes into an isolated tissue surviving primarily on osseous backflow . Subperiosteal techniques that preserve the blood supply to this tissue is considered in a sequential plan of cleft management . The authors were able to perform apf repair in 13 cases through this technique in our study with a predictable healing result in 77% cases . Another approach utilized by jackson for the management of small- and moderate - sized fistulae involved a combination of buccal and palatal flaps along with bone grafting . However, such an intervention was performed in fewer number of patients in this study . Another attempt made by the surgeons to repair apfs with secondary mucolized tissue involved creating a vomer flap in small figure of three which was associated with significantly improved healing outcome in all the cases . Perhaps, an exclusive study on fate of vomer flap would provide more precise details regarding the predictability of this technique . Henceforth, the authors have made an effort to put forward treatment options for apf depending on their location by means of a simplified algorithm [figure 5]. For an apf, exclusively in alveolus buccal flap or crevicular flap can be used if patients' age is over 11 years, whereas for patients' younger than this secondary alveolar bone grafting with or without prior orthodontics or buccal flap appear to be more suitable . During management of fistulas extending to hard palate or into the hard palate, selection of the treatment modality depends on a major extent on the tissue quality . In case of normal palatine tissue, whereas in cases with secondary mucolised tissue, if it is present over greater segment, a vomer flap is advocated; if it is bilateral, tongue flap is advised; and in case of lesser segment, rotation of the greater segment is recommended . The current study was based on a stringent protocol regarding the selection of a surgical treatment plan based on anatomical location of fistula and the quality of surrounding tissue . Postoperative follow - up of maximum number of cases revealed minimal complication or recurrence rate . Additional factors that could be included in future studies for similar clinical cases include speech, etiology of fistula, and width of palate at the time of primary closure . Furthermore, we believe that a long - term follow - up of at least 6 months would be better to provide more reliable result
Osteoporosis and parkinson s disease (pd) are two chronic diseases among many that can lead to a downward spiral in a patient s quality of life, and they particularly affect elderly people . Osteoporosis is a systemic and metabolic skeletal disease characterized by increased risk of bone fragility due to a reduction in bone mass and microarchitectural deterioration of bone tissue1,2,3 . Pd is a chronic, slowly progressive neurodegenerative disease characterized by symptoms of rigidity, akinesia / bradykinesia, resting tremor, postural instability, and gait disorders . Recognized as the second most common neurodegenerative disorder after alzheimer s disease, pd has a great impact on quality of life, as it significantly affects the ability to carry out daily living activities due to both motor and non - motor symptoms and complications of medical treatment5 . Pd patients are at increased risk of falling compared with the general population, since they exhibit progressive symptoms such as reduced arm swing and balance and walking problems, especially at later stages of the disease6, 7 . As the risk of fall steps up, falls more frequently occur, and the rates of complications from falls such as fracture increase . Indeed, many studies have revealed that fracture risk, hip fracture being the most common, is increased in patients with pd8,9,10,11 . Osteoporosis - related fractures, especially hip fractures, impair quality of life as the independent mobility level worsens, and ultimately, the patient becomes bed - ridden . These fractures can lead to death by the end - stage of the disease10, 12 . Considering these potential negative effects, the importance of osteoporosis and lower bone mineral density (bmd) levels of pd patients are well understood . However, to our knowledge, no studies to date have investigated the bmd, vitamin d levels, and osteoporosis frequency of a pd population by comparing male and female patients with controls separately . The aim of this study was to compare bmd, vitamin d levels, and osteopenia and osteoporosis rates among male and female patients with pd and controls . Patients aged 5585 years who consecutively admitted to the movement disorders outpatient clinic of our institute between april 2014 and october 2014 were allocated to the study if their medical treatments were optimized, they were diagnosed with pd according to the uk parkinson s disease society brain bank diagnostic criteria13, and their pd stages were stage 4 or below; that is, they were still able to walk or stand unassisted or benefited from use of only one mobility aid, as per hoehn and yahr (h&y) staging . Patients were excluded if they had a history of any chronic diseases that could affect bone metabolism, had a history of lumbar spine and/or proximal femur fractures, had had an arthroplasty, or had been treated with corticosteroids, antiepileptics, bisphosphonates, hormone replacement therapy or vitamin d replacement therapy in the last year . The participants body mass indices (kg / cm) were determined based on demographic data collected from them including their age, gender, height, and weight . Following this, the properties of pd for each patient, such as disease duration and daily levodopa dosages, were recorded . In addition, the pd severity scale was determined for each participant based on the h&y classification14 . Unified parkinson s disease rating scale (updrs) part iii was utilized to assess the severity of the motor symptoms of pd15 . If the patients had fluctuations, they were assessed as being in the on state, namely medicine active . The control group was formed from individuals who were admitted to the musculoskeletal disorders outpatient clinic of our institute during the study interval . Individuals were excluded if they had a history of metabolic bone disease, had a history of lumbar spine and/or proximal femur fractures, had had an arthroplasty, or had been treated with corticosteroids, antiepileptic, bisphosphonates, hormone replacement therapy or vitamin d replacement therapy in the last year . The institutional ethics committee of our institute approved the study, and signed informed consent forms were obtained from all participants . The bmd (kg / cm) of the skeletal sites was measured by a trained x - ray technician using a hologic qdr-4500a dual - energy x - ray densitometer (hologic, bedford, ma, usa). The dual - energy x - ray absorptiometry (dxa) device was calibrated daily and weekly using the appropriated phantom methods . Measurements were made at the following sites: lumbar spine (l14), femoral neck, and total hip . Is defined as the number of standard deviations above or below the mean for the 20- to 40-year - old healthy adult reference population of the same sex and ethnicity . According to world health organization (who) criteria, osteopenia is diagnosed when a patient s t - score is between 1.0 and 2.5 standard deviations, and osteoporosis is diagnosed when the t - score is less than or equal to 2.5 standard deviations . Fasting (at 8.0010.00 a.m.) 10 cm peripheral venous blood samples were collected from the participants, placed in gel - containing tubes, and then centrifuged at 1200 g for 10 min to analyze the serum for 25(oh)d level . A commercial elisa kit (immuno - biological laboratories, minneapolis, mn, usa) was used to measure the serum 25(oh)d level, with the normal range being 11.142.9 ng / ml . A 25(oh)d level below 20 ng / ml was considered to indicate vitamin d deficiency, one between 20 and 30 ng / ml was considered to indicate vitamin d insufficiency, and one higher than 30 ng / ml was considered to be normal . Data were presented as means standard deviation (sd) or counts (percentages). Means were compared using independent t - tests for two groups . Statistical significance was based on a 5% level . Statistical analyses were performed using medcalc for windows, version 15.6 (medcalc software, ostend, belgium). One hundred fifty - two pd patients were assessed for allocation to the study at the movement disorders clinic over the course of seven months . A total of 37 patients were excluded from the study (previously had lumbar surgery and/or total hip replacement surgery (n=14), previously received a medical treatment on account of osteoporosis diagnosis (n=19), or previously had deep brain stimulation surgery (n=4)). The study was performed with the remaining 115 pd patients in the pd group and with 117 age- and gender - matched individuals in the control group . No difference was observed between the pd group and controls according to demographic characteristics (table 1table 1.demographic data for the parkinson s disease patients and controls and clinical characteristics of the parkinson s disease patientssubjectsmalesfemalespd (n=47)controls (n=47)pd (n=68)controls (n=70)age (years)67.2 10.2 * 66.8 5.867.1 10.0 * 67.4 6.6weight (kg)79.1 12.9 * 74.5 11.771.5 12.7 * 73.9 10.6bmi (kg / m)28.3 4.0 * 26.9 4.029.8 5.5 * 31.1 4.4pd: parkinson s disease; bmi: body mass index . * not significant). Pd: parkinson s disease; bmi: body mass index . * not significant the mean age of pd onset was 67.2 10.2 years for men and 67.1 10.0 years for women . The mean duration of pd was 85.1 62.6 months for men and 58.5 50.9 months for women . The mean levodopa dose was 450.5 214.5 mg / day for men and 327.9 326.8 mg / day for women . The mean updrs part iii score was 16.2 8.4 for men and 17.0 9.6 for women . Table 2table 2.bone mineral density and t - scores of the parkinson s disease patients and controlssubjectsmalesfemalespd (n=47)controls (n=47)pd (n=68)controls (n=70)lumbar spinebmd (g / cm)0.986 0.194 * 1.098 0.1700.804 0.1321.004 0.201t - score0.91 1.750.59 1.372.24 1.171.16 1.65femoral neckbmd (g / cm)0.741 0.1280.871 0.1480.650 0.1230.810 0.108t - score2.07 1.181.43 1.232.34 1.121.36 0.87total femurbmd (g / cm)0.901 0.148 0.995 0.1570.771 0.137*0.929 0.129t - score1.27 1.130.54 1.341.69 1.060.56 1.09vitamin d (ng / dl)12.6 7.620.6 11.211.7 6.8*18.6 11.4pd: parkinson s disease; bmd: bone mineral density . p<0.05; * * p<0.001 shows the mean bmd, t - scores, and vitamin d levels of the pd patients and controls . The mean bmd was significantly lower in the lumbar spine (p=0.003 for male, p<0.001 for female), femoral neck (p<0.001 for both), and total femur (p=0.004 for male, p<0.001 for female) in both male and female pd patients compared with the controls . Male pd patients femoral neck and total hip t - scores were found to be lower than male controls (p=0.012 and p=0.005, respectively). Female pd patients also had lower mean t - scores in the lumbar spine, femoral neck, and total femur (p<0.001 for all). The mean vitamin d levels were significantly lower in both male and female pd patients compared with the controls (p<0.001 for both). * p<0.05; * * p<0.001 the rates of osteopenia and osteoporosis in the lumbar spine, femoral neck, and total hip of male participants were not statistically different compared with the male controls . However, the osteopenia and osteoporosis rates in female pd patients in the lumbar spine, femoral neck, and total femur were found to be higher compared with the female controls (table 3table 3.osteoporosis and osteopenia in parkinson s disease patients and controlssubjectsmalesfemalespd (n=47)controls (n=47)pd (n=68)controls (n=70)lumbar spine (n,%) osteoporosis7 (14.9)5 (10.6)32 (47.1)15 (21.4)osteopenia17 (36.2)17 (36.2)26 (38.2)33 (47.1)normal23 (48.9)25 (53.2)10 (14.7)22 (31.4)femoral neck (n,%) osteoporosis14 (29.8)7 (14.9)39 (57.4)6 (8.6)osteopenia24 (51.1)29 (61.7)19 (27.9)43 (61.4)normal9 (19.1)11 (23.4)10 (4.7)21 (30.0)total femur (n,%) osteoporosis6 (12.8)5 (10.6)16 (23.5)10 (14.3)osteopenia20 (42.6)25 (53.2)38 (55.9)32 (45.7)normal21 (44.7)17 (36.2)14 (20.6)28 (40.0)vitamin d (n,%) deficient42 (89.4)29 (61.7)63 (92.6)50 (71.4)insufficient2 (4.3)7 (14.9)4 (5.9)12 (17.1)sufficient3 (6.4)11 (23.4)1 (1.5)8 (11.4)pd: parkinson s disease . * p<0.05; * * p<0.001). In addition, vitamin d deficiency and insufficiency were identified in 89.4% and 4.3% of the male pd patients and 92.6% and 5.9% of the female pd patients, respectively, which were significantly higher rates than those of their control counterparts (p=0.008 for male, p=0.004 for female) (table 3). There are three primary outcomes of this study: (1) lumbar spine, femoral neck, and total hip bmd values were lower in the pd patients group, irrespective of gender, than the control group . (2) higher rates of osteoporosis and osteopenia of the lumbar spine, femoral neck, and total hip were ascertained in female pd patients by comparison with the controls, whereas a significant difference did not exist for these rates between male pd patients and the controls . (3) as weighty factors in the development of osteoporosis, lower vitamin d levels and higher rates of vitamin d insufficiency were found for both male and female pd patients in comparison with the controls . It was also determined that the bmd levels were higher for the men than the women in both the study group and the control group . It was reported that osteoporosis and osteopenia were present in 91% of female pd patients and 61% of male pd patients and that female patients had 7.3% lower bmd levels than their age - matched controls and concordantly 2.6 times the fracture risk17, 18 . In women it has been shown that estrogen can promote apoptosis of bone - resorbing osteoclast and can lead to inhibition of osteoclastic bone resorption19 . The disappearance of a premenopausal inhibitory effect of estrogen on osteoclastic bone resorption after the postmenopausal stage is a major cause of lower bmd level and higher rates of osteoporosis in women . Immobilization is a leading factor explaining the decreased bone density and the increased risk of osteoporosis in patients with pd . In particular, immobilization becomes more apparent as the ambulation level reduces with pd progression . Besides, restriction of daily living activities in pd patients and transpiring motor symptoms such as bradykinesia, rigidity, postural instability, and gait disturbance form a basis for immobility . Rigidity can be considered to have a positive impact on bmd . On the other hand, that comes to a deadlock since pd increases muscle weakness and reduces mobility, but decreased mobility increases muscle weakness all the more . Lumbar bmd levels are independently associated with the strength of the trunk muscles rather than trunk rigidity20, whereas the specific cause of muscle weakness is not known in pd21, 22 . Bone tissue is sensitive to the mechanical environment that it belongs to; that is, bone tissue is constantly exposed to mechanical stimulation by muscle contraction and body movement that applies forces to the bone . In response to mechanical stresses on the bone tissue, osteocytes stimulate bone remodeling by producing molecular signals stimulating osteoblasts and osteoclasts23 . Subnormal mechanical stress as a result of reduced mobility leads to bone mass loss depending on duration and dosage . Sato et al . Analyzed the effect of immobilization - induced hypercalcemia on bone metabolism in pd patients and found that increased serum calcium levels correlated negatively with updrs part iii, suggesting the presence of immobilization - induced bone resorption with resultant hypercalcemia in patients with pd25 . In the present study, the bmd values for the femoral region of interest showed greater statistically significant differences than those for the lumbar spine in the pd patients compared with the controls group . The reason for this might be the decreased downward axial mechanical loading in the femoral region due to decreased mobility26 . Another consideration for low bmd values in pd patients may be hyperhomocysteinemia due to l - dopa treatment . It has been shown that patients with pd have higher serum homocysteine concentrations than controls, while the serum homocysteine levels were found to be independently associated with the bmd of the proximal femur27 . Homocysteine can induce differentiation of osteoclasts and apoptosis of osteoblasts28,29,30; thus, it is rational to infer that pd patients are readily affected by osteoporosis . Vitamin d deficiency is a major cause of lower bmd levels and a higher risk of osteoporosis and osteopenia in pd patients when compared with controls . The recent studies indicating that pd patients have lower levels of 25(oh)d relative to controls31,32,33 . It has also been demonstrated that a low vitamin d concentration was associated with bone loss21 . The prevalence of vitamin d deficiency in pd patients is higher compared with that of patients with alzheimer s disease32 . This suggests that there is a specific relationship between vitamin d deficiency and pd . Calcitriol (1,25(oh)2d) can be synthesized in neurons and microglia as a means of activating 1-hydroxylase . It was suggested that sustained inadequate vitamin d intake leads to a chronic loss of dopaminergic neurons and plays an important role in the pathogenesis of pd31 . Vitamin d status is a significant factor of skeletal integrity, and inadequate serum 25(oh)d level is correlated with weakness of muscles and increased incidences of falls and related fractures34 . It can be concluded that vitamin d deficiency in pd patients may result in decreased bmd values and increased risk of osteoporosis . Immobilization - induced hypercalcemia can cause suppression of 1,25-(oh)2d in the kidney by partially inhibiting parathyroid hormone secretion25 . Furthermore, sunlight deprivation due to immobilization may also lead to vitamin d deficiency in patients with pd35 . This study showed that vitamin d levels both in male and female pd patients were lower compared with those of the controls . The study elucidates that all pd patients regardless of gender have high vitamin d deficiency and insufficiency rates . Malnutrition is a quite common problem, specifically in patients with an advanced stage of pd . Hand - eye coordination disorders, dysphagia, intestinal hypermotility, depression, cognitive deficits, and the side effects of antiparkinsonian medications increases the risk of malnutrition . The reduction in bmi due to malnutrition may produce negative effects on bmd in the case of axial loading, although the present study could not observe any differences in bmi for either gender between pd patients and the control group . Additionally, regarding reduced bmd, a lower body fat content has also been found to be associated with lower estriol secretion in women16 . Also, malnutrition adversely affects the stability of vitamin d, and it results in deficiency of folic acid and vitamin b12, which leads to increased homocysteine . To the best of our knowledge, this is the first case - control study on bmd scores, vitamin d levels, and osteoporosis in pd patients that has separately compared male and female groups with controls . Firstly, h&y stage 5 patients were not included in the study; that is, the negative impact of stage 5 disease on bmd was avoided . In connection with this, higher rates of osteoporosis and osteopenia might have been ruled out . Secondly, it was not a population - based study . Therefore, the osteopenia and osteoporosis rates, as well as vitamin d levels, could be different from those of the general population . Thirdly, we did not collect data about dietary habits (e.g., smoking, alcohol consumption, or receipt of a vitamin d - fortified diet), activities of daily living, and amount of sunlight exposure, which might affect vitamin d levels and bmd scores . The main finding of this study was that all the pd patients had lower bmd levels, both in the lumbar and femoral regions, compared with the controls regardless of gender . In addition, the frequency of osteoporosis was only higher in female pd patients . Consequentially, screening for osteoporosis is needed as part of evaluations of patients diagnosed with pd in order to establish preventive measures to limit the future risk due to osteoporotic fractures, which may reduce qualify of life and even increase mortality.
Epithelial - mesenchymal transition (emt) and its reverse process (met) are physiological processes operative since the first days of the embryological development [1, 2]. In adults, this phenotypic transition is often just a physiological response to a call for tissue regeneration, as in the case of normal wounds [3, 4]. In fact, the cells deriving from emt, the myofibroblasts, smooth muscle cells alike, are specialized fibroblasts highly active in the production of extracellular matrix components (collagens, elastin, fibronectin, metalloproteinase, etc . ). Unfortunately, emt is often exacerbated during pathological events such as organ fibrosis, where the process occurs out of control and is irreversible . Recently, it has been pointed out that this differentiating process could be at the basis of the spreading of many epithelial cancers, since the cells resulting from emt acquire a much higher mobility potential [6, 7]. Among the many organ pathologies characterized by a fibrotic outcome, the renal nephrotic syndrome (ns) is one of the most studied and with the highest social impact . In the last few years, many new aspects of the pathogenesis of the disease have been unveiled . Now it is clear that, at least in the genetic and familiar forms, but also in many acquired cases, the filtration defect of ns depends on the quantitative and/or qualitative alteration of the podocyte slit - diaphragm protein components . The slit - diaphragm is a very specialized cell junction, with a selective filtration capacity, which preserves the organism from losing important proteins into the preurine filtrate . The major culprits have been identified in nephrin (nphs1) and podocin (nphs2) [813], among the many others . Nephrin is a large membrane protein with an igg like ectodomain, belonging to the igg superfamily proteins, while podocin is an integral membrane protein with one transmembrane domain and a c - terminal cytoplasmic tail . The nephrin transmembrane domain and most of the cytoplasmic tail are homologous to the corresponding regions of the stomatin family proteins . Nephrin and podocin interact physically and functionally, linking the cell membrane to the cytoskeleton and directing its reorganization [15, 16]. Most of the genetic forms of ns seem to carry mutations impairing the expression or the function of these two proteins, although impairment of all the other slit - diaphragm components can induce several degrees of proteinuria, up to severe ns or focal segmental glomerulosclerosis (fsgs) [1719]. As a result, the glomeruli become unable to perform their selective filtration function, leading to massive proteinuria . On the other hand, also external causes can induce damage to the podocytes, like infective, metabolic, and immunological agents . In these cases, as well, the slit - diaphragm seems to be the pathogenetic target . Besides the loss of the filtration function, ns develops a progressive and persistent fibrosis leading to the loss of functional glomeruli . So far, fibrosis has been mainly seen as consequence of the inflammatory and immunological response due to the involvement of nonresident cells, often present in the disease . Still, many investigators agree that these factors can account for no more than 5060% of the observed fibrosis . In the case of tubular interstitial sclerosis, the other major outcome of many renal pathologies, the fibrosis has been partially attributed to the intrinsic ability of the renal tubular epithelial cells to undergo emt [2022]. Usually, this is seen as consequence of increased activity of tgf-1 [23, 24] and other cytokines produced during these diseases ., a very reasonable and suggestive hypothesis is that the podocyte itself might be active producer of the sclerotic tissue in the ns, by undergoing emt (personal observation). Other earlier and more recent investigations, respectively, showed the ability of the podocyte to express low levels of -sma and undergo emt [2628] although, in the some cases, the authors referred to it as glomerular epithelial cells, not univocally identifying them as podocytes . Thus, we reasoned that, if the slit - diaphragm were pathologically altered, like for the nephrin genetic deficiency, this would lead to an impairment of the cell contact machinery . The loss of the cell contacts is, indeed, one of the triggering mechanisms of emt [3032]. Therefore, the nephrin deprived podocyte itself might undergo emt, contributing to the consequent sclerotic outcome . In the present paper, we tested our hypothesis and showed that a conditionally immortalized human podocyte cell line is able to undergo emt upon tgf-1 treatment, like previously shown in a murine model . Conditionally immortalized wild type and nephrin - mutated human podocytes cell lines (kindly supplied by saleem, ma, academic and children's renal unit, university of bristol, bristol, uk) were developed by transfection with the temperature - sensitive sv40-t gene, as previously described [33, 34]. The nephrin - mutated podocyte cell line was deprived of nephrin by genetic manipulation that inserted a constitutive 121delct frame shift mutation in nephrin transcript . After transfer to the nonpermissive temperature (37c), they enter growth arrest and express markers of differentiated in vivo podocytes . The cells were grown at the appropriate temperature, with 5% co2 in a humidified incubator in a growth medium composed of rpmi 1640, 10% fetal bovine serum (fbs), 1% glutamine, 1% antibiotics, 10 mg / ml insulin, 5.5 mg / ml transferrin, and 5 ng / ml sodium selenite (purchased as a mix from sigma - aldrich, st . Louis, mo, usa, product code i-3146). Where indicated, the cells were starved for 48 hr in 1% fbs and treated with 5 ng / ml of tgf-1 (cod . 100 - 21), which was purchased from pepro tech, rocky hill, nj, usa . In the experiment in absence of calcium, the cells were grown as indicated above but in calcium - free rpmi 1640 (gibco, oh, usa) as medium and 1% fbs . Total cell lysates were prepared in ripa buffer (50 mm tris - hcl, 150 mm nacl, 1% triton x-100, and 1 mm ethylen - diamine - tetra - acetic acid, edta), added with a protease cocktail and the phosphatase inhibitor cocktails i and ii (sigma, mo, usa). Typically, 30 g of the total cell lysates was separated on sds - page . Where indicated, the cell extract was fractionated in membranes, cytoplasm, and nuclei as following . After extensive washing in pbs, the cells were scraped in isotonic buffer (40 mm hepes, ph 7.5, 5 mm mgcl2, and protease and phosphatase inhibitors). Then they were passed several times through a 21-gauge needle and frozen and thawed for three times . The nuclear fraction was pelleted at 720 g for 10 min at 4c and resuspended in ripa buffer . 30 min at 4c, and the pellet containing the membrane fraction was resuspended in ripa buffer . Then, the proteins were blotted to a pvdf immobilon - p membrane, which was previously prepared according to the manufacturer's protocol (millipore, bedford, ma, usa). After blocking in tbs/1% bsa (0.1 m nacl, 0.05 m tris - hcl, ph 7.4, and 1% bsa, ph 7.5) for 2 hr, the membrane was incubated overnight or for at least 3 hr with the indicated antibodies . Then, the blots were incubated with the proper alkaline phosphatase conjugated secondary antibodies and developed with nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (nbt / bcip) reagents (roche diagnostics gmbh, mannheim, germany). The cells were grown to subconfluence on cover glass (bdh, milan, italy) fixed with 4% paraformaldheyde . The primary antibodies were used at a 1: 10 to 1: 20 dilution in the blocking solution as indicated in the relative figures . The goat snai-1 (homologous of drosophila snail) (sc-10433) and anti - fsp1 (also known as mts-1) (sc-19949) antibodies were purchased from santa cruz biotechnologies, (santa cruz, ca, usa). The secondary antibodies were conjugated with alexa fluor 488 dye (molecular probes, eugene, or, usa) and used at 1: 100 dilution . The confocal laser - scanning microscopy was performed using a focal scanning system laser co mrc 1024 er (biorad, usa). Expression levels of -sma, wt-1, nephrin, and proteins were evaluated by indirect immunofluorescence and confocal microscopy analysis on renal biopsy from patient with nephrotic syndrome of finnish type homozygous for p.s569r (c.1707c> a) nphs1 mutation and from wild type (wt) subject . Briefly, the slides were incubated overnight with the primary antibodies at 4c (mouse anti--sma, sigma - aldrich, 1: 100; rabbit anti - wt-1, sc-192, santa cruz biotechnologies, 1: 100; guinea pig anti - nephrin, progen biotechnik, 1: 100), washed in pbs, and then incubated with goat anti - mouse igg - alexa fluor 488, goat anti - rabbit igg - alexa fluor 555, and chicken anti - guinea pig igg - alexa fluor 488, respectively . The slides, mounted with an antifading aqueous medium (gel / mount, biomeda), were examined under a fluorescence microscope equipped with appropriate filters (leica tcs sp5; leica, wetzlar, germany). Confocal images were taken at 500 nm intervals through the z - axis of the section, encompassing a total of 4 m in depth . Images from individual optical planes and multiple serial optical sections were analyzed, and the images were sequentially scanned . Negative controls were performed by replacing the primary antibody with nonimmune serum at equivalent concentration . The cells were collected by scraping with a rubber policeman, after previous wash with pbs . The cells were pelleted at 600 g for 10 min, resuspended in 200 l/100 mm plate of 20 mm hepes, ph 7.9, 1 mm edta, 1 mm dithiothreitol, and protease and phosphatase mix (see section 2.2), and kept at 4c for 15 min . Of 1% np40 to obtain a final concentration of 0.2% np40 and incubated at 4c for 15 min . The pellet (p1) mostly containing the unbroken nuclei was resuspended in 1 vol . Of bls (20 mm hepes, ph 7.9, 1.5 mm mgcl2, 0.2 mm edta, 0.5 mm dithiothreitol, 0.5 mm pmsf, 80 mm nacl, and 25% glycerol). Of bhs (as in bls, but containing instead 0.9 m nacl) was slowly added and incubated at 4c for 30 min under moderate shaking . The assay was performed as previously described using 25 g of nuclear extract proteins in a total volume of 20 l containing 20 mm tris - hcl, ph 7.5, 0.1 m nacl, 0.35 mm dithiothreitol, 0.5 mm edta, 0.5 mm pmsf, 10% glycerol, and 0.5 l of 2 mg / ml of double stranded poly(di - dc) (1 g / reaction). The sequences of the oligonucleotides used as double - strand probe was the following: nf-b plus: 5-agt tga ggg gac ttt cc cag gc-3; nf-b plus: 5- gcc tgg gaa agt ccc ctc aac t-3. The oligonucleotides utilized were labeled at their 5 ends with (p)atp (nen life science) using t4 polynucleotide kinase . The rabbit anti - p65 (c-20) antibody was purchased from santa cruz biotechnologies . The binding to the double - stranded oligonucleotides was performed by incubating at 4c for 30 min 10 ftmoles of p - labeled probes with the previous nuclear extract . Where indicated, 1 l of antibody anti - p65 the dna - protein complexes were separated by electrophoresis on a 5% polyacrylamide gel and detected by autoradiography of the dried gel . One reproduced the normal podocyte features (wild type podocytes: wt), while the other was genetically deprived of nephrin with a constitutive 121delct frame shift mutation in nephrin transcript (nephrin - mutated podocytes: neph). The neph clone showed in this paper is representative of other tested nephrin deprived clones, since they all gave similar results and the experiments were always performed with at least 3 other similarly obtained clones, to rule out a casual clone effect . Both lines were immortalized with a retroviral construct carrying the sv40 large t antigen gene containing both the tsa58 and the u19 mutations . The immature podocytes were able to differentiate into nonreplicating mature podocytes only at nonpermissive temperature . In figure 1 (left panel), the effects of tgf-1 treatment on wild type human podocytes are shown . Assuming an emt process behind such a phenotype, we tested the podocytes for the expression of -sma . The western blot in the right panel of figure 1 shows that tgf-1, indeed, induced a strong 10-time increase of -sma already after 48 hr of treatment and was persistent even after five days . Noteworthy, it is the presence of discrete amounts of -sma in the untreated podocytes, as previously described in these cells . Another report showed that -sma is expressed in primary murine podocytes and seems to play a role in their maturation . Yet, although constitutively expressed, in our experiments, tgf-1 significantly triggered -sma expression in an emt process fashion . To better define whether we were in the presence of a typical emt, we searched for a set of other specific emt markers . Therefore, we looked at the downregulation of p - cadherin, typically expressed in podocytes, instead of the classical e - cadherin . In figure 2(a), we showed that p - cadherin was almost completely absent after tgf-1 treatment, being strongly downregulated already after 48 hr . In regard to cadherin behavior, an interesting finding was the switch to n - cadherin (figure 2(b)), which is often observed during emt occurring in metastasizing epithelial tumors . Screening for other emt markers, we looked at loxl2, a member of lox family [41, 42], recently identified as an upstream inhibitor of the proteasome - dependent degradation of snail [43, 44], which in turn, down regulates e - cadherin . In this case too, we found an important upregulation (figure 2(c)). The same increased expression was detected for slug, transcriptional partner of snail-1 [45, 46] (figure 2(d), right panel). Figure 2(e) also showed confocal images of snail-1 and fsp-1 (mts-1), specific fibroblast marker . The figure showed their expressions in basal conditions or after 5 days of tgf-1 treatment and confirmed that they were upregulated as well . Taken together, these results suggest that we were in presence of a typical epithelial - mesenchymal transition . Since the typical nephrotic syndrome is characterized by a qualitative and/or quantitative impairment of proteins of the slit - diaphragm, mainly nephrin and/or podocin, we thought that the syndrome might configure a condition of cell contact loss, which by itself has been described as a possible emt trigger [30, 31]. To test our hypothesis, we analyzed the nephrin - mutated podocytes for the same myofibroblast markers, with and without tgf-1 treatment . First, we noticed that they significantly differed at a phenotypic level from the wild type podocytes (compare figure 1, right panel wt and figure 3(a) neph). Western blots indicated a much higher expression of -sma as compared to the wild type podocytes (figure 3(b)). In addition, fibronectin and vimentin were constitutively highly expressed (figures 3(c)-3(d)), while they were almost absent or weakly expressed in the control cells . Interestingly, in the same figure (panel (e)) we show that these typical mesenchymal markers, like vimentin and -sma, failed to be upregulated by tgf-1, probably because they are already expressed at high levels, compared to the ones achieved by the wild type podocytes when triggered by this cytokine . Taken together, this evidence suggests that the nephrin deficiency itself induced an emt process . To test whether those features depended on the rupture of the homophilic contacts between podocytes, due to the absence of nephrin, we reproduced a similar condition in the wild type podocytes, by growing them in ca - free medium . According to our hypothesis, the wild type podocytes in absence of ca showed a significant upregulation of -sma (figure 4). It was already evident after 2 days of ca deprivation, reaching the same extent of tgf-1, or even higher, after 5 days (from about 2 times to 5 times after 2 and 5 days). Then, we decided to study the consequences of nephrin deficiency on the podocyte intracellular signaling . Since the emt induces loosing of cell contacts, of which the downregulation of e - cadherin is the most typical consequence (p - cadherin in the podocytes case), we looked at the signaling downstream of it . As evinced by its expression and cellular distribution, we found, indeed, a significant activation of -catenin . Figure 5(a) showed, in fact, a nuclear increase of -catenin translocation and its overall higher expression (figure 5(a): membrane + nucleus). However, the activation of -catenin was not induced by tgf-1 in wild type podocytes (data not shown), in contrast with previous reports . Based on these findings, we analyzed the nuclear levels of tcf-4 and lef-1, two of the main transcription factors cooperating with -catenin at transcription level . In figure 5(b), it is shown that nuclear tcf-4 and lef-1 were increased, respectively, three and nine times . Wondering about the consequences at cell cycle level of e - cadherin--catenin signaling perturbation, we investigated the functional status of two important tumor suppressor and cell proliferation regulators, often acting in coordination: p53 and prb . Figure 5(c) shows that p53 phosphorylated form is increased, both as fraction of total p53 (phospho - p53/p53 total) and as absolute amount, both in the cytoplasm and nucleus (phospho - p53/gapdh or /histone however, the expression of total p53 (phosphorylated and unphosphorylated) between the wild type and nephrin - mutated podocytes was not significantly changed (p53 total / gapdh or /histone panel (d) of the same figure shows also a sensible increased phosphorylation of prb in nephrin - deficient podocytes, meaning an overall less active status . Investigating other main signaling pathways, in agreement with a recent report, we found also a significant upregulated expression and activation of nf-b p65 (figure 6(a), left panel). In fact, most of the increase of p65 is localized in the nuclear compartment (figure 6(a), right panel). In the same figure, the emsa for the nf-b consensus sequence is also shown, confirming an increase of p65 binding to its dna target in nephrin - mutated versus wild type podocytes (figure 6(b), left panel). In figure 6(b), right panel, the p65 identity in the major dna - protein complex detected is also confirmed, since a specific anti - p65 antibody induced a supershift of the radiolabelled band . Since nf-b is central to many transduction pathways, we wondered whether the results regarding p53 and prb, and maybe -catenin, were in some way dependent on nf-b activation . To this purpose, we blocked the ib- proteasome - dependent degradation with bay-117082, an inhibitor of ib- phosphorylation, inducing, therefore, degradation of nf-b . In these conditions, we screened both podocyte cell lines for -catenin, ph - p53, ph - rb, and some of the mesenchymal markers which are overexpressed in nephrin - mutated podocytes . In figure 7, it is shown that bay-117082 lowered p65 expression as expected, but also p53 phosphorylation was diminished and -sma was down - regulated in both podocyte cell lines . In the figure, we showed that even vimentin level was almost shut down by the treatment in the nephrin - mutated podocytes, indicating a sort of reversion of emt . Conversely, prb phosphorylation and -catenin nuclear translocation remained unaffected, as well as the nuclear level of tcf-4, a -catenin target . Strangely, bay-117082 upregulated tcf4 in the wild type podocytes . Thus, not all the altered intracellular signaling of the nephrin - mutated podocyte can be ascribed to nf - kb activation, since -catenin and prb are not affected . It does, though, have an effect on the mesenchymal transformation, since -sma and vimentin are down - regulated upon its inhibition . This has been described in other models [4952]. To reinforce our original hypothesis, we tested whether also these altered pathways could be reproduced in the wild type human podocytes by ca deprivation . Figure 8, indeed, shows that -catenin nuclear translocation rate and p53 and prb phosphorylation levels mirrored their behavior in nephrin - mutated podocytes . At this point, it is evident that nephrin deprivation and, therefore, cell contact rupture must induce a wide and complex series of cross - interacting signals probably emt - dependent . A natural question arising from this study is whether these in vitro findings reflected a physiopathological occurrence in human chronic nephropathies, dependent on nephrin impairment, like for nphs1 . To answer the question, we analyzed by immunofluorescence and confocal microscopy renal biopsies from patients with nephrotic syndrome of finnish type . The one shown here is homozygous for p.s569r (c.1707c> a) nphs1 mutation . The results in figure 9 demonstrated that -sma was highly expressed in the glomeruli of the tested nphs1 biopsy, as compared to a kidney biopsy from a normal tissue . In figure 9, panel (a) (compare (a) versus (b)), we showed the absence of nephrin expression in the nphs1 glomeruli, as expected . The panel (c) of the same figure showed the colocalization of -sma with wt1, typical podocyte marker, to indicate its specific podocyte expression (compare (a), (b), and (c) versus (d), (e), and (f)). Such a colocalization is better shown in panel (d), which is an enlargement of a particular of panel (c). In panel (b) of figure 9 the negative control reaction for the immunofluorescence of -sma and wt1 is shown, as indicated . In panel (e) of the same figure, we showed the expression of -sma in a different renal pathology that is not based on nephrin impairment, the iga nephropathy . In this case, the expression was much less pronounced as compared to the biopsy of a healthy subject and, more importantly, did not seem to affect the podocyte area of the glomerulus . The p - cadherin was down - regulated, and the expression of -sma, typical myofibroblast marker, was upregulated and so all the major players of emt . Our study agrees with other investigator findings in similar mouse cell systems [28, 29, 39]. We believe that our findings corroborate the hypothesis of podocyte emt as a putative mechanism underlying the sclerotic reactions that characterize human nephrotic syndromes and other glomerular - based pathologies . More interestingly, we showed that genetic ablation of nephrin induced a constitutional emt as well, with most of the above indicated molecular hallmarks . Interestingly, tgf-1 barely affected, if at all, the expression of mesenchymal markers in nephrin - mutated podocytes, just as expected in a myofibroblast phenotype . These results prompted us to test the hypothesis that the impairment of the slit - diaphragm, and in general, of the cell contacts, might have a role in nephrin deficiency - dependent emt . In fact, the rupture of the intercellular junctions by ca deprivation induced, in the human wild type podocytes, phenotypic and molecular expression changes very similar to the emt observed in the nephrin - mutated podocytes . This finding might explain as well glomerulosclerosis occurring in nephrotic syndromes in which components of the slit - diaphragm other than nephrin are absent or functionally impaired . A more detailed investigation on the molecular consequences of nephrin ablation showed complex alterations of critical pathways, involving -catenin, nfb, p53, and prb . Considering the indubitable upsetting of the slit - diaphragm induced by nephrin ablation and emt - dependent downregulation of p - cadherin, we postulated that -catenin, among others, was likely the most affected protein in the network between the cell membrane and the cytoskeleton . In fact, many investigations reported concomitant -catenin activation upon e - cadherin knocking down or inhibition [5355]. Although primary -catenin activation seems to be able to indirectly downregulate e - cadherin, by activating snail, while it is known that snail, in turn, can also downregulate nephrin, in our model, it is more likely that -catenin activation is secondary to the cell contact impairment and consequent loss of cadherin . However, wnt-1/-catenin pathway has been recently highly considered in the podocytopathies as a new important pathogenetic or favoring factor to be taken into account [57, 58]. Definitely, it is more difficult to understand the p53 activation and prb downmodulation by hyperphosphorylation . We do not know yet what their role is in the economy of this kind of podocyte damage, mainly because we did not find any apoptosis increase . However, the absence of apoptosis could still be explained by the survival signaling coming from -catenin activation pathway . As matter of fact, a case of p53 activation and increase of prb phosphorylation has been described as dependent on nutlin-3, a p53 indirect activator . Moreover, previous observations indicated -sma to be positively regulated by p53 . Also, p53 activation in aggressive fibromatosis together with -sma expression, and in another case, rb depletion, both induced e - cadherin downregulation and emt . Adding further complexity to the observed pathway picture, we confirmed the activation of nf-b, in agreement with a recent investigation on the same nephrin - deficient cell line . Based on the presented findings, we would like to express a different point of view at this regard, since we believe that nf-b activation is more likely dependent on the emt / p - cadherin downregulation process, as previously noted in a model of malignant melanoma, rather than being induced directly by nephrin ablation . However, hussain et al ., while finding a nf-b activation, did not describe a nephrin deficiency - dependent emt, like in our case . As matter of fact in fact, bay-117082-dependent inhibition of nf-b affected negatively p53 phosphorylation, as well as vimentin and -sma expressions . We are in presence of a complex signaling network that must be triggered at the slit - diaphragm level, but it is hard to say what is due just to nephrin absence and what is dependent on the cell contact impairment as whole . It could well be that nephrin ablation on one side and cell contacts loss on the other trigger a cascade of overlapping and interfering signals, probably orchestrated by the emt process . Our in vitro study found also confirmation in kidney biopsy from a patient carrying a nphs1 mutation . This is very strong evidence that the in vivo pathology follows a pathway that is not different from what we observed in vitro . Nevertheless, we are fully aware that more cases of nphs1 should have been analyzed, but due to the difficulties to find those cases, we were not able to perform more immunofluorescence experiments . Though, it is quite suggestive that only the nephrotic syndrome caused by nephrin deficiency showed high levels of -sma, while in a iga nephropathy biopsy, the slight increase of -sma was mainly detected outside the glomerular area . Finally, we showed that ca deprivation reproduced the described pathway alterations even in the wild type podocytes . Although we are aware that ca deprivation might induce more complex metabolic effects, the impairment of the cell contacts is one of the most evident consequences, and this method has been previously used to the same purpose [30, 31]. Therefore, we feel that this latter result is reinforcing our hypothesis that the cell - cell interactions might play an important and probably primary role in the phenomena here described.
Herpes zoster is a reactivation of the latent varicella zoster virus in the dorsal spinal ganglion that is initiated by triggering factors such as trauma, aging, an immunocompromised state, malignancy, and radiotherapy . When reactivated, the virus replicates, resulting in inflammation, host cell death, and transportation of the virus along the axonal length of the sensory nerves to the skin . Patients clinically present with early symptoms of dysesthesia, tingling sensations, numbness, and itching, followed by the characteristic appearance of a rash in a few days . Vesicle formation is seen along the course of the nerve in a week to 10 days . These vesicles transform into bullae and eventually burst open to form scabs . When the trigeminal ganglion is involved, the pain produced by the infection can mislead clinicians into diagnosing the disorder as trigeminal neuralgia or odontalgia . Herpes zoster may produce complications such as blindness as seen in trigeminal opthalmicus1, acute or chronic encephalitis, myelitis, retinitis, autonomic dysfunction, motor neuropathies, guillain - barr syndrome, hemiparesis, and cranial or peripheral nerve palsies2 . More common complications include bacterial superinfection of the scabs by staphylococcus aureus or streptococcus pyogenes, scarring, and hyperpigmentation . Individuals infected with human immunodeficiency virus (hiv) show a higher prevalence for zoster infection than the general population1 . Although a reduction in immunity is considered the rationale for this feature, it has not been well documented in the literature that individuals on anti - retroviral therapy (art) experience a condition known as immune reconstitution inflammatory syndrome . During the first two months post - art or due to the change to a highly active antiretroviral therapy (haart), the patient's immunity tends to improve and respond better to latent infections, resulting in the manifestation of the infection . Also called immune recovery syndrome, haart is characterized by an increase in the cd4 count and a significant reduction in the viral load345 . The national aids control organisation (naco), a government initiative launched in 1992 to prevent and control hiv infection in india, estimated the epidemiological status of hiv / aids in 2012 as 20.89 lakh . The immunocompromised state in hiv patients makes them susceptible to various infections, including shingles or herpes zoster . A study carried out from 2004 to 2005 in karnataka, india, confirmed that out of 90 reported herpes zoster infections, 37.77% were sero - positive cases of hiv6 . Thoracic and lumbar dermatomes are commonly affected, and the trigeminal nerve is involved in 13% of patients7 . Currently, no evidence - based pharmacological strategy or a definitive guideline table for the prevention of immune reconstitution inflammatory disease can be recommended, as none of the pharmacological clinical trials have been completed . Clinical trials involving the use of maraviroc, a ccr5 chemokine receptor antagonist and a nonsteroidal anti - inflammatory drug (nsaid), are currently ongoing . Statins, vitamin d, and corticosteroids have also been suggested as other possible immune recovery inflammatory disease prevention strategies8 . Since the early initiation of art is not always possible due to a lack of awareness and economical limitations, a late presentation along with advanced hiv leads to high rates of opportunistic infections in these patients . Herpes zoster is a reactivation of the latent varicella zoster virus in the dorsal spinal ganglion initiated by triggering factors such as trauma, aging, an immunocompromised state, malignancy, and radiotherapy . The virus is transported along the axonal length of the sensory nerves to the skin and clinically presents with early symptoms of dysesthesia, tingling sensations, numbness, and itching; a characteristic rash appears after a few days . Vesicle formation is seen along the course of the nerve in a week to 10 days . These vesicles transform into bullae and eventually burst open to form scabs . When the trigeminal ganglion is involved, the pain produced by the infection can mislead clinicians to diagnose it as trigeminal neuralgia or odontalgia . The national aids control organisation (naco), a government initiative launched in 1992 to prevent and control hiv infection in india, estimated the epidemiological status of hiv / aids in 2012 as 20.89 lakh . The immunocompromised state in hiv patients makes them susceptible to various infections, including shingles or herpes zoster . A study carried out from 2004 to 2005 in karnataka, india, confirmed that out of 90 reported herpes zoster infections, 37.77% were sero - positive cases of hiv6 . Thoracic and lumbar dermatomes are commonly affected, and the trigeminal nerve is involved in 13% of patients7 . Currently, no evidence - based pharmacological strategy or a definitive guideline table for the prevention of immune reconstitution inflammatory disease can be recommended, as none of the pharmacological clinical trials have been completed . Clinical trials involving the use of maraviroc, a ccr5 chemokine receptor antagonist and a nonsteroidal anti - inflammatory drug (nsaid), are currently ongoing . Statins, vitamin d, and corticosteroids have also been suggested as other possible immune recovery inflammatory disease prevention strategies8 . Since the early initiation of art is not always possible due to a lack of awareness and economical limitations, a late presentation along with advanced hiv leads to high rates of opportunistic infections in these patients . Herpes zoster is a reactivation of the latent varicella zoster virus in the dorsal spinal ganglion initiated by triggering factors such as trauma, aging, an immunocompromised state, malignancy, and radiotherapy . The virus is transported along the axonal length of the sensory nerves to the skin and clinically presents with early symptoms of dysesthesia, tingling sensations, numbness, and itching; a characteristic rash appears after a few days . Vesicle formation is seen along the course of the nerve in a week to 10 days . These vesicles transform into bullae and eventually burst open to form scabs . When the trigeminal ganglion is involved, the pain produced by the infection can mislead clinicians to diagnose it as trigeminal neuralgia or odontalgia . A 75-year - old male patient visited the hospital with a chief complaint of pain in the entire right half of his face for the past eight days . The patient revealed a history of extraction of the right maxillary third molar tooth eight days before at a private clinic . His pain was severe in intensity and continuous in frequency, radiating to the forehead on the same side . The patient had been on art two months earlier for a period of three months, which was revealed only after the results of the investigations were obtained . There was history of a loss of appetite and weakness for the past six months . On examination, multiple cutaneous vesicles and scabs covered the same side of his cheek, ear, upper and lower lips, chin, and submandibular region. (fig . 1) mild diffuse swelling on the right half of the lips was noted, and generalized ulceration of the right buccal and labial mucosa was present. (fig . 2, 3) ulcerations on the anterior two - thirds of the right half of the tongue, lingual mucosa, tonsillar pillars, and the soft palate were also present . The ulcers were irregular and more than a centimeter in size and were covered with slough . Herpes simplex is the most common skin infection that presents a clinical picture similar to herpes zoster; others include impetigo, contact dermatitis, folliculitis, scabies, insect bites, papular urticaria, candidal infection, and dermatitis herpetiformitis . Therefore, laboratory testing with polymerase chain reaction, direct fluorescent antibody staining, and immunoglobulin m enzyme - linked immunosorbent assay (elisa) have proven useful for such cases . A history of or exposure to the zoster virus or the clinical presentation of a rash with a dermatomal pattern helps distinguish varicella infection from disseminated herpes zoster . In this case, the patient had a distinct pattern of lesions along the sensory distribution of the maxillary and mandibular nerves . His laboratory report showed a marked decrease in his lymphocyte count, and he tested positive for hiv . Anti - inflammatory drugs such as corticosteroids are reserved for mild to moderate symptoms of pain . Any interruption of art during the phase of opportunistic infection is not recommended unless it is a life - threatening opportunistic infection because the cessation of art can lead to art resistance . Since the patient was immunocompromised, the recommended oral dosage of 1,000 mg of acyclovir three times a day for seven days was prescribed . The patient was treated for the next two days and then was discharged against medical advice since he wanted to be admitted to a government hospital . A 75-year - old male patient visited the hospital with a chief complaint of pain in the entire right half of his face for the past eight days . The patient revealed a history of extraction of the right maxillary third molar tooth eight days before at a private clinic . His pain was severe in intensity and continuous in frequency, radiating to the forehead on the same side . The patient had been on art two months earlier for a period of three months, which was revealed only after the results of the investigations were obtained . There was history of a loss of appetite and weakness for the past six months . On examination, multiple cutaneous vesicles and scabs covered the same side of his cheek, ear, upper and lower lips, chin, and submandibular region. (fig . 1) mild diffuse swelling on the right half of the lips was noted, and generalized ulceration of the right buccal and labial mucosa was present. (fig . 2, 3) ulcerations on the anterior two - thirds of the right half of the tongue, lingual mucosa, tonsillar pillars, and the soft palate were also present . The ulcers were irregular and more than a centimeter in size and were covered with slough . Herpes simplex is the most common skin infection that presents a clinical picture similar to herpes zoster; others include impetigo, contact dermatitis, folliculitis, scabies, insect bites, papular urticaria, candidal infection, and dermatitis herpetiformitis . Therefore, laboratory testing with polymerase chain reaction, direct fluorescent antibody staining, and immunoglobulin m enzyme - linked immunosorbent assay (elisa) have proven useful for such cases . A history of or exposure to the zoster virus or the clinical presentation of a rash with a dermatomal pattern helps distinguish varicella infection from disseminated herpes zoster . In this case, the patient had a distinct pattern of lesions along the sensory distribution of the maxillary and mandibular nerves . His laboratory report showed a marked decrease in his lymphocyte count, and he tested positive for hiv . Anti - inflammatory drugs such as corticosteroids are reserved for mild to moderate symptoms of pain . Any interruption of art during the phase of opportunistic infection is not recommended unless it is a life - threatening opportunistic infection because the cessation of art can lead to art resistance . Since the patient was immunocompromised, the recommended oral dosage of 1,000 mg of acyclovir three times a day for seven days was prescribed . The patient was treated for the next two days and then was discharged against medical advice since he wanted to be admitted to a government hospital . A reactivation of the zoster virus in the nerve ganglia can be precipitated by many triggering factors, such as aging, trauma, immunosuppression, malignancy, and radiotherapy . The patient in our case report had a history of trauma following a tooth extraction, which triggered the reactivation of the latent virus . Although these drugs should theoretically be started within 72 hours of onset of rash, they should be initiated even in cases with delays, as they reduce the duration of viral shedding and also the severity and progression of the infection . Corticosteroids, analgesics, and topical agents such as lignocaine are adjuvants that can be administered to reduce the pain and severity of the infection . Patients who experience insomnia due to the discomfort may also benefit from tricyclic anti - depressants . The diagnosis of herpes zoster should prompt the clinician to consider hiv testing if risk factors, such as an age of less than 50 years or multi - dermatomal lesions, are present . He belonged to an older age group (75 years), and only his facial skin was affected, suggesting that clinicians cannot always narrow down the probability of hiv infection based upon its relation with risk factors . It is imperative that every patient with zoster infection be tested for hiv infection regardless of their absence of risk factors . It is a lesser - known fact that art causes a manifestation of latent infections345 such as herpes zoster as a result of immune recovery, as seen in this case report . A high vigilance for the patient by the treating physician during the first few months of art, during which there is a peak incidence of immune recovery inflammatory disease, is recommended . All patients who present with a zoster should be questioned regarding their history of art.
Migraine is a periodic, often pulsatile unilateral headache, which is common in all ranges of ages . Migraine headaches usually begin in the early adulthood although it can begins as late as fifth decade of life . A mendelian pattern was not observed in both types of migraine but familial occurrence is common in both types and especially in patients with aura . Migraine is a common disorder and prevalence of it is approximately 6.5 percent in men and 18 percent in women . In many women, migraine attacks tend to occur in premenstrual period and in some women migraine attacks occur solely in this period . Episodes of headaches usually persists 472 hours and nausea, vomiting, photophobia, phonophobia and fatigue are common symptoms that are usually observed in this disorder . Pain is usually severe and needs treatment with analgesics and sometimes bed rest is necessary . Migraines have many risk factors, some patients experience severe attacks after especial foods such as cheese, chocolate, onion or especial foods rich in tyramine . Obesity is a serious problem in our life which is growing more and more specially in city dwellers . Obesity has lots of complications such as heart failure, sudden death, early fatigue ability and osteoarthritis . Many methods are used for migraine headaches treatment such as chemical drugs, psychotherapy, injection of botulinum toxin, behavioral modifications, psychotherapy, chiropractic, acupuncture and biofeedback techniques . Some of these treatments are not accepted by all experts but there is consensus upon drugs . Treatment are divided into two phases, acute treatment of pain and preventive treatment . In the first phase, we usually use analgesics (nsaids), serotonin agonists and ergots derivatives and in the second phase the most preferred drugs are beta blockers, tricyclic antidepressants and anticonvulsants . Migraine is very important disease with high prevalence which makes many patients to spend a lot of time in bed for resting . Although there are many various treatments, some of them are not tolerated in all patients . Further investigation is recommended to find better options for the treatment of migraine headaches . In this study, we aimed to evaluate the effects of weight on the preventive treatment of migraine headaches . This was a prospective experimental study conducted on 203 patients who were referred for evaluation of headache to the neurology clinic in the period from 2009 to 2010 . Subjects were categorized in four groups according to their body mass index (bmi), <24.9, 24.9 - 29, 29 - 34.9 and> 35 . After the description of methods and aims of study all patients who had more than 3 episodes of migraine headaches each month and voluntarily asked for preventing treatment were selected for study . All patients were adults between 18 to 45 years old who had migraine headaches according to international headache society criteria (ihs). Visual analogue scale (vas) and 6-point behavioral rating scale (brs-6) were two standard international scales which were used for evaluation of the severity of pain . Brs-6 is a standard score for pain based on clinical symptoms which is divided from 0 to 5 . All patients were evaluated for the frequency, duration and severity of pain in the beginning of study and at the end of 4, 6 and 8 weeks . Selected patients were treated with nortriptyline 0.6 mg / kg / d and propranolol 1mg / kg / d . Dosage was increased gradually over four weeks from the beginning of study to the end of 4 week . Individuals who had any contraindication for use of these drugs were not included in this study . Mean age of the patients was 30.5 7.1 years ranged from 15 to 45 years . Weight, height and bmi of participants were 80.4 14.1 kg, 1.67 0.07 meter and 28.5 4.1, respectively . Pain frequency decreased from 8.1 2.3 attacks per month at baseline to 2.9 2.6 attacks per month in 8th week . Pain duration decreased from 16.1 6.1 hour at baseline to 11.7 5.2, 7.2 5.8 and 6.6 5.8 in 4th, 6th and 8th weeks, respectively . Vas also showed a decreasing pattern from 73.3 15.2 mm in 1st week to 56.8 16.2 mm, 40.3 20.9 mm and 32.7 23.9 mm in 4th, 6th and 8th weeks, respectively . It reduced to 2.72 1.04 in 4th week, 1.84 1.07 in 6th week and 1.40 1.20 in 8th week . Different demographic characteristic of patients, pain duration, frequency and severity of pain measured with vas and brs-6 scales are summarized in tables 1 to 3 . Variations of visual analogue scale in different body mass index groups during the weeks of intervention behavioral rating scale (brs) in different body mass index groups during eight weeks of intervention pain frequency and duration in different body mass index groups throughout the study period data are presented as mean standard deviation visual analogue scale variations in different groups in different weeks of study data are presented as mean standard deviation behavioral rating scale in different weeks of study in four groups of body mass index data are presented as mean standard deviation the aim of this study was to evaluate the effects of bmi on the treatment of migraine headaches . As mentioned earlier, all patients were classified in four groups according to their bmi, and all were treated with the similar dosage of drugs . Age, height and weight were different in all four groups and increased with different bmi in different groups . The mean frequency of pain was similar in all groups at the beginning but there was significant difference at the end of study . The mean duration of pain had significant difference in all groups at the beginning and at the end of 8 week . Mean vas score did not show statistically significant difference between bmi groups at the beginning . However, the difference was statistically significant at the end of the study in favor of better response to treatment in lower bmi . Brs-6 score showed similar pattern . In spite of direct influence of bmi on pain duration, it had no influence on pain frequency . However, comparing the influence of treatment on bmi in different groups showed that both frequency and duration had better outcome in patients with lower bmi . The findings of this study were in agreement with studies by bigal et al . And lipton et al . Because their studies showed association between bmi and migraine headache, although these studies had different methods . The findings of this study did not support mattsson and tietjen et al . Which did not find direct influence of bmi on migraine headaches . The mechanism by which obesity affects on migraine or its treatment is unknown but mechanism that influence on body weight may simultaneously influence on migraine . Lower activity of sympathetic and higher activity of parasympathetic systems and secretion of special neuropeptides such as neuropeptide y or melanocortins might be common factors . This study showed that obesity has a direct influence on the treatment of migraine headaches . Consequently, it should be recommended to patients to reduce their weight for better response to the treatment . In addition, physicians must be careful about migraine drugs which make a tendency for increased appetite.
The agricultural food production in developing countries and the mediterranean region suffers severely from the infestation of the parasitic weeds striga and orobanche sp.1, 2 seeds of these parasitic weeds can germinate only when a chemical stimulant exuded by the roots of host plants, such as maize, sorghum and millet come into contact or are in close proximity . This process leads to the development of a radicle from the seeds which then attaches itself to the root of the host plant through haustorium formation, thus enabling the germinated seed to extract nutrients, water and salts from the host plant with the consequence that its growth and production of a crop is severely affected.3 yield losses up to 90% have been reported.2 the chemical stimulants that were found to be responsible for triggering the germination of striga and orobanche seeds are the naturally occurring germination stimulants, collectively termed strigolactones (sls). The first sl, namely, strigol 1 was isolated as early as 1966 from the root exudate of cotton plants. [4a] its detailed structure was established about 20 years later.4 currently, a series of about 20 natural sls have been isolated and characterized,5, 6, 7 which are subdivided in two families, namely, one having the bc/2 stereochemistry as in (+) strigol (1) and the other one as in ()orobanchol (2) (figure 1). The control of parasitic weeds is extremely difficult due to the biological complexity of these weeds when coupled to their hosts, and hitherto no general method is available.5, 6, 7, 8, 9 an exciting and appealing option is that of suicidal germination approach, which involves applying an exogenous sl to the soil in absence of a host resulting in the germination of the seeds, but due to lack of nutrients the germinated seeds will die . Natural sls cannot be used for this approach as they have a too complex structure for a multigram synthesis.10 hence simpler sl analogues with an appreciable bioactivity are needed for this purpose . Early examples of such sl analogues are the gr compounds, e.g. Gr24 (figure 1).5, 6, 11 for a rational design of sl analogues a structureactivity relationship study provides valuable insight . Systematic simplification of sls revealed that the bioactiphore resides in the cd part of the molecule (figure 2).6, 7, 12 in this model there is a considerable structural freedom in the apart of the molecule . Model for designing sl analogues with germination activity . Using this model several new sl analogues with a high germination activity were designed, synthesized and subjected to bioevaluation . Typical examples are nijmegen1 (4),6, 7, 13 ketone derived analogue 5 6, 7, 14 and ketoenolderived sl analogue 6 6, 7, 15 (figure 3). Some representative sl analogues with germinating activity . A successful suicidal germination was reported in 1976,16 but this approach for weed control was abandoned, because of lack of funds and also because of the presumed instability of sl analogue under field conditions.17, 18, 19 about 30 years later the method was successfully used in tobacco infested by orobanche.7, 20 some of the new sl analogues, e.g 5 and 6, were evaluated in pot experiments and shown to be potential suicidal germinating agents.21 hence, suicidal germination is a realistic option and there is a need for identifying optimal germinating agents to be applied in an effective combat of parasitic weeds.20 the model shown in figure 2 was further detailed by varying the substituents at the position 3 and 4 of the dring.22 replacing the hydrogen at c2 of the dring with a methyl group was not investigated at that time as it was considered not to be relevant . However, now there is new information available about the mode of action of sls, namely that the enzymatic detachment of the dring plays a key role in the molecular cascade leading to germination (figure 4).23 the question now arises whether a methyl at the c2 position, indicated by an arrow in figure 4, will affect this process . This paper addresses this problem and deals with the preparation of some sl analogues having a methyl group at c2 of the dring and their bioevaluation . Gr 24 and nijmegen1 were taken as the target molecule for the introduction of a methyl group at c2 of the dring . The preparation of abcpart of gr24 (7) and sheehan aldehyde 10 was conveniently accomplished using literature procedures.24, 25 for the coupling of the dring, the general strategy26 was followed, as outlined in scheme 1 . The required hydroxy and chlorobutenolides 9 and 12 were prepared as shown in scheme 2, 27 by condensation of pyruvic acid and with acetone and butenone, respectively . The scaffold 7 upon treatment with chlorobutenolides 9a and 9b gave the dring modified sl analogues 8a and 8b, respectively . In a similar manner, scaffold 10 was converted into analogues 11a and 11b in moderate yields which were not optimized in this stage . The geometry of the enolic double bond in these analogues could not be deduced unambiguously from the nmr spectra . Therefore, an xray diffraction analysis was performed for analogue 11b (figure 5). There is only one mention28 of a compound having a methyl group at c2 of the dring, namely the compound 8b . It was prepared in a different manner, namely from a hydroxybutenolide obtained by reaction of methyllithium with 2,3dimethylmaleic anhydride29 followed by treatment with thionyl chloride30 and subsequent coupling28 with the enolate derived from the abc scaffold 7 . An observation of great practical importance is that the coupling of the an enolate with a chlorobutenolide proceeds in most cases highly satisfactory, in contrast to the coupling with the corresponding bromo butenolide which often is problematic . For instance, for the synthesis of sl analogue 5 derived from tetralone the coupling with bromobutenolide could not be realized, whereas with the chlorobutenolide the product formation took place smoothly . In actual practice we now convert the bromide into the chloride by treatment with lithium chloride in dimethylformamide in order to ensure a successful coupling reaction . The newly prepared sl analogues were bioassayed using seeds of s. hermonthica and o. cernua . The results are summarized in bar diagrams shown in figure 6 and figure 7 (see also the supporting information). The biodata reveal that the biological potency of the newly synthesized sl analogues with a dring substituted at c2 is comparable to, or somewhat lower than, that of natural isomers of gr24 (3) and nijmegen 1 (4). Therefore, this study suggests that structural changes in the dring moiety of gr 24 and nijmegen1 can be used as an alternative strategy to procure these materials in a rather economical manner, because the synthesis of hydroxybutenolide modified at c2 (9a and 9b) can be obtained from very cheap starting materials, namely pyruvic acid, acetone or 2butanone using simple and short reaction pathways . Bar diagram representation of percentages of the germinated seeds of s. hermonthica after exposure to various concentrations of compounds 8a, 8b, 11a, 11b and gr 24 (3) as a positive control . Bar diagram representation of percentages of the germinated seeds of o. cernua after exposure to various concentrations of compounds 8a, 8b, 11a, 11b and gr 24 (3) as a positive control . The newly prepared sl analogues were bioassayed using seeds of s. hermonthica and o. cernua . The results are summarized in bar diagrams shown in figure 6 and figure 7 (see also the supporting information). The biodata reveal that the biological potency of the newly synthesized sl analogues with a dring substituted at c2 is comparable to, or somewhat lower than, that of natural isomers of gr24 (3) and nijmegen 1 (4). Therefore, this study suggests that structural changes in the dring moiety of gr 24 and nijmegen1 can be used as an alternative strategy to procure these materials in a rather economical manner, because the synthesis of hydroxybutenolide modified at c2 (9a and 9b) can be obtained from very cheap starting materials, namely pyruvic acid, acetone or 2butanone using simple and short reaction pathways . Bar diagram representation of percentages of the germinated seeds of s. hermonthica after exposure to various concentrations of compounds 8a, 8b, 11a, 11b and gr 24 (3) as a positive control . Bar diagram representation of percentages of the germinated seeds of o. cernua after exposure to various concentrations of compounds 8a, 8b, 11a, 11b and gr 24 (3) as a positive control . The sl analogues having either the gr 24 or nijmegen1 basic skeleton and modified drings were readily obtainable by coupling with the appropriate chlorobutenolides modified at c2 . The biological data reveal that the bioactivity is hardly affected by the methyl substituents at c2 . The coupling protocols with the butenolides shown in scheme 2 can be added to the arsenal of methods for preparing a large variety of sl analogues . It should be noted that the new sl analogues 8a, 8b, 11a and 11b having a methyl group at c2 of the dring, are racemates . . Detailed conclusion on the influence of the c2methyl group in the dring on the germination stimulating activity can only be drawn when the respective enantiopure single diastereomers and enantiomers of these analogues are tested . However, for the purpose of obtaining simple and cheap suicidal germination stimulants for use in the field, the procurement of these compounds as racemates is satisfactory . The results of the bioassays are relevant for the understanding of the mode of action of sls.23 hnmr spectra were recorded on a bruker ac 100 spectrometer (100 mhz), ac 300 (300 mhz), and a bruker am400 (400 mhz), respectively, using me4si (tms) as the internal standard . Cnmr spectra were recorded on a bruker ac 300 (operating at 75 mhz) and am 400 (operating at 100 mhz) spectrometers with cdcl3 (= 77.0 ppm), [d6]acetone (29.206 and 206 ppm) as standards . Elemental analyses were conducted on a carlo erba instruments chnso ea 1108 elemental analyzer . Mass spectra were recorded using a double focussing vg 7070e mass spectrometer in the mode indicated, or a varian saturn 2 gc ms iontrap system . Gc ms separations were carried out on a fusedsilica capillary column (db5, 30 m 0.25 mm) and helium was used as the carrier gas . Packard hp 5890 gas chromatograph using a capillary column (25 m) hp1 with nitrogen (2 ml / min, 0.5 atm) as the carrier gas . Thinlayer chromatography (tlc) was carried out on merck precoated silica gel 60 f254 plates (0.25 mm) using the eluents indicated . Spots were visualized using a uv lamp, or with a potassium dichromate spray (prepared from 7.5 g of k2cr2o7 in 250 ml of h2o containing 12.5 ml 1 m h2so4) followed by heating at 140 c . Column chromatography was performed on silica gel (kieselgel, merck) using eluents indicated . The compounds 9a and 9b were prepared by previously described procedures.27, 28 3{(e)1[(2,4dimethyl5oxo2,5dihydro2furanyl)oxy]methylidene}3,3a,4,8btetrahydro2hindeno[1,2b]furan2one (8a): a stirred solution of the tricyclic lactone 7 (562 . 2 mg, 3.23 mmol) in ethyl formate (20 ml) was treated with small pieces of metallic sodium (81.7 mg, 3.55 mmol) whilst being maintained under a stream of nitrogen . After 1.5 h the mixture was concentrated in vacuo and anhydrous dmf (10 ml) was added to the residue . Then the dmf solution was cooled (0 c) and treated dropwise with a solution of the chlorobutenolide 9a (710.0 mg, 4.85 mmol) in anhydrous dmf (5 ml). The mixture was allowed to reach room temperature and then set aside for 24 h. the mixture was concentrated in vacuo, the residue was treated with water (50 ml) and ethyl acetate (30 ml), the separated aqueous layer was extracted with ethyl acetate (2 20 ml), the combined organic layers were washed with water, dried (mgso4) and then concentrated in vacuo . The resultant crude material was purified by column chromatography (hexane / ethyl acetate, 1:1, v / v) to give a mixture of unchanged compounds 7 and 9a (125.8 mg) and the desired compound 8a (514.8 mg, 51%), m.p . 200202 c; h nmr (cdcl3, 400 mhz): = 7.517.27 (m, 4 h, arh + = cho), 6.88 (s, 1 h, ch=), 5.96 (d, j = 7.9 hz, 1 h, h8b), 3.963.91 (m, 1 h, h3a), 3.45 (dd, j 4,3a, trans = 9.3, j = 16.9 hz, 1 h, h4), 3.18 (dd, j 4,3a, cis = 3.1, j = 16.9 hz, 1 h, h4), 2.02 (s, 3 h, ch3), 1.82 (s, 3 h, ch3) ppm . C nmr (cdcl3, 100 mhz): = 171.4, 169.7, 147.6, 144.9, 142.6, 138.8, 134.9, 130.0, 127.4, 126.4, 125.2, 113.1, 106.3, 85.8, 38.8, 37.3, 23.8, 10.6 ppm . Intensity (%)]: 313 ([m + 1], 0.4); 312 ([m], 0.4); 202 ([m + 1 111], [c12h9o3], 39.6); 111 ([m 201], [c6h7o2], 100). It should be noted that the product obtained consists of a mixture of two racemic diastereoisomers . 3{(e)1[(2,3,4trimethyl5oxo2,5dihydro2furanyl)oxy]methylidene}3,3a,4,8btetrahydro2hindeno[1,2b]furan2one (8b): a stirred solution of tricyclic lactone 7 (417.0 mg, 2.40 mmol) in ethyl formate (20 ml), while maintained under nitrogen, was treated with small pieces of metallic sodium (60.6 mg, 2.64 mmol) and then set aside for 2 h. the mixture was concentrated in vacuo and anhydrous dmf (10 ml) added to the residue . The thus obtained solution was cooled (40 c) and treated dropwise with a solution of chlorobutenolide 9b (512.1 mg, 3.12 mmol) in anhydrous dmf (5 ml). The mixture was then processed in the manner described above to afford a mixture of unchanged compounds 7 and 9b (66.4 mg) and the desired compound 8b (377.4 mg, 48%) after column chromatography (hexane / ethyl acetate, 1:1, v / v), m.p . 213217 c; h nmr (cdcl3, 400 mhz): = 7.507.16 (m, 4 h, arh + = cho), 5.96 (d, j = 7.9 hz, 1 h, h8b), 3.953.92 (m, 1 h, h3a), 3.42 (dd, j 4,3a, trans = 9.3, j = 16.9 hz, 1 h, h4), 3.20 (dd, j 4,3a, cis = 3.1, j = 16.9 hz, 1 h, h4), 1.98 (s, 3 h, ch3), 1.90 (s, 3 h, ch3), 1.59 (s, 3 h, ch3) ppm . C nmr (cdcl3, 100 mhz): = 171.3, 169.8, 155.3, 147.4, 142.6, 138.8, 130.0, 127.9, 126.4, 125.2, 113.3, 107.5, 85.8, 38.8, 37.3, 22.5, 10.6, 8.7 ppm . Intensity (%)]: 349 ([m + na], 23.0), 327 ([m + 1], 62.5); 203 ([m 123], 38.1); 201 ([m125], [c12h9o3], 4.9); 125 ([m 201], [c7h9o2], 100). C19h18o5 (326.35): calcd . Methyl (z)3[(2,4dimethyl5oxo2,5dihydro2furanyl)oxy]2(1,3dioxo1h2isoindolyl)2propenoate (11a) (2menijmegen1): a stirred and cooled (60 c) solution of sheehan aldehyde 10 (400.0 mg, 1.62 mmol) in anhydrous dmf (10 ml), while maintained under nitrogen, was treated with potassium tertbutoxide (199.7 mg, 1.78 mmol), followed by a solution of chlorobutenolide 9a (261.0 mg, 1.78 mmol) in anhydrous dmf (5 ml). The mixture was allowed to attain room temperature and set aside for 24 h, and processed in the same manner as described above to afford compound 11a (286.7 mg, 50%) after column chromatography (hexane / ethyl acetate, 1:1, v / v), m.p . 182184 c; h nmr (cdcl3, 300 mhz): = 7.907.70 (m, 4 h, arh + = cho), 6.93 (s, 1 h, ch=), 3.76 (s, 3 h, och3), 2.00 (s, 3 h, ch3), 1.73 (s, 3 h, ch3) ppm . C nmr (cdcl3, 100 mhz): = 169.9, 166.1, 163.3, 150.5, 145.8, 134.2, 133.7, 132.2, 123.8, 106.6, 106.3, 52.3, 23.5, 10.5 ppm . Intensity (%)]: 375 ([m + nh4], 39.3). Intensity (%)]: 247 ([m + 1 111], [c12h8no5], 97.3); 246 ([m 111], [c12h7no5], 14.6); 111 ([m 246], [c6h7o2], 100). C18h15no7 (357.32): calcd . C 60.51, h 4.23, n 3.92; found c 60.47, h 4.21, n 3.93 . Methyl (z)2(1,3dioxo2,3dihydro1h2isoindolyl)3[(2,3,4trimethyl5oxo2,5dihydro2furanyl)oxy]2propenoate (11b) (2,3me2nijmegen1): a stirred and cooled (40 c) solution of sheehan aldehyde 10 (716.9 mg, 2.90 mmol) in anhydrous dmf (10 ml), while maintained under nitrogen, was treated with potassium tertbutoxide (358.2 mg, 3.20 mmol), followed by a solution of chlorobutenolide 9b (261.0 mg, 1.78 mmol) in anhydrous dmf (8 ml). The mixture was allowed to attain room temperature, set aside for 72 h, and processed as described above to afford the desired compound 11b (360.2 mg, 34%) after column chromatography (hexane / ethyl acetate, 1:1, v / v), m.p . 175176 c; h nmr (cdcl3, 400 mhz): = 7.917.60 (m, 4 h, arh + = cho), 3.75 (s, 3 h, och3), 1.97 (s, 3 h, ch3), 1.89 (s, 3 h, ch3), 1.67 (s, 3 h, ch3) ppm . C nmr (cdcl3, 100 mhz): = 170.2, 166.1, 163.3, 156.9, 150.2, 134.2, 132.2, 126.8, 123.8, 107.7, 105.9, 52.3, 22.2, 10.5, 8.6 ppm . Ms / fab [m / z, rel . Intensity (%)]: 394 ([m + na], 61.6), 372 ([m + 1], 60.3), 371 ([m], 13.2), 246 ([m 125], [c12h8no5], 17.1); 125 ([m246], [c7h9o2], 100). C 61.45, h 4.61, n 3.77; found c 61.17, h 4.47, n 3.71 . Xray diffraction analysis of compound 7: crystals of 11b suitable for xray diffraction studies were obtained by slow cooling of the solvent (isopropyl ether).
Existence of non - inflamed or inflamed vermiform appendix in an inguinal hernia is named amyand's hernia in honor to the surgeon claudius amyand who successfully performed first perforated appendicitis . A 69-year - old turkish male patient with a slight right groin pain and swelling was presented to our clinic, and found to have a slightly tender and reducible right inguinal hernia . He underwent surgery under general anesthesia, and a adhesive caecum and an inflamed appendix were explored within the hernia sac . Adhesions were divided by sharp dissection and appendectomy was performed . After carrying out a lichtenstein hernioplasty, he recovered uneventfully, and neither complication nor recurrence was detected during 52 months of follow - up . Although occurrence of an appendicitis in an inguinal hernia is rare, a surgeon should be vigilant for facing with it even in elective cases . Treatment can be provided only surgically, but surgical treatment is not standard except from appendectomy . In our opinion, application of mesh hernia repair should depend on the degree of inflammation of appendix and the presence of incarceration of hernia sac with a suitable antibiotic admission for 3 - 5 days postoperatively . Claudius amyand (1660 - 1740), also known as surgeon - in - ordinary or sergeant surgeon to king george ii of england, defined an acutely inflamed appendix located in the inguinal canal in an 11-year - old boy on 6 december 1735 . Then he treated himself by an appendectomy, which is the first recorded successful appendectomy in the world . Actually, he described an appendicitis in the inguinal canal (later, it is named as amyand's hernia), and performed first successful appendectomy at the same time in 1735 . The surgeon defined the operation as most complicated and perplexing, and pathology revealed a chronically inflamed appendix included within the inguinal hernia sac and perforated by a previously swallowed pin . After the successful appendectomy, so much of hernia bag had been detached from the skin . The boy recovered uneventfully and discharged with a truss, he was ordered to wear it for some time, to confirm cure . This case was published in philosophical transactions of the royal society of london . In the present report, we purposed to present a rare case of inguinal hernia consisting the appendicitis and adhesive caecum in a 69-year - old man . A 69-year - old turkish man presented with slight right groin pain and swelling, but did not complain about nausea, vomiting or abdominal pain . On the physical examination, a slightly tender and reducible right inguinal hernia was detected . It was no abnormality except for a minimal tenderness at the right lower quadrant of the abdomen . The mobile bowel was observed inside the right inguinal channel and the scrotum, but no any mass or abnormality was detected in the testes in the preoperative scrotal ultrasonography . At the operation, an adhesive caecum and an inflamed appendix with appendiceal fecalith were explored within the hernia sac (figure 1). A drawn image of an adhesive caecum and an inflamed appendix with an appendiceal fecalith within the sac of right inguinal hernia after splitting the sac perioperatively . . Then an appendectomy was performed, and the remnant stump and caecum were brought back into the abdominal cavity . The hernia repair was achieved by carrying out a lichtenstein hernioplasty using prolene mesh and prolene sutures . However, the congestion and focal lenfocytic infiltration was seen in serosal portion of the appendix (figure 2). Although mucosal and submucosal part was in normal histological appearance, congestion and focal lenfocytic infiltration was seen in serosal portion of the appendix (haematoxylin & eosin, original magnification, 100). The probability of a person to have the disease of acute appendicitis in her / his life is 8%, and occurrence of an asymptomatic appendix within an hernia sac is a rare entity accounting for 1% . However, the incidence of an acute appendicitis in a sac of inguinal hernia is an even less common, it is about 0.13% [24]. Ryan reported in 1937 that 11 of 8692 cases (0.13%) of appendicitis were located in external hernia sacs . Thomas et al reported only 7 acute appendicitis occurring in an external hernia sac during 8 years . However, ages of that kind of cases ranges from 3 weeks to 88 years . The incidence of acute appendicitis in an hernia sac is reported between 0.008% and 1% in the english - language literature . An inflamed appendix is an emergency condition . In the case of amyand's hernia, nonetheless, to our knowledge, weber is the unique surgeon who suggested that he was detected it preoperatively . In the evaluation of groins and scrotum similarly, ultrasound imaging did not give additional information preoperatively except for the exhibition of the sliding hernia in the present case . Strangulated hernia, strangulated omentocele, acute hydrocele, richter's hernia, testicular tumor with hemorrhage, inguinal adenitis, and epidydimitis may be concerned in the differential diagnosis of amyand's hernia . Appendectomy through the herniotomy with primary hernia repair using the same incision is used for the treatment of hernial appendicitis . In the presence of appendix vermiform in the sac of hernia, however, in case of inflamed appendix in the sac, appendectomy without mesh hernia repair is suggested by some authors . Nonetheless, some prefer to perform both appendectomy and mesh hernia repair in the treatment of inflamed appendix in the inguinal hernia sac, and they recommended the intravenous broad - spectrum antibiotics for at least 3 - 5 days to prevent a possible mesh infection . Additionally, torino et al irrigated the inguinal area with antibiotics . In our case, even if the appendix was congested and inflamed with an adhesive caecum inside the sac, the sac was not incarcerated and no any abscess was observed . Therefore, we performed an appendectomy with mesh hernia repair, did not irrigate the inguinal area with antibiotics, and postoperatively admitted an intravenous broad - spectrum antibiotic for 3 days . The appendicitis within an amyand's hernia is rare, but misdiagnosis is not seldom when it occurs . Although it can be treated by surgery only, surgical treatment is not standard except from appendectomy . In our opinion, application of mesh hernia repair should depend on the degree of inflammation of appendix and the presence of incarceration of hernia sac . Awareness of this condition would be helpful in the preoperative diagnosis of both exigent and elective cases.
Samuel darling in 1906 is a dimorphic fungal organism found in moist fertile soil contaminated by bird or bat droppings . Although endemic in east and central us states bordering the ohio river valley and the lower mississippi river, in europe some rare autochthone cases in italy, near the po valley, have been described . Transmission regularly occurs via inhalation of h. capsulatum spores from the soil, and primary infections in the immunocompetent host are generally asymptomatic or present as a self - limiting flu - like illness . If infection cannot be cleared, as in immunocompromised patients with disabled cellular immunity, the organism continues to reproduce intracellularly and disseminates throughout the body via lymphatic and hematogenous circulation, cumulating in a state called disseminated histoplasmosis . Disseminated histoplasmosis manifests most prominently in the digestive tract, from mouth to anus, beside its well - described symptoms and manifestations, such as pulmonary disease, skin lesions, fever, and encephalopathy . Untreated disseminated histoplasmosis usually leads to death within a few weeks . Therefore, accurate diagnosis and appropriate treatment, we present the case of an obviously nonimmunocompromised patient from a nonendemic area infected by h. capsulatum, and we thus describe the first case with a disseminated disease course in austria . A 67-year - old man was referred to our department of otorhinolaryngology in december 2014 for the evaluation of anorexia, painful dysphagia, hoarseness, diarrhea, and a history of unintentional weight loss of about 15 kg (equaling approximately 20% of his previous body weight of roughly 80 kg) in the last 3 months, accompanied by intermittent fever, night sweats, and chills . His past medical history consisted of stable coronary heart disease and well - controlled chronic obstructive pulmonary disease caused by long - term tobacco smoking (about 30 pack - years). He was a retired engineer employed at a local steel company, operative in africa and middle and south america, where the patient was involved in construction work and soil - disruptive activities for several months . However, there was no history of far distance travelling during the last 6 years . On admission, the patient was in an alarming general condition, cachectic with a wasting syndrome, requiring intermittent parenteral nutrition . The most obvious clinical observation was an extremely painful stomatitis and angular cheilitis with ulcers involving the tongue and the palate (fig 1a, b). Laboratory data on admission included an unremarkable blood count with slightly elevated c - reactive protein of 3.8 mg / dl, an increased lactate dehydrogenase of 264 u / l, and a mildly elevated angiotensin - converting enzyme level of 64 screening for tumor markers and autoimmune disease as well as testing results for hiv, hepatitis, and tuberculosis were negative . Chest x - ray demonstrated an emphysematous lung with reticulonodular parenchymal changes (fig 2a). Further evaluation for a suspected occult malignant disease, bearing in mind the long - term tobacco consumption, included a whole - body positron emission tomography scan, which detected an additional hypermetabolic process in the ascending colon (fig 2b). This prompted us to perform an endoscopic evaluation of the colon and the terminal ileum . Macroscopically, rather small ulcerous lesions and erosions presented on a diffuse edematous mucosa with capillary congestion in the terminal ileum and ascending colon upon endoscopic examination (fig 2c). Histological examination of the biopsies excluded a malignant process but rather described a polypoid mucosa infiltrated by a kind of granulomatous inflammatory process with the presence of prominent histiocytes . Intracellularly, these foam cells harbored abundant intracellular round - shaped fungal elements with a characteristic halo sign, about 24 m in diameter, highly indicative of h. capsulatum (fig 2d). Biopsies of the tongue and a computed tomography - guided puncture of the lung confirmed the presence of round intracellular fungal forms using periodic acid - schiff staining (fig 1c, d). Diagnosis was finally established from an oral re - biopsy by panfungal polymerase chain reaction, performed at a reference center at the medical university of vienna, confirming positivity for h. capsulatum . Liposomal amphotericin b (1 mg / kg body weight) was administered to the patient, and constitutional symptoms markedly improved during the next few days . After 4 weeks of treatment, the patient was discharged from hospital and switched to oral itraconazole . On follow - up 6 months later (fig 3), the patient continued to improve and subjectively reported that his voice was much stronger and that his dysphagia and gastrointestinal symptoms had completely resolved . Particularly, conditions with compromised cellular immunity affecting t cells are prone to propagation of the disease, a scenario often resembling miliary tuberculosis . Only a small proportion of exposed individuals (<0.1%) may develop disseminated disease . Aids, htlv-1, hepatitis c, renal failure, long - term corticosteroid use or therapy with biologicals, and older age are well - characterized risk factors . Notably, up to 40% of patients presenting with disseminated histoplasmosis do not have any obvious risk factors . Endogenous reactivation of old foci during an immunocompromised state might be the trigger of dissemination . In our patient, the underlying chronic obstructive pulmonary disease might have been a risk factor for primary infection of the lung; however, we could not delineate any commonly acquired or inherited immune deficiency states often associated with disseminated histoplasmosis . Thus, reactivation years after the last stay in endemic areas, performing soil - disruptive activities, might just be attributed to an age - related immune system rundown, an already previously described promoting factor for dissemination . In clear contrast to the initial presentation of acute histoplasmosis, which almost exclusively involves the respiratory tract, disseminated histoplasmosis most prominently affects the gastrointestinal tract . Typical sites with abundant lymphatic tissue, such as the terminal ileum, are mostly affected, presenting on endoscopic examination as segmental inflammation, ulcerative lesions, and also polypoid masses, often incorrectly attributed to malignancy, inflammatory bowel disease (e.g., crohn's disease and ulcerative colitis), or appendicitis . In the majority of patients, gastrointestinal manifestation takes a subclinical disease course . Nonetheless, endoscopy is an important diagnostic tool for the assessment of dissemination . Therefore, it appears crucial for clinicians to be aware that an oropharyngeal or laryngeal presentation of histoplasmosis may be the only apparent clinical sign of dissemination . In addition, a differential diagnostic consideration should include tuberculosis, other fungal diseases, such as coccidioidomycosis and blastomycosis, and viral infection, such as coxsackievirus or herpes virus infection . Microscopically, diffuse lymphohistiocytic infiltrates with fungal elements about 24 m in size detected within the cytoplasm of macrophages are pathognomonic . Granulomatous inflammatory processes are often associated with elevated angiotensin - converting enzyme serum levels, and previous reports even suggested exclusion of histoplasmosis, especially during acute disease states, before establishing the diagnosis of sarcoidosis . Although serological testing for antibodies to h. capsulatum revealed a negative result in our case, this seems to be in accordance with previously published reports of diminished sensitivity of serological tests in disseminated disease, especially during immunocompromised states . Periodic acid - schiff staining is considered the gold - standard technique for microscopically based diagnosis, but sensitivity might be hampered by sampling errors, lack of expertise, or the impossibility to obtain representative material from severely ill patients . Although sensitivity of polymerase chain reaction is highly dependent on the type of isolate (culture vs. direct clinical), its impact lies in the reliable diagnostic differentiation between different fungal specimen . Lipid formulation of amphotericin b at 35 mg / kg body weight per day, intravenously, for 12 weeks, followed by itraconazole at 200 mg 3 times daily for 3 days and then 200 mg twice daily for a total of 12 weeks p.o ., is the accepted gold - standard therapy for disseminated histoplasmosis . Considering the severe illness of the patient, we decided for a prolongation of intravenous antifungal therapy up to 4 weeks . When clinical response was observed, antifungal therapy was replaced by oral itraconazole, and this treatment was continued for up to 1 year . As disease monitoring by the detection of the h. capsulatum antigen was impossible due to its exclusive availability in the usa, response to therapy was monitored on a clinical basis, showing a dramatic symptomatic improvement . Disseminated histoplasmosis is an important differential diagnostic consideration in patients with systemic illness, especially when oropharyngeal and gastrointestinal lesions are dominating . The rising incidence of disseminated histoplasmosis in nonendemic areas emphasizes the need for a thorough examination of the patients travelling history . Due to its heterogeneous clinical presentation, a close interaction between clinicians across the borders of their subdisciplines and between pathologists and microbiologists is mandatory for establishing the right diagnosis.
Levothyroxine (l - t4) is used worldwide as replacement therapy for patients with hypothyroidism, and for the last few years it has been the third most commonly dispensed therapy in the united states . Synthetic l - t4 as a therapeutic agent was first used in the 1950s and was widely adopted as the primary agent for thyroid hormone replacement, replacing the natural desiccated thyroid extract that had been used over the previous 50-year period . Although replacement therapy with levothyroxine has been prescribed for more than 60 years and is generally considered straightforward, cross - sectional surveys of patients on treatment with levothyroxine demonstrate that between 40% and 48% are either overtreated or undertreated [3, 4]. Firstly, a number of factors may interfere with intestinal absorption of l - t4, including food, dietary fibre, coffee, drugs, gastric or intestinal resection, and disease . As a result, current guidelines recommend that l - t4 is taken in a fasting state at least 30 minutes before breakfast [68]. Secondly, it is not always reliable to follow medical advice, especially for drug therapy; this is especially relevant for l - t4 therapy as a significant number of patients have problems in postponing their breakfast by 3060 minutes to ensure that they take l - t4 in the fasting state . In the therapeutic scenario the recent introduction of nontablet l - t4 formulations, such as liquid and soft gel capsules, would seem to raise doubts as to this recommendation . The results of a recent randomized, double - blind, placebo - controlled crossover trial showed that a liquid l - t4 formulation can be ingested during each patient's normal breakfast, mixed with tea, coffee, milk, cappuccino, orange juice, and so forth, thus potentially improving therapeutic compliance . This study, the titi study (tirosint-versus - tiche study, ibsa italia), looked to extend these findings by investigating whether an l - t4 soft gel capsule formulation could also be safely and effectively administered to stable euthyroid patients during each patient's normal breakfast without any malabsorption . Eligible patients were selected by a search in the database for those treated and followed up at the thyroid unit of the department of clinical and experimental sciences, university of brescia, italy . Eligibility (search) criteria were as follows: (a) treatment of hypothyroidism with liquid l - t4 (ibsa farmaceutici italia srl ., lodi, italy) ingested during each patient's normal breakfast; (b) stable levothyroxine replacement over the last 6 months; (c) complete personal medical history; and (d) details of current drug therapy and any previous therapy . In order to avoid any possible confounders (i.e., the need to increase l - t4 therapy in patients affected by hashimoto's thyroiditis during the study span) we enrolled only patients who had previously undergone total thyroidectomy for proven benign goitre . All participants had to maintain the same breakfast habits and any ongoing therapy for the entire period of the study . All patients were assessed for thyroid - stimulating hormone (tsh), free t4 (ft4), and free t3 (ft3) levels and were switched from liquid l - t4 to the soft gel capsule formulation at the same dosage of l - t4, ingested during each patient's normal breakfast . Serum concentrations of tsh, ft4, and ft3 were once again established after six months . All patients gave their informed consent to participate in the study, conducted in accordance with the declaration of helsinki . Serum concentrations of ft4 (normal range 8.019.0 pg / ml, analytical sensitivity 1 pg / ml), ft3 (normal range 2.44.7, analytical sensitivity 0.35 pg / ml), and tsh (normal range 0.44.5 miu / l, analytical sensitivity 0.004 miu / l) were measured using a fully automated architect i2000 analyzer (abbott diagnostics, abbott park, il, usa) based on chemiluminescent magnetic immunoassay . Tsh, ft4, and ft3 levels distribution levels were nonnormally distributed and were not normalized by the usual procedures of data transformation; in these cases results are presented as median with minimum and maximum values . Comparisons between continuous variables were performed by paired samples t - test or related samples by the wilcoxon signed rank test, as appropriate . Of the 2371 assessed patients with hypothyroidism, 60 (51 females and 9 males, aged 47.7 11.2 years) were eligible for the inclusion criteria and were enrolled for the study . All patients were euthyroid based on test results conducted 6 months before recruitment whilst receiving stable liquid l - t4 therapy ingested with breakfast (mean dosage 106.25 24.28 g / day). A detailed description of breakfast composition, particularly related to insoluble fibres and/or soya milk, was obtained in each subject (table 1). The thyroid hormonal profiles during administration of liquid l - t4 six months before recruitment, at enrolment and after 6 months' treatment with l - t4 soft gel capsules, are shown in table 2 . There were no differences in tsh levels at the three different time points, but during treatment with the soft gel capsule formulation ft3 and ft4 levels were significantly lower than those found 6 months before recruitment and at enrolment with the liquid l - t4 formulation . A subgroup analysis was conducted on the last 30 consecutive patients using data after three months of treatment with l - t4 soft gel capsules (table 3). There were no significant differences in hormone levels between 6 months before recruitment, enrolment, and at 3 months . Significant reductions in ft3 and ft4 levels were observed between 3 and 6 months' treatment with l - t4 soft gel capsules whereas there was a slight increase in tsh value . The main finding of this study is that both the liquid and soft gel capsule formulations of l - t4 can be taken with breakfast . Priority should however be given to the liquid l - t4 formulation in those patients where even a small change in ft4 and ft3 levels is to be avoided . Current guidelines for the treatment of hypothyroidism recommend ingesting levothyroxine in the fasting state 6030 minutes before breakfast or at bedtime, at least three hours after the evening meal, for optimal and consistent absorption [68]. This recommendation is based on study data demonstrating that concomitant ingestion of food [1215], fibre, soya products, and coffee is associated with higher serum tsh values in hypothyroid subjects treated with l - t4 as compared with the fasting state . Taking l - t4 with coffee, or with water followed by coffee within a few minutes, results in a poor tsh response in many patients . The recent introduction of new nontablet formulations of l - t4, such as soft gel capsules and a liquid, could resolve this problem . In one small study of hypothyroid patients, vita et al . Observed that the absorption of a soft gel preparation of l - t4 (tiche capsules, ibsa, switzerland) was not impaired by taking it with coffee . The same authors also reported that the problem of incomplete absorption of l - t4 caused by proton pump inhibitor - induced increases in gastric ph was not observed with the l - t4 soft gel formulation . Demonstrated that there is therapeutic equivalence in liquid l - t4 administration at breakfast or 10 min beforehand . We have previously observed that taking l - t4 at 30 minutes before having breakfast was not associated with alterations in optimal tsh, ft4, and ft3 concentrations in a group of patients taking their dose of a liquid l - t4 formulation (tirosint, ibsa, italy) with coffee . More recently, we clearly demonstrated in a randomized, placebo - controlled double - blind crossover trial that the thyroid hormone profiles are almost identical after the administration of the same dose of oral liquid l - t4 either with breakfast or in the fasting state (30 minutes before breakfast)., no previous study has made comparisons between soft gel capsule and liquid formulations of l - t4 taken at breakfast time . The clinical relevance of the significant reduction in ft4 and ft3 serum levels after switching from liquid to soft gel capsule l - t4 is not clear . Nevertheless, further analysis of data on the last 30 patients would appear to indicate a progressive reduction in ft4 and ft3 levels after the switchover as time passed . At three months from switching over from liquid to soft gel capsule l - t4, tsh, ft4, and ft3 levels were superimposable on those found at recruitment, but reductions were seen over the next 3 months of therapy (i.e., by 6 months). The tsh had again slightly increased but the change was not of statistical significance . Based on these findings, it would be reasonable to assume that absorption of the soft gel capsule l - t4 formulation gradually diminishes as time passes when ingested with breakfast . A longer follow - up during soft gel capsule treatment could yield further information on the trend of thyroid hormonal profile and the clinical significance of the hormonal changes demonstrated by this study . For this reason, large longitudinal prospective studies are needed to clarify this important issue . In conclusion, the results of this study suggest that, in general, both the liquid and soft gel capsule formulations of l - t4 can be taken with breakfast . Priority should however be given to the liquid l - t4 formulation in those patients where even small changes in ft4 and ft3 levels need to be avoided, such as those receiving suppressive therapy for thyroid cancer and/or cardiopathic patients receiving substitutive l - t4 therapy.
Physical mixtures of benfotiamin at three different mass ratios (1:1, 1:2, 1:3 and 1:4) were prepared . The prepared mixtures were then filled in glass bottles, sealed and stored in a dessicator until further use . A mixture of drug and polymers in three different mass ratios were wetted with water and kneaded thoroughly for 30 minutes in a glass mortar . The dissolution study of pure drug, physical mixture and solid dispersion was carried out by using usp dissolution apparatus (type 2) at 100 rpm at temperature of 37 0.5c using 900 ml volume of ml p. 1.2 and p. 7.4 the volume withdrawn was replaced by fresh volume of dissolution medium to maintain constant volume of medium . The filtered samples were analyzed spectrophotometrically at 226 nm and the drug release was determined . The phase solubility studies were performed to determine stoichimetric proportions of benfotiamin and carriers- pvp k-30 and hpmc . The effects of polymers concentration at room temperature on solubility are shown in figure 1 . Results of concentration of carriers on solubility of benfotiamin the plot of drug solubility against polymer concentrations at room temperature indicated a linear relationship between drug and polymer solution . Both the type shows al type of plot i.e. The solubility of benfotiamine increased with increasing carrier concentration . Dissolution of the pure drug, physical mixtures as well as solid dispersions of benfotiamin with pvp k-30 (equivalent to 40 mg) was tested in acidic buffer (ph 1.2) and phosphate buffer (ph 7.4) for a period of 60 minutes . Dissolution of the pure drug, physical mixture and solid dispersion prepared by kneading method in ratio of 1:4 was found to be 36.63%, 41.63 and 99.72% in 50 minutes in acid buffer medium . Pure drug and physical mixtures shows almost same release, whereas the solid dispersion (1:4) shows 00% drug releases in one hour . The solid dispersion prepared using ratio 1:1, 1:2 and 1:3 showing corresponding drug releases that is 79.29%, 83.30% and 95.12% in 60 minutes as shown in figure 2 . In - vitrodissolution profile of benfotiamin with pvp k-30 in ph . Increasing the drug carrier ratio from 1:1 to 1:4 improved drug release profiles observed in for all formulations in case of kneading method with pvp k-30 and hpmc but the drug release rate was higher in 1:4 ratio for both the polymers . The drug release was found to be better in solid dispersions prepared with pvp k-30 as compared to those prepared with hpmc.
In 1998 kononen and colleagues published in nature medicine, the results of a high throughput analysis of tumor cases, named tissue micro array (tma) technology, via an ingenious substantial modification of a previous method, of transferring cores from several donor blocks into a virgin unique acceptor as replicated samples identified by the position in the rows and columns of the array . Three thousand and counting publications later, the tma technology has fulfilled the promises of high throughput analytical ability . The inbuilt geometry of the design and the unsophisticated execution has favoured a practical approach: common sense, histotechnology expertise and unwritten tips rather than written standards . It is therefore not surprising that the intrinsic analytical error rate has never been addressed and the standards for design and construction, either for diagnosis or for research, have never been defined, except for cooperative large - scale studies, or for the data exchange specification. [46] notably, none of these methodology suggestions addresses how to control for errors in matching individual samples on the slide with its own data or how safely track those data, except for a quote about asymmetry in order to prevent map orientation confusion . Published data about the analytical error rate in tma are lacking; unpublished estimates suggests that 1 in 5,000 to 10,0000 samples may be misidentified, even using printed blocks and barcode - driven sample ids (stephen m hewitt, personal communication). More frequently, lack of correspondence between cored - out samples and the original block are attributed to tissue heterogeneity, totally ignoring the contribution of sample switching . The technique is evidently assumed to be totally accurate in identifying each and every single spot; the occurrence of tissue heterogeneity and the high - throughput ability masks and overcomes small individual error - driven experimental noise . Therefore formal demonstration that superimposing a tma design onto the tma slide image results in validation of the individual components has never been made . As a consequence, reports of diagnostic use of the tma, where positive identification of a patient is crucial, are scarce . This report addresses and solves error control issues and provides scientific ground to the use of the tma technology for diagnostic but for industrial or scientific purposes as well . The laboratory information system (lis) in use in our department (athena, noemalife spa, bologna, italy) identifies each object with an unique identification (i d) number, including an 8 digit numeric slide i d . Patient and specimen data are entered into the lis, creating unique ids for accession number, procedure, specimen, tissue block, tissue slides, etc . The lis interface was modified to print its unique i d onto the label of each slide as a code 128c high density linear barcode, via a label printer (datamax m4206). We chose to use the tissue slide instead of the tissue block for barcoding because the slide is the item on which the pathologist marks areas to be sampled . Matching the slide with the corresponding block is a standard procedure in histology, which entails two separate and qualitatively distinct procedures totalling 6 seconds or less: matching first the complete ids on the label and on the block, then matching the shape of the tissue on the slide with the one on the corresponding block . The pathologist would chose which sample and which area of the sample would be arrayed and a set of marked, barcoded slides would be forwarded to the technician in charge of arraying . We next generated a query on the lis database (based on oracle 10.2), which extracts from the database five items (slide i d, block number, case number, patient i d and date in which the sample was received), when prompted by the i d scanned on the tissue slide [figure 1]. The query is exported as an excel file, to be acquired by the tissue arrayer software in the donor block database . Note that none of the records we chose to be inserted in the file allows identification of the patient by a third unauthorized party . The amount of information extracted from the lis is limited only by the dimension of the field in the database . Tma construction through individual case segmentation flow process the figure depicts the flow of samples, images and data through the process . Note that there is no access to the wsi database which does not pass through a barcode reader check (the wand symbol). A final reconciliation of the data contained in the design with the individual cores in the wsi is done by barcode - driven match between the wsi and its own unique design barcode reading was accomplished with a common hand held linear barcode reader . The file containing the 5 informative items of the donor tissue, is drag into the open window of the galileo tma program (tissue microarrayer model tma galileo ck4500; integrated systems engineering srl, milano, italy), populating automatically all the donor block fields . Next, before the design of the tma begins, the galileo software requests that the i d of the tma acceptor block is inserted; this is accomplished by barcode scanning a 5 digit alphanumeric unique tma i d, previously generated by a tma database separated from the lis, printed and inserted into the back of the virgin acceptor block [figure 1]. The same barcode i d is then printed together with alphanumeric date, stain, etc on a label glued on each section cut from that tma [figure 1]. The tma array is purposely designed with an intrinsic asymmetry, i.e. Transformation of the geometry by reflection along the axis generate new, readily distinguishable geometric forms . Care is taken so that the asymmetry produced by inserting or emptying spots is not lost upon accidental loss of single spots or rows on the slide . During the tma design construction with the galileo software, the design is a bi - dimensional matrix of n m positions, in which each full spot may be considered 1 and the empty one 0 . We define as i and j the indexes identifying the matricial design and a () each element, which is then a (i, j)=0 \| 1 where 0<i <m e 0<j <n . By applying to the matrix each of the 7 possible transformations [table 1], it is possible to calculate the robustness of the asymmetry by calculating the ensuing superimpositions of full and empty spots and ranking the safety of the design . A simple computer program can easily loop through all the possible transformations and count the number of spots that do not overlap after each change . A rank of 0 discards the design because there is at least one symmetric transformation, thus a chance of error [figure 2]. Listing of the possible transformations of the tma design consisting of a square array, a design less favourable than a rectangular one ranking the asymmetry of the tma design the figure depicts pair wise comparisons (top, bottom) of tma design with sectors (left) and solid (right). In all of them, empty spots are marked as 0, filled ones as 1 . Left of each design are listed the seven transformations () and the calculated rank of that transformation (see text). A rank of o in any permutation will result in discarding the design, because of symmetry the source of asymmetry is consistently placed in one fixed position (e.g. Lower right corner), to facilitate the correct orientation of the tma image before segmenting . The use of random placement of the samples makes the use of sectors unnecessary; in addition, empty rows or lines within the design run the risk of misidentify spots in case the tma section is particularly wavy or disrupted . Upon tma completion, two types of files are generated: an excel file containing the tma design, the list of all samples and relative data and an xml file, in which the same data are coded in a format readable by the software of the whole slide scanner . The galileo tma software natively generates files in the tma des (data exchange specification) xml format . The xml file is imported in spectrum (aperio technologies, vista, ca, usa) in a designed database, and each xml tag is addressed to a designed field in the database . The aperio slide scanner acquires the whole slide image (wsi) of the tma and the tma barcode on the label [figure 1] and stores both in the spectrum database . Additional information can be added to the software by the investigator, before or after the image analysis . To ensure exact matching of all patient's i d data with the corresponding sample images, the spectrum software was modified so that the match of the i d in the design and the i d on the wsi [figure 1] is highlighted, leaving to the operator the choice to hit the segment command . Without a match, once the correct rotation of the wsi is defined by placing the source of asymmetry in the pre - defined corner, the grid of the design is adjusted to accommodate the shearing that may take place when the 4 m section is placed on the glass slide and to fit all the available spots on the wsi . The laboratory information system (lis) in use in our department (athena, noemalife spa, bologna, italy) identifies each object with an unique identification (i d) number, including an 8 digit numeric slide i d . Patient and specimen data are entered into the lis, creating unique ids for accession number, procedure, specimen, tissue block, tissue slides, etc . The lis interface was modified to print its unique i d onto the label of each slide as a code 128c high density linear barcode, via a label printer (datamax m4206). We chose to use the tissue slide instead of the tissue block for barcoding because the slide is the item on which the pathologist marks areas to be sampled . Matching the slide with the corresponding block is a standard procedure in histology, which entails two separate and qualitatively distinct procedures totalling 6 seconds or less: matching first the complete ids on the label and on the block, then matching the shape of the tissue on the slide with the one on the corresponding block . The pathologist would chose which sample and which area of the sample would be arrayed and a set of marked, barcoded slides would be forwarded to the technician in charge of arraying . We next generated a query on the lis database (based on oracle 10.2), which extracts from the database five items (slide i d, block number, case number, patient i d and date in which the sample was received), when prompted by the i d scanned on the tissue slide [figure 1]. The query is exported as an excel file, to be acquired by the tissue arrayer software in the donor block database . Note that none of the records we chose to be inserted in the file allows identification of the patient by a third unauthorized party . The amount of information extracted from the lis is limited only by the dimension of the field in the database . Tma construction through individual case segmentation flow process the figure depicts the flow of samples, images and data through the process . Note that there is no access to the wsi database which does not pass through a barcode reader check (the wand symbol). A final reconciliation of the data contained in the design with the individual cores in the wsi is done by barcode - driven match between the wsi and its own unique design barcode reading was accomplished with a common hand held linear barcode reader . The file containing the 5 informative items of the donor tissue, is drag into the open window of the galileo tma program (tissue microarrayer model tma galileo ck4500; integrated systems engineering srl, milano, italy), populating automatically all the donor block fields . Next, before the design of the tma begins, the galileo software requests that the i d of the tma acceptor block is inserted; this is accomplished by barcode scanning a 5 digit alphanumeric unique tma i d, previously generated by a tma database separated from the lis, printed and inserted into the back of the virgin acceptor block [figure 1]. The same barcode i d is then printed together with alphanumeric date, stain, etc on a label glued on each section cut from that tma [figure 1]. The tma array is purposely designed with an intrinsic asymmetry, i.e. Transformation of the geometry by reflection along the axis generate new, readily distinguishable geometric forms . Care is taken so that the asymmetry produced by inserting or emptying spots is not lost upon accidental loss of single spots or rows on the slide . During the tma design construction with the galileo software, the design is a bi - dimensional matrix of n m positions, in which each full spot may be considered 1 and the empty one 0 . We define as i and j the indexes identifying the matricial design and a () each element, which is then a (i, j)=0 \| 1 where 0<i <m e 0<j <n . By applying to the matrix each of the 7 possible transformations [table 1], it is possible to calculate the robustness of the asymmetry by calculating the ensuing superimpositions of full and empty spots and ranking the safety of the design . A simple computer program can easily loop through all the possible transformations and count the number of spots that do not overlap after each change . A rank of 0 discards the design because there is at least one symmetric transformation, thus a chance of error [figure 2]. Listing of the possible transformations of the tma design consisting of a square array, a design less favourable than a rectangular one ranking the asymmetry of the tma design the figure depicts pair wise comparisons (top, bottom) of tma design with sectors (left) and solid (right). In all of them, empty spots are marked as 0, filled ones as 1 . Left of each design are listed the seven transformations () and the calculated rank of that transformation (see text). A rank of o in any permutation will result in discarding the design, because of symmetry the source of asymmetry is consistently placed in one fixed position (e.g. Lower right corner), to facilitate the correct orientation of the tma image before segmenting . The use of random placement of the samples makes the use of sectors unnecessary; in addition, empty rows or lines within the design run the risk of misidentify spots in case the tma section is particularly wavy or disrupted . Upon tma completion, two types of files are generated: an excel file containing the tma design, the list of all samples and relative data and an xml file, in which the same data are coded in a format readable by the software of the whole slide scanner . The galileo tma software natively generates files in the tma des (data exchange specification) xml format . The xml file is imported in spectrum (aperio technologies, vista, ca, usa) in a designed database, and each xml tag is addressed to a designed field in the database . The aperio slide scanner acquires the whole slide image (wsi) of the tma and the tma barcode on the label [figure 1] and stores both in the spectrum database . Additional information can be added to the software by the investigator, before or after the image analysis . To ensure exact matching of all patient's i d data with the corresponding sample images, the spectrum software was modified so that the match of the i d in the design and the i d on the wsi [figure 1] is highlighted, leaving to the operator the choice to hit the segment command . Without a match, once the correct rotation of the wsi is defined by placing the source of asymmetry in the pre - defined corner, the grid of the design is adjusted to accommodate the shearing that may take place when the 4 m section is placed on the glass slide and to fit all the available spots on the wsi . We took a mathematical approach to the question on how to validate matching the grid containing the patient's i d and data (the tma construction design) with the image of the tma stained section . (intuitively, it has no nontrivial isometries), provided it satisfies two, easy to check, conditions . We set the following standard notation: is the real line, the plane endowed by the euclidean distance, [a, b] is the straight segment from the point a to the point b in . We prove the following statement: let r be a finite, hence discrete, set of points in, such that the two following two properties hold . Let lpq be the line through the points p, q; consider the set s of all the segments [p, q], p, q r with the property that one of the two connected components of lpq has empty intersection with . Then we require the existence of a segment [a, b] s whose length is unique among the lengths of the other segments in s.let [a, b], a, b r be the unique segment of length n in s. then r should not be symmetrical with respect to the axis perpendicular to [a, b] through its midpoint . Let lpq be the line through the points p, q; consider the set s of all the segments [p, q], p, q r with the property that one of the two connected components of lpq has empty intersection with . Then we require the existence of a segment [a, b] s whose length is unique among the lengths of the other segments in s. let [a, b], a, b r be the unique segment of length n in s. then r should not be symmetrical with respect to the axis perpendicular to [a, b] through its midpoint . Then if: is an isometry such that (r)= r, must be the identity function . We recall than an isometry in is a function f such that de(x, y)=de(f(x), f(y)) for all (x, y), where de is the euclidean distance in . They include (and are in fact generated by) all reflections, rotations and translations . This statement is false if we require to be a homeomorphism (i.e. Bijective and bicontinuous function) only . Proof: being an isometry, it sends lines in lines, thus ([a, b]) is a segment connecting the two points (a) and (b) in r. because of the continuity of, there exists a connected component of l(a)(b) with empty intersection with r, since so does lab . Therefore [(a), (b)] belongs to the set s, as we are assuming (r)=r . The length of such a segment must be the same as the length of [a, b], again because is an isometry . By the unicity of the segments in s of length n (condition (1) on r), it must be [(a), (b)] = [a, b] and restricted to [a, b] is an isometry onto [a, b]. Either of the following cases must necessarily happen: (a) = a and (b) = b. then restricted to the line lab is an isometry onto lab itself that fixes two points, therefore it must be the identity on the same line . This leaves only one non trivial possibility for as map on: a reflection along the lab axis . But in that case, it is impossible that (r) = r, since would swap the connected components of lab and the points in r not in lab are all contained in one component . This leaves = identity on the whole plane as the only possibility. (a) = b and (b) = a. then must have at least a fixed point, which must necessarily be the middle point m of [a, b] since is assumed to be an isometry . Thus it must either be a rotation by around m, in which case (r) r because swaps the connected components of lab, or else a reflection through the line perpendicular to [a, b] through m. however, the condition (2) on r implies that (r) r for such an isometry . (a) = a and (b) = b. then restricted to the line lab is an isometry onto lab itself that fixes two points, therefore it must be the identity on the same line . This leaves only one non trivial possibility for as map on: a reflection along the lab axis . But in that case, it is impossible that (r) = r, since would swap the connected components of lab and the points in r not in lab are all contained in one component . (a) = b and (b) = a. then must have at least a fixed point, which must necessarily be the middle point m of [a, b] since is assumed to be an isometry . Thus it must either be a rotation by around m, in which case (r) r because swaps the connected components of lab, or else a reflection through the line perpendicular to [a, b] through m. however, the condition (2) on r implies that (r) r for such an isometry . This shows that the only possible isometry of such that (r) = r is the identity function . Think of an array of specimens placed in glass slide as the finite set r satisfying the conditions (1) and (2). Assume the shape of the slide to be unknown and that the array is placed randomly on it . This result implies that, choosing any labelling of the specimens and applying an unknown sequence of integrity - preserving transformations to the slide, to recover the initial labelling of the specimens it suffices to move the slide around until the shape of the array matches the original one . The conditions (1) and (2) are isometry invariant, meaning that, they hold for a set r if and only if they hold for (r), where is any isometry of . As a consequence, the array of specimens, when glued on the slide, will be strongly asymmetrical, provided the initial (computer generated) one was and all the processing involved in the slide preparation can be assumed to transform the array only just through isometries . The easiest examples of lattices satisfying the conditions (1) and (2) are rect - angular ones with sides lengths c d with an extra specimen added in a length c boundary row . For that array we can take n to be c + 1 and immediately verify that the conditions are fulfilled . Appropriately removing blocks of points from one corner of a rectagular array, with sufficiently many rows and columns, provides an example of strongly asymmetrical array which preserves this property in case of deletion of a few columns, rows and points . Validation of the tma design and the wsi match (a) is a graphic rendering of the two properties set forth before analyzing the match between the tma design and the wsi; (b) represents the four possible isometric alignments of the tma design and the wsi, only one of which represents the identity of both the implementation of a barcode - driven error control of the design and execution of a tma for patient diagnosis requested approximately 4,000.00 to modify the lis program, in order to print the slide i d as a linear barcode in addition to the customary alphanumeric identifiers . The generation of the query to extract from the lis a sample data by barcode scanning costs approx . The modification of the aperio spectrum segmentation interface to match the ids on the slide and in the database was part of this project . The speed of the tma construction and the workflow did not change apparently after the implementations were put in place, mostly because of the deep automation already in place; it actually gained speed and smoothness of operation by leaving to the machine the tedious but necessary task of sample checking . We took a mathematical approach to the question on how to validate matching the grid containing the patient's i d and data (the tma construction design) with the image of the tma stained section . (intuitively, it has no nontrivial isometries), provided it satisfies two, easy to check, conditions . We set the following standard notation: is the real line, the plane endowed by the euclidean distance, [a, b] is the straight segment from the point a to the point b in . We prove the following statement: let r be a finite, hence discrete, set of points in, such that the two following two properties hold . Let lpq be the line through the points p, q; consider the set s of all the segments [p, q], p, q r with the property that one of the two connected components of lpq has empty intersection with . Then we require the existence of a segment [a, b] s whose length is unique among the lengths of the other segments in s.let [a, b], a, b r be the unique segment of length n in s. then r should not be symmetrical with respect to the axis perpendicular to [a, b] through its midpoint . Let lpq be the line through the points p, q; consider the set s of all the segments [p, q], p, q r with the property that one of the two connected components of lpq has empty intersection with . Then we require the existence of a segment [a, b] s whose length is unique among the lengths of the other segments in s. let [a, b], a, b r be the unique segment of length n in s. then r should not be symmetrical with respect to the axis perpendicular to [a, b] through its midpoint . Then if: is an isometry such that (r)= r, must be the identity function . We recall than an isometry in is a function f such that de(x, y)=de(f(x), f(y)) for all (x, y), where de is the euclidean distance in . They include (and are in fact generated by) all reflections, rotations and translations . This statement is false if we require to be a homeomorphism (i.e. Bijective and bicontinuous function) only . Proof: being an isometry, it sends lines in lines, thus ([a, b]) is a segment connecting the two points (a) and (b) in r. because of the continuity of, there exists a connected component of l(a)(b) with empty intersection with r, since so does lab . Therefore [(a), (b)] belongs to the set s, as we are assuming (r)=r . The length of such a segment must be the same as the length of [a, b], again because is an isometry . By the unicity of the segments in s of length n (condition (1) on r), it must be [(a), (b)] = [a, b] and restricted to [a, b] is an isometry onto [a, b]. Either of the following cases must necessarily happen: (a) = a and (b) = b. then restricted to the line lab is an isometry onto lab itself that fixes two points, therefore it must be the identity on the same line . This leaves only one non trivial possibility for as map on: a reflection along the lab axis . But in that case, it is impossible that (r) = r, since would swap the connected components of lab and the points in r not in lab are all contained in one component . This leaves = identity on the whole plane as the only possibility. (a) = b and (b) = a. then must have at least a fixed point, which must necessarily be the middle point m of [a, b] since is assumed to be an isometry . Thus it must either be a rotation by around m, in which case (r) r because swaps the connected components of lab, or else a reflection through the line perpendicular to [a, b] through m. however, the condition (2) on r implies that (r) r for such an isometry . (a) = a and (b) = b. then restricted to the line lab is an isometry onto lab itself that fixes two points, therefore it must be the identity on the same line . This leaves only one non trivial possibility for as map on: a reflection along the lab axis . But in that case, it is impossible that (r) = r, since would swap the connected components of lab and the points in r not in lab are all contained in one component . (a) = b and (b) = a. then must have at least a fixed point, which must necessarily be the middle point m of [a, b] since is assumed to be an isometry . Thus it must either be a rotation by around m, in which case (r) r because swaps the connected components of lab, or else a reflection through the line perpendicular to [a, b] through m. however, the condition (2) on r implies that (r) r for such an isometry . This shows that the only possible isometry of such that (r) = r is the identity function . Think of an array of specimens placed in glass slide as the finite set r satisfying the conditions (1) and (2). Assume the shape of the slide to be unknown and that the array is placed randomly on it . This result implies that, choosing any labelling of the specimens and applying an unknown sequence of integrity - preserving transformations to the slide, to recover the initial labelling of the specimens it suffices to move the slide around until the shape of the array matches the original one . The conditions (1) and (2) are isometry invariant, meaning that, they hold for a set r if and only if they hold for (r), where is any isometry of . As a consequence, the array of specimens, when glued on the slide, will be strongly asymmetrical, provided the initial (computer generated) one was and all the processing involved in the slide preparation can be assumed to transform the array only just through isometries . The easiest examples of lattices satisfying the conditions (1) and (2) are rect - angular ones with sides lengths c d with an extra specimen added in a length c boundary row . For that array we can take n to be c + 1 and immediately verify that the conditions are fulfilled . Appropriately removing blocks of points from one corner of a rectagular array, with sufficiently many rows and columns, provides an example of strongly asymmetrical array which preserves this property in case of deletion of a few columns, rows and points . Validation of the tma design and the wsi match (a) is a graphic rendering of the two properties set forth before analyzing the match between the tma design and the wsi; (b) represents the four possible isometric alignments of the tma design and the wsi, only one of which represents the identity of both the implementation of a barcode - driven error control of the design and execution of a tma for patient diagnosis requested approximately 4,000.00 to modify the lis program, in order to print the slide i d as a linear barcode in addition to the customary alphanumeric identifiers . The generation of the query to extract from the lis a sample data by barcode scanning costs approx . The modification of the aperio spectrum segmentation interface to match the ids on the slide and in the database was part of this project . The speed of the tma construction and the workflow did not change apparently after the implementations were put in place, mostly because of the deep automation already in place; it actually gained speed and smoothness of operation by leaving to the machine the tedious but necessary task of sample checking . The focus of our work is on areas which have been taken as granted or obvious and therefore not scientifically investigated such as asymmetry of the design, requirements of the tma design layout, matching of the design with the image and validation of the correspondence between data and image for individual spots . We have sought to control and minimize the source of i d errors through the process . This aspect has not been addressed before because of the high data yield achievable with the tma technique, with reduces the impact of sample heterogeneity (and for that matter, switching individual samples) on the significance of the results within large cohorts of patients . Switching individual samples or patients has little bearing if one looks e.g. At the impact of a phenotype on prognosis, particularly if no clinical decisions have to be made on each and every single patient within that cohort . To obtain error control we have used barcode readings throughout the entire process [figure 1]. Scanning a linear barcode is not error - free, however it considerably reduces the source of keystroke errors . Random placement of core replicas within the tma, a recommended practice, requires a compact design . This makes redundant the use of sectors, introduced both to shape the asymmetry and to facilitate the visual identification of different groups of samples within the tma . Sectors use up tma space and do not necessarily guarantee asymmetry (see examples in figure 2). The randomness of core placement further highlights the obligate handling of the tma on wsi scanners: traditional microscopes do not apply anymore . One additional question we addressed was the validation of the correspondence between the i d and the image of individual cores, obtained upon matching the whole design geometry with the geometric matrix composed of core sections images . We applied the laws of geometry, which states that once the vectorial transformation of an intrinsically asymmetric matrix (e.g., the wsi) is coincident with another given matrix (e.g., the tma design) the vector pointing to an individual component has the same value, thus the points coincide and the i d of each and every component is validated . A graphic rendering of the grid, which includes lines connecting all the individual positions in the grid, is of paramount importance to verify the isometries while adapting the grid upon the tma image . There are other issues which we did not address; one is the amount of human intervention needed during the segmentation (or de - arraying) procedure and the efficiency required . This issue has been examined by a handful of papers, one of which, compares a custom made de - arraying system with the one which has been used for the present study . However, equivocal placement of individual cores may still be judged by a human eye . Another issue we did not solve is how and where a robust asymmetry should be enforced during the process . Our feeling is that asymmetry is too important for safely identify individual samples / patients and that it should be enforced at some point . One last non - preventable source of analytical error may occur when the combination of loss of cores from the periphery of the section and the deformation of the paraffin sheet upon collecting the section from the water bath results in an equivocal assignment of a core to a row or a line . If each sample is represented twice or three times within the tma, there should be no loss of analysis for that specimen . Lastly, whether is done automatically or under human supervision, the asymmetry of the design should be visible in the grid superimposed to the wsi before segmenting . Ideally, spots left empty for the asymmetry should be automatically recognized as an additional safeguard against matching the wrong grid with the wrong wsi . We have applied for the first time a scientific approach to the i d error control to the construction and data analysis with the tma technology, we have validated the process of identifying the wsi of single samples / patients in the tma, setting diagnostic standards for the use of the tma . While all these aspects seem redundant for the research use of the tma technology, they are mandatory for any use of it which involves patient's care . However, controlling for analytical errors in experiments in science and industry is a golden standard and we advocate the use of our solutions in all fields using the tma high throughput technology.
Gastroesophageal reflux (gor) affects a large number of infants and children . For the majority, the condition is self - limiting and most patients improve spontaneously over the first years of life . Medical therapy is the first line treatment of choice, but surgery remains an option if this fails to control reflux adequately . Indications for surgical treatment include failed medical therapy, the presence of reflux - related respiratory complications, or failure to thrive . In older children, in whom reflux is unlikely to resolve spontaneously, there is generally a reluctance to accept lifelong proton pump inhibitors (ppis) and quality of life issues become more prominent . The recognition that the placement of gastrostomy tubes for feeding significantly increases the risk of gor, together with evidence that reflux may play an important role in the occurrence of apnoeic or bradycardic episodes, sudden death, recurrent chest infections, and chronic reactive airway disease, has also resulted in a large increase in antireflux surgery . Laparoscopic antireflux surgery is now firmly established as a useful tool in the management of gor in children, as it is associated with less morbidity and a more rapid recovery, particularly in children with neurological impairment [46]. Several antireflux procedures have been described, but which of these techniques offer the best outcomes in children has been open to debate . Nissen's fundoplication was established prior to the advent of laparoscopic surgery, so this technique was the first to be adapted for laparoscopic surgery and, consequently, has been the most widely used by laparoscopic surgeons [79]. Nevertheless, a high incidence of mechanical side effects has been reported, such as dysphagia, an inability to belch or vomit, and gas bloating, all of which are likely due to the establishment of a supercompetent gastrooesophageal sphincter by the 360-degree wrap . As a result, alternative fundoplication techniques have been developed by boix - ochoa, toupet, and others . However, which of these techniques offer the best outcomes in children is still under debate . One of the modifications of the laparoscopic nissen technique, using a partial anterior wrap, was described by watson in 1991 . This method is associated with reduced mechanical side effects in adults, but its efficacy in children has not yet been firmly established . The aim of this study was to evaluate the short- and long - term outcomes of laparoscopic watson fundoplication in children and infants in terms of durability and the incidence of mechanical complications, especially in those children with neurological impairment . This was a prospective observational study of 76 children and infants who had laparoscopic watson fundoplication performed by a single surgeon at the royal aberdeen children's hospital and the royal alexandra children's hospital in brighton between 1995 and 2014 . Demographic data, investigations, operative technique, and all reported complications were collected and analysed using microsoft excel . All patients were investigated with barium swallow and 89% had preoperative endoscopy (see table 1). Where necessary, the degree of reflux was clarified by oesophageal ph and manometry studies, where acid reflux was defined by a demeester score> 14.72 . All patients received a single dose of 25 mg / kg co - amoxiclav (or 30 mg / kg cefotaxime if allergic to penicillin). Laparoscopy utilised a four - port technique with an additional epigastric stab incision to accommodate a nathanson liver retractor . A 10 mm port was used in older children and a 5 mm port in small children and infants, to accommodate either a 0-degree or 30-degree laparoscope . Once pneumoperitoneum was established, two instrument ports were placed either side of the umbilicus for triangulation using either 5 mm or 3.5 mm ports for older or younger children, respectively . A further 5 mm or 3.5 mm port was placed near the left anterior axillary line to retract the gastrooesophageal junction . In children, spatial restrictions necessitate accessing the hiatus by dissection of the lesser omentum in the initial instance . Adhesions between the oesophagus and right crus were then released and the inferior oesophagus mobilised by extended dissection into the posterior mediastinum . The phreno - oesophageal ligament was divided anteriorly before the oesophagus was brought down into the peritoneum, taking care to avoid damage to the anterior vagus nerve . Next, the fundus was drawn over to the patient's right with division of phreno - fundal adhesions to release the left crus . A nylon tape was then used as a sling and held via the left anterior axillary port . Once 4 - 5 cm of tension - free oesophagus was brought down into the peritoneum, the crural repair was performed with two or three 2/0 ethibond sutures . The angle of his was recreated by suturing the fundus to the left anterior oesophageal hiatal rim . Further fundal tissue was then rolled over the anterior oesophagus and sutured to the mid portion and right angle of the anterior hiatal rim to create a 180-degree anterior wrap . Finally, three sutures were passed from the rolled - over fundus through the right oesophageal wall to the right crural repair . Half circle needles were used in younger children and infants and ski needles in older children . Postoperatively, patients were allowed clear fluids via oral or gastrostomy routes on the same day as surgery and allowed feeds the following day . Follow up included standard post - operative outpatient appointments at 6 weeks, 3 months and, where possible, at least one annual review . Treatment failure was defined as the continued or renewed dependence on ppis, histamine h2 receptor antagonists, or regular antacids for reflux symptoms as well as wrap failure with reoperation . Thirty - three (34.2%) were neurologically impaired, one suffered from cystic fibrosis, and 6 were born with oesophageal atresia + / tracheooesophageal fistula . Fifteen patients (19.7%) had previously undergone abdominal or thoracic surgery or percutaneous endoscopic gastrostomy (peg) placement . One child had previously undergone nissen fundoplication by a nonpaediatric specialist and represented with recurrent symptoms and a further patient underwent a failed boix - ochoa fundoplication by a paediatric surgeon . Contrast studies in these patients confirmed failure of the wrap and recurrence of hiatus hernia . The majority of children (n = 68; 89.4%) also underwent upper gastrointestinal endoscopy and 44 (57.9%) patients had ph studies . Median postoperative stay was 2 days (mean 3 days; range 119 days). Of the 76 patients undergoing surgery, 21 (27.6%) had a gastrostomy placed at the same time . Immediate complications included 4 minor liver injuries (5.25%), 6 minor bleeds (7.9%), and 1 bleed requiring a postoperative transfusion . In the short- to medium - term, one patient developed a wound infection and one had an early port site hernia . Postoperatively, dysphagia was reported in 9 cases (11.8%), 5 of whom had confirmation of relative narrowing at the cardiooesophageal junction at the level of the wrap on barium swallow . The median duration of follow - up was 30 months (range 043 months). One child developed late dysphagia and was found to have a recurrent stricture proximal to the wrap on barium swallow, requiring endoscopic balloon dilatation . Reflux symptoms (arching, postfeed cough, or vomiting) were reported in 14 cases (18.4%), but only 2 had acid reflux confirmed by ph studies . Two cases developed a small hiatus hernia managed medically, 3 cases suffered recurrent retching again managed medically, and a high risk older cerebral palsy patient with respiratory compromise had a gastrojejunostomy placed for breakthrough chest aspiration after spinal surgery . A patient who had undergone a tracheooesophageal fistula (tof) repair as a neonate and who had been listed for fundoplication due to frequent apnoeas, thought to be reflux - related, continued to experience apnoeas postoperatively, but at a reduced frequency . Laparoscopic fundoplication for the management of gor in infants and children has become one of the most frequently performed operations in paediatric surgery, as its effectiveness and safety have been well established . Nissen's technique remains the most commonly performed antireflux operation in children . In adult practice, laparoscopic anterior fundoplication appears to be associated fewer adverse events, especially mechanical complications, than nissen fundoplication [1517]. The advantages should, in theory, apply to paediatric practice as well . To date, however, only a limited number of patients series with children or infants undergoing anterior fundoplication have been published [14, 1821]. Comparison of complications and treatment failures are hampered by the lack of precise definitions of symptoms, such as gas bloat and early dysphagia . Even treatment failure is variously defined as wrap breakdown on contrast swallow, recurrence of symptoms, persistent or recurrent reflux on ph studies, and reinstitution of medical therapy or reoperation . In our unselected consecutive series of patients, postoperative dysphagia is seen in approximately 12% of cases, but it is nearly always transient and rarely requires oesophageal dilatation . Although 18.4% reported ongoing or recurrent reflux, this figure appears to be satisfactory relative to other reports [1217]. Furthermore, the incidence of early dysphagia in our series falls between that reported by other authors for nissen fundoplication [10, 17], although there are marked differences in study design . Over a third of the children in our series had neurological comorbidities . The outcome of antireflux surgery in children with neurological impairment is known to be inferior to that of neurologically intact children [46]. Many of these children are accepted for surgical intervention on the basis of vomiting or failure to thrive, which may be due to causes other than reflux . More thorough preoperative investigations and distinction of centrally induced retching from true reflux might have altered the decision to proceed to surgery in some of our patients . Indeed, in adults, most surgeons would now be reluctant to perform antireflux surgery without convincing evidence of high volume reflux on endoscopy, contrast swallow, and/or ph - manometry studies . Despite the fact that reliable ph studies are difficult to perform in children, especially in those unable to self - report symptom events, consideration should be given to obtaining such objective evidence prior to surgery, especially if the indications are nonspecific . This study confirms that the technique of laparoscopic watson fundoplication is safe in infants and children, including those with significant neurological disability . Adverse events, although relatively common, were generally very mild and tended to be transient.
Periodontal diseases (pds) constitute a series of infections caused by the microorganisms that colonize sites at or below the gingival margin . These infections commonly lead to periodontal inflammation and often result in the destruction of the supportive periodontal tissues . The organisms that cause these diseases reside in unique structures termed biofilms that offer partial protection to the colonizing organisms from the defense mechanisms of the host, as well as from the antimicrobials used for treatment . The primary goal of conventional periodontal therapy is the alteration of subgingival biofilms present on periodontally diseased sites that could be associated with the progressive destruction of the supportive periodontal tissues . It is well documented that mechanical therapy combined with oral hygiene instruction is effective in achieving this goal . Comparisons between manual and power - driven approaches to plaque and calculus removal and their effects on clinical outcomes show that both are equally effective . Over the last 3 decades, locally delivered, anti - infective pharmacological agents have been employed in attempts to treat local bacterial infections associated with gingivitis and periodontitis . Techniques of the local anti - infective therapy with pharmacological agents have varied widely, both in the methods of drug delivery and in the pharmacological agents employed . Drug delivery methods initially included oral rinses, then irrigation devices, followed by subgingival irrigation using syringes or powered irrigation devices . More recently, drug delivery has involved the incorporation of anti - infective drugs in sustained - release vehicles enabling subgingival administration of the drug . These sustained - release vehicles overcome many of the problems inherent in other drug delivery methods, such as rinsing or irrigation . These systems exhibit shortcomings including inadequate or unpredictable penetration of the periodontal pocket by the drug, rapid clearance of the drug from the pocket resulting in inadequate time of the exposure of subgingival bacteria to the drug and poor patient compliance . Local anti - infective therapy has also employed an eclectic variety of pharmacological agents from topical antiseptics to broad - spectrum antimicrobials . Chlorhexidine, tetracycline, minocycline, metronidazole, doxycycline, as well as other antimicrobials and antiseptics have been used in several forms . Two systematic reviews with the meta - analysis of data from studies evaluating the effect of locally delivered antimicrobials as an adjunctive therapy to scaling and root planing (srp) revealed evidence of improved clinical outcomes in terms of the greater pd reduction and/or clinical attachment gain compared to srp alone . This was particularly true with the use of tetracyclines, although the clinical significance remains questionable . The aim of the present study was to compare the clinical and microbiological effects of scaling and root planing with hand instruments to a non - surgical treatment with the use of an ultrasonic device combined with the application of locally delivered doxycycline . The subjects for the present cohort study were recruited from the postgraduate clinic of the department of preventive dentistry, periodontology and implant biology, school of dentistry, aristotle university of thessaloniki, greece between september 2004 and march 2005 . The ethical committee of the school of dentistry of aristotle university of thessaloniki, greece approved the study protocol and all participating patients signed an informed consent at the beginning of the study . The patients (n = 20) comprising the control group were the same as in our previous study . Another twenty adult patients with generalised advanced chronic periodontitis were recruited for the study following a screening examination of 28 patients by one examiner (i.v . ), which included full mouth probing and radiographic examination, in order to comprise the control group . The inclusion and exclusion criteria were as follows: (i) subjects must be adults between 18 and 70 years of age . (ii) subjects must have at least 4 sites with initial probing pocket depth (ppd) 5 mm in at least 2 quadrants, demonstrating bleeding on probing . (iii) subjects must not have received any periodontal treatment during the previous 6 months . Clinical examinations were performed at the screening examination, 3 and 6 months after treatment and involved assessment of ppd, clinical attachment level (cal), gingival bleeding index (gbi) and plaque index . The measurements of ppd and cal were performed at six sites per tooth with a manual periodontal probe (hu - friedy pcp - unc 15, hu - friedy, chicago, il, usa) to the nearest millimetre . Prior to the study, the intra - examiner variability test was carried out to assess the accuracy of examiner's measurements . In order to assure the reproducibility of measurements, all recordings regarding ppd and cal were repeated after a period of 30 minutes . In the event of a difference of> 2 mm between the two measurements, the mean of pair of the two closer measurements was evaluated for further analysis . The patients fulfilling the necessary prerequisites were assigned into 2 groups of 20 patients each: control group (scaling and root planing with hand instruments - srp) and experimental group (ultrasonic debridement + doxycycline - ud + doxy). At all time points, the outcomes of research were assessed blind and the examiner (i.v . ), k.p .) Who were also unaware of the treatment that the patient had received (coded samples). Experimental design and treatment procedures at the screening examination, a full mouth measurements of clinical parameters were recorded and intraoral radiographs were taken . After a period of 1 week (baseline examination), subgingival plaque samples were taken from 6 preselected sites from each patient . The sites were selected according to their initial probing depth and were divided in 3 categories: (i) 2 sites with ppd 4 mm (shallow pockets), (ii) 2 sites with ppd> 4 to 6 mm (moderate pockets) and (iii) 2 sites with ppd> 6 mm (deep pockets). No furcation, endo - periodontic defects or third molars were included in the study material . Microbiological sampling at the same sites, as at the baseline examination, and a full mouth clinical recordings were repeated at 3 and 6 months after the baseline . At the same session, supragingival scaling was performed with hand instruments and ultrasonics, and oral hygiene instructions (ohi) were given by the examiner . The ohi included twice - daily tooth brushing, using the modified bass technique, and once - daily inter - dental cleaning with inter - dental brushes . At the next appointment, two weeks after the baseline examination, the allocated intervention was initiated . Hand instrumentation of the whole dentition was performed at weekly intervals in three to four sessions by using gracey curettes under the local anaesthesia (sg 3/4, 11/12, 13/14 and after five curettes sas 3/4, 11/12, 13/14, hu - friedy pcp - unc 15, hu - friedy, chicago, il, usa). Patients of the experimental group received debridement of whole dentition in 3 - 4 sessions, under the local anaesthesia, at weekly intervals, utilizing a piezoelectric ultrasonic device (ems piezon, ems, nyon, switzerland) with a and p instruments (swiss instrumentspm, ems, nyon, switzerland) under water irrigation . The teeth were treated until a smooth, appropriately debrided surface was achieved . In the four deeper, preselected pockets 8.8% doxycycline gel (atridoxtm, atrix laboratories, inc ., collins, co, usa) was applied at the completion of therapy and after the first re - examination at 3 months . In both groups the endpoint of the smoothness was judged by the supervisor (i.v . ), who decided upon the completion of root instrumentation by using a periodontal probe (hu - friedy pcp 11, hu - friedy, chicago, il, usa) and an explorer (hu - friedy wilkins - tufts 17/23, hu - friedy, chicago, il, usa). Collins, co, usa) is a subgingival controlled - release product composed of a two syringe mixing system . Syringe a contains 450 mg of the atrigel delivery system, which is a bioabsorbable, low viscosity polymeric formulation composed of 36.7% poly(dl - lactide) (pla) dissolved in a biocompatible carrier of 63.3% n - methyl-2-pyrrolidone (nmp). The product, when mixed, is a pale yellow, viscous liquid with a concentration of 8.8% doxycycline hyclate . The two syringes are coupled together mixing the two components for 100 cycles . By the use of 23-gauge cannula attached to the delivery system the test product was slowly introduced into the periodontal pocket, starting from the base of pocket, until it reached the gingival margin . Following withdrawal of the cannula tip, a curette was used to pack any overflow of the drug down into the pocket . No periodontal dressing or adhesive was used . Upon contact with the crevicular fluid, the liquid product solidifies and the patients were instructed not to perform oral hygiene measures at the treated areas for 1 week . Microbiological evaluation after the isolation with cotton rolls, drying and removal of supragingival plaque, the subgingival samples were taken with a sterile gracey curette (hu - friedy, chicago, il, usa), were subsequently placed individually in 200 l of te buffer (tris hcl 10 mm, edta 1 mm, ph = 7.5) and stored after the treatment with an alkali solution (0.5 m naoh) at - 4 oc . The microbiological samples were evaluated separately for 3 bacterial species using the " checkerboard " dna - dna hybridisation technique as described by socransky et al . . The subgingival species used for development of digoxigenin - labelled whole genomic probes were porphyromonas gingivalis (fdc 381), tannerella forsythia (fdc 338) and treponema denticola (td1). The primary analysis was " per protocol " and included all patients who attended the final examination . Data were entered into an excel sheet database (ms office excel 2000; microsoft corporation, redmond, wa, usa). Mean and standard error of the mean (sem) were calculated for every parameter . Levene's test for the quality of error variance was applied in order to check the homogeneity of clinical parameters at the baseline . The analysis was made for plaque index, gbi, ppd and cal based on full mouth measurements (the third molars were not included). A further analysis was performed for ppd and cal for the three different categories, according to the initial pocket depth . The first category comprised pockets with initial pocket depth lesser or equal to 4 mm, the second pockets with initial pocket depth> 4 to 6 mm and the third pockets with pocket depth greater than 6 mm . Bacterial species were quantified following the formation of reference curve, which allowed the conversion of chemiluscent signals to total bacterial counts (total lab v2005, nonlinear dynamics ltd, newcastle upon tyne, uk). The homogeneity of two groups at the baseline for microbiological parameters was checked using the mann - whitney test . Averaged bacterial scores from each subject were averaged for each group and compared at all time - points . A further comparison at the three examinations was made for the sites with initial ppd> 4 mm (moderate and deep pockets), which were those that received the drug in the control group . The differences over time within groups for both clinical and microbiological results were analysed with the non - parametric test wilcoxon signed ranks . The comparison between control and control group was performed using the mann - whitney test . All statistical analysis was carried out with the aid of statistical software (spss version 12.0, spss inc ., at the baseline examination 40 patients entered the study (20 in the srp group, 20 in the ud + doxy group), from which 33 subjects completed the 6 month protocol (16 in the srp group, 17 in the ud + doxy group, mean age 50.46, range 37 - 69 years). Four patients did not return for any re - examination, while another three did not attend the final examination . One patient moved, three patients started work, preventing visits to the clinic and the other three were unwilling to finish the study for personal reasons . The flowchart of the patients is illustrated in figure 1 and the characteristics of the patient sample that completed the study are summarised in table 1 . The initial statistical analysis revealed no statistical differences between the two groups at the baseline examination . The intra - examiner variability test demonstrated that the reproducibility of the measurements of ppd and cal within 1 mm was 90% . Epidemiological characteristics of the patient sample (mean sem, per protocol analysis) ppd = probing pocket depth; cal = clinical attachment level . An average of 69.1% of all pockets in the srp group and 71.5% in the ud + doxy group had initially ppd 4 mm . At the 6 month examination in the srp group the shallow pockets represented 84.9%, the moderate pockets 11.3% and the deep pockets only 3.8% of all pockets . In the ud + doxy group the oral hygiene status, as assessed by the plaque index, during the course of study is shown in table 2 . At the baseline the mean full - mouth plaque scores were 88% in the srp group and 81% in the ud + doxy group . A statistically significant decrease to 26% in the control group and to 37% in the control group was recorded at the 6 month examination (wilcoxon signed ranks test, p <0.05). No statistically significant differences were observed between the two groups at any interval (mann - whitney test, p> 0.05). Mean plaque index and gingival bleeding index scores (mean sem [mm]) at different examination intervals for control and experimental group patients statistically significant difference from baseline (wilcoxon signed ranks test, p <0.05). Gingival bleeding index (gbi) a statistically significant reduction in gbi scores was observed in both treatment groups following treatment . Gbi indices also the improved following a similar pattern in both groups; at the 6 month re - examination the gbi was reduced from 59% to 33% in the srp group and from 61% to 32% in the ud + doxy group (wilcoxon signed ranks test, p <0.05, table 2). No statistically significant difference in gbi between the two groups was observed at any examination interval (mann - whitney test, p> 0.05). Probing pocket depth (ppd) changes in the ppd are presented in table 3 . A marked mean ppd reduction was observed for both treatment modalities at the 3 month re - examination (0.88 mm for the srp and 0.94 mm for the ud + doxy group, wilcoxon signed ranks test, p <0.05). During the following observation period (3 to 6 month examination) no statistically significant differences were observed between the two groups at any interval (mann - whitney test, p> 0.05). The ppd measurements were further analysed for the three different categories of initial pocket depth . For the shallow (ppd 4 mm), the moderate (> 4 to 6 mm) and the deep pockets (ppd> 6 mm) the results are presented in table 3 . In the deep pockets an additional, statistically significant reduction was observed in ppd between 3 and 6 months in the experimental group (wilcoxon signed ranks test, p <0.05, table 3). Probing pocket depth (ppd) scores for the various ppd categories at different examination intervals for control and experimental group patients statistically significant difference from baseline (wilcoxon signed ranks test, p <0.05). Statistically significant difference from 3 months (wilcoxon signed ranks test, p <0.05). No statistically significant differences were observed between groups (mann - whitney test, p> 0.05). Clinical attachment level (cal) a statistically significant improvement of cal was revealed for both groups at the 3 month examination (0.50 mm for the srp group and 0.46 mm for the ud + doxy group respectively, wilcoxon signed ranks test, p <0.05, table 4). Statistically significant differences between the two groups were not recorded at any interval (mann - whitney test, p> 0.05). In the srp group the 6 month result remained significantly better compared to the baseline score (wilcoxon signed ranks test, p <0.05), whereas in the ud + doxy group the mean cal score did not demonstrate a significant difference in comparison with the baseline value (wilcoxon signed ranks test, p> 0.05). Clinical attachment level scores for the various probing pocket depth categories at different examination intervals for control and experimental group patients statistically significant difference from baseline (wilcoxon signed ranks test, p <0.05). No statistically significant differences were observed between groups (mann - whitney test, p> 0.05). A similar analysis as for the ppd measurements was made for the cal measurements as well . The results for the shallow, moderate and deep pockets are shown in table 4 . The differences between 3 and 6 month re - examinations and the baseline examination were statistically significant for the two groups (wilcoxon signed ranks test, p <0.05), whereas no statistically significant, inter - group differences were observed (mann - whitney test, p> 0.05). The results for all the investigated species and for all of the six sites per patient are summarised in table 5 . At the 3 month of examination, this decrease was statistically significant only for porphyromonas gingivalis (wilcoxon signed ranks test, p <0.05). At the 6 month re - examination, a statistically significant difference with baseline was observed in the experimental group for the treponema denticola (wilcoxon signed ranks test, p <0.05), attributable to a further reduction in numbers of this species between the re - examinations . No statistically significant differences were found between the two groups at any time interval (mann - whitney test, p> 0.05). Mean numbers (x105, mean sem) of the 3 microorganisms' species tested in various initial probing pocket depths at different examination intervals statistically significant difference from baseline (wilcoxon signed ranks test, p <0.05). Pg = porphyromonas gingivalis; tf = tannerella forsythia; td = treponema denticola . The frequency distribution revealed an increase in the percentage of sites with less or equal to 105 microorganisms for porphyromonas gingivalis, tannerella forsythia and treponema denticola and a subsequent decrease in the percentage of sites with more than 105 microorganisms (table 6). Frequency distribution (%) of sites depending on various microorganisms species concentration tested at different examination intervals pg = porphyromonas gingivalis; tf = tannerella forsythia; td = treponema denticola . The results of further analysis that was performed for the sites that had initial ppd> 4 mm, are presented in table 5 . A statistically significant decrease was found for porphyromonas gingivalis in both groups at the 3 month examination (wilcoxon signed ranks test, p <0.05). No statistically significant differences were found between the two groups at any time interval (mann - whitney test, p> 0.05). The findings of the present study indicate that the adjunctive use of doxycycline failed to significantly improve the therapeutic outcome of mechanical treatment . In a previous study, the hand instruments were compared to the ultrasonics in the treatment of chronic periodontitis, but no difference was found between the two treatment modalities . The addition of locally delivered doxycycline in the treatment protocol of present study did not seem to offer any beneficial effect to the patients treated with ultrasonic debridement in comparison to the conventional hand instrumentation alone . A marked reduction in every clinical parameter was observed for both treatment modalities in this study . Overall a statistically significant mean reduction in ppd of 0.88 mm and a mean cal gain of 0.59 mm was recorded for the srp group . The corresponding values for the ud + doxy group were 0.92 mm and 0.31 mm . Cal scores at the 6 month re - examination did not show a statistically significant difference compared to baseline scores in the experimental group . In the deep pockets an additional, statistically significant, reduction was observed in ppd between 3 and 6 months . This could be explained by the fact that the test drug was applied to only 4 pre - selected pockets and therefore it would be very difficult to provide a true clinical benefit as the measurements were performed for the whole dentition . The question of whether the adjunctive use of locally delivered antimicrobials to mechanical treatment offers an improved clinical and microbiological outcome over srp alone remains unanswered . A number of studies have reported only limited improvement of cal and ppd recordings when comparing the combined treatment protocol (srp plus locally delivered antimicrobials) to conventional mechanical treatment alone [17 - 20]. Generally a ppd reduction of 1 - 1.5 mm in moderate pockets (4 - 6 mm of initial ppd) and of 2 - 2.5 mm in deeper sites has to be expected after mechanical root instrumentation [21 - 26]. This occurs concomitantly with a cal gain of approximately 0.5 mm and 1.5 mm in the moderate and the deep pockets respectively . Any additional pocket reduction or cal gain, following the administration of the drug, would therefore represent a true clinical benefit and hence may reduce the need for additional periodontal surgery . Previously published studies referring on the utilization of locally delivered doxycycline polymer focused on its effects when used as a monotherapy . Comparable efficacy of the 2 treatments in the terms of reduction in pocket depth and gain in clinical attachment level was reported in two parallel multicenter studies comparing the locally delivered doxycycline to the srp alone . In another multicenter study, heijl et al . Using a split mouth design, observed changes in probing depth comparable to this study for each treatment . The difference between the responses to scaling alone and scaling combined with tetracycline fibers were small and not statistically significant . In a systematic review, the reviewers generally found modest differences for ppd favouring the combined treatment . These, even when statistically significant, ranged from 0.1 mm to 0.5 mm, and were of little clinical importance . Effects for cal gains were smaller and statistical significance was less common . In very accurately designed study two different approaches were evaluated for the non - surgical treatment of moderate periodontal pockets . Traditional srp was compared to ultrasonic debridement followed by 8.5% doxycycline hyclate administration in sites deeper than 5 mm . At the 3 month examination better results regarding ppd and cal were observed in the ultrasonic debridement group than in the srp group . A retreatment of non - responding sites (ppd> 5 mm) consisting of ultrasonic debridement combined with doxycycline application in the srp group at this time point lead to a similar clinical outcome for both groups 6 months after baseline . Taking into account the reduced time needed for ultrasonic treatment the authors concluded that simplified, subgingival debridement in conjuction with doxycycline application could be a justified approach for deeper periodontal pockets . Although, in the present study the time required for the instrumentation was not specifically recorded, the therapists felt that ultrasonic debridement was less time consuming than hand instrumentation . The patient - centered outcome variables, like reduced treatment time, are considered of importance in the evaluation of treatment outcome today . Simplified, less time and effort demanding procedures like ultrasonic debridement combined with locally delivered antimicrobials, i.e. Doxycycline hyclate, could represent a reliable alternative in treating moderate periodontal pockets or can be an option for patients where a surgical intervention is not indicated . In this respect the findings of the present study are in accordance with those of the wennstrom et al . Adjunct use of the locally delivered drugs seems to have a beneficial effect in smokers and in retreatment of the patients with persisting periodontal pockets . The adjunctive use of locally delivered doxycycline in smokers may offer some benefit for the active and supportive periodontal treatment in both clinical and microbiological findings . Initial studies on maintenance patients have shown that the local application of tetracyclines seems to offer improved results [19 - 34], although more recent data failed to support those findings . In accordance with that, tomasi et al . Concluded that locally delivered doxycycline failed to improve the healing outcome following the re - instrumentation with a piezo - ceramic ultrasonic device of periodontal pockets with post - therapeutic depth greater or equal to 5 mm . At the 3 month re - examination both therapeutic approaches resulted in a statistically significant reduction of the number of porphyromonas gingivalis only . A profound, yet not statistically significant, reduction was observed for tannerella forsythia and treponema denticola as well, but in no case was eradication of the periopathogenic species found . Following the administration of drug, an adjunct effect of doxycycline on the presence of treponema denticola no statistically significant differences were observed between the two groups regarding the microbiological parameters . The findings are in accordance with earlier studies comparing scaling and root planing alone with scaling and root planing followed by the application of locally delivered drugs in initial and supportive periodontal treatment . In contrast, goodson et al . In a large, multicenter trial showed a greater reduction in the numbers and proportions of red complex bacteria following the adjunctive use of minocycline microspheres . In order to be effective antimicrobials must reach their target site and be maintained there in sufficient concentrations long enough for their antimicrobial effect to occur . The concentration required for efficacy is often estimated from the minimum inhibitory concentration (mic), although this has substantial shortcomings; in particular, the fact that mic is assessed in vitro whereas the drug is active in vivo in a constantly changing host environment . Other problems related to estimating effective drug concentrations in vivo are the large number of periodontal subgingival organisms and the large variation in the mics of isolates . Defining the minimally effective concentration is complex and most dosages are based on in vitro experiments in which bacteria were grown under planktonic conditions . It is known that bacteria are organized into biofilms when present in the periodontal pocket . These biofilms will probably require a significantly higher concentration of antimicrobial to kill those bacteria where cargill et al . Found that legionellae in biofilms were 135 times more resistant to iodination when compared to microorganisms growing in non - organised or planktonic fashion [42 - 44]. This is a strong indication that locally delivered antimicrobials should be used adjunctively with mechanical instrumentation to disrupt the biofilms . A concern in the use of antimicrobials in the treatment of chronic periodontitis is the risk for the emergence of an antibiotic - resistant complex of species . This issue in relation to the use of locally applied, sustained - release doxycycline was evaluated in a recent study of walker et al . . It was concluded that doxycycline treatment did not result in a change of actual number of resistant bacteria or in the acquisition of antibiotic resistance . According to the findings of present study the adjunctive use of locally delivered doxycycline did not seem to add any beneficial effect to the active treatment of chronic periodontitis . Only in deep pockets an additional improvement was observed between 3 and 6 months in the experimental group, which however was not strong enough to provide a statistically significant difference between the two groups . In conclusion, it is suggested that more long - term studies focusing on the outcome of the combined therapy during supportive periodontal treatment are needed, where more favourable results could be expected, especially in the case of deep pockets.
It is postulated that these cause luminal obstruction resulting in subsequent acute inflammation of the mucosa . Appendicoliths are composed of inspissated faecal material, mucus with trapped calcium phosphate and inorganic salts . Dropped appendicoliths from appendicectomy are rare, but the surgeon and radiologist ought to consider them as a potential source of intra - abdominal complications . Until recently, the presence of an appendicolith was considered 100% specific for the diagnosis of acute appendicitis . However, 13% of patients without acute appendicitis showed an appendicolith on ct (2). Presence of an appendicolith is also associated with a high incidence of perforation, particularly in children . Appendicoliths can be detected by plain abdominal radiographs in 10 to 15% of patients with acute appendicitis . With the recent popularity of ultrasound and the appendicoliths can drop at the time of resection of the appendix, during forceful extraction through the umbilical port or if the appendix perforates . Complications associated with retained faecoliths following appendicectomy are becoming more prevalent with the increased use of the laparoscopic technique, similar to the increase in dropped gallstones following the introduction of laparoscopic cholecystectomy (2). The time between laparoscopic appendicectomy and representation with abscess ranged from 2 months to 4 years (2). The natural history of dropped appendicoliths is not yet known but there is some early evidence that they have a significant association with abscess formation, with the appendicolith acting as a nidus for infection . Ct - guided percutaneous drainage of intra - abdominal abscess secondary to retained appendicoliths is only successful in the short term . Formal surgical drainage and removal of the appendicolith is required for long - term success (2). Ultrasound has been described as a useful adjunct for intra - operative localisation of the appendicolith (4). A 17-year - old female presented to the emergency department, with intermittent right upper quadrant pain, nausea and vomiting . She was previously fit and well, with her only background history being a laparoscopic appendicectomy for perforated appendicitis the previous year, aged 16 . Abdominal examination revealed tenderness to palpation in the right upper quadrant and no palpable masses . Bloods tests revealed raised inflammatory markers . Computed tomography of the abdomen was performed and revealed a 43 cm structure of low attenuation posterolateral to segment 6 of the liver with surrounding irregular wall and a central 15 mm high density lesion, suggestive of a peritoneal foreign body, possibly a retained appendicolith from previous surgery (figure 1). Abdominal ct scan showing a radio - opaque faecolith in the right upper quadrant (arrowed) it was felt that this could be removed via ultrasound guidance through a small intercostal incision under local anaesthetic . This was attempted but failed secondary to an iatrogenic pneumothorax and failure to localise the lesion . Once the peritoneum was opened purulent fluid discharged and on exploration of the cavity the faecolith was found (figure 2). The patient s post - operative course was uneventful and she was discharged home the following day in a stable condition, afebrile and with normal serum inflammatory markers . Appendicectomy, especially in cases of perforated appendicitis, is associated with postoperative abscess formation in up to 20 per cent of cases (1). Though this treats the abscess, it does not necessarily treat the root cause and nidus of infection the appendicolith . In cases of retained appendicolith, failure to do so may result in recurrent intra - abdominal abscesses, wound infection and occasionally fistula formation (5). Retained appendicolith has been reported in different sites including the pelvis, gluteal region, hepatorenal pouch (of morrison) and subhepatic region . It can be detected by ultrasound or computed tomography scan as fluid collection containing a focus of high attenuation which may be single or multiple . Occasionally, plain radiograph film may show these retained appendicolith, though the sensitivity is only 10% (1). The literature reveals few cases of subhepatic abscesses due to retained appendicolith and most of them required operative intervention, whether laparoscopic or via the open approach . A high index of suspicion ought to be maintained in patients presenting with recurrent intra - abdominal sepsis and a history of appendicectomy . The risk of retained appendicolith may be reduced by ensuring that the distal end is intact after division of the appendix . The authors advise gentle manipulation when handling an acutely inflamed appendix to avoid appendicolith spillage . In the event of spillage, the appendicolith ought to be retrieved, as failure to do so may lead to subsequent complications . Definitive treatment depends on removal of appendicolith, and the authors advocate that this can be safely accomplished via the laparoscopic method.
Exposures to ambient air pollution and secondhand smoke (shs) have been implicated as health risks, particularly for some conditions such as asthma (chilmonczyk et al .,; roemer et al .,; peters et al ., 1997; vedalet al .,; ostro et al ., 2001; wallace et al ., 2003; measurement error is inherent to air pollution studies because most have used fixed (central) monitors at a distance from study subjects . Even for studies that have used personal monitors,, chapter 6, 2006; buonaccorsi, chapter 5, 2010; buzas and stefanski, 1996) can be used to minimize bias in health - effect estimates due to measurement error by employing data from both fixed and personal monitors as well as other variables related to exposure . Regression calibration methods are well established and have been extended for longitudinal or clustered data within recent decades, most commonly by employing linear mixed models (bartlett et al ., 2009); nonlinear mixed models, ko and davidian, 2000; or generalized linear mixed models (glmm; carroll et al ., 1997; wang et al .,,). Specific extensions for rciv using glmms have also been made (li and wang, 2012). These articles provide framework for estimation but do not give explicit forms for specific models with interaction terms . Other recent measurement error methods articles involve interaction terms, but not longitudinal data or instrumental variables (e.g., wong et al .,; huang et al ., 2005). We previously derived explicit forms of rciv estimators and variances for longitudinal models with interaction terms between the unobservable predictors, that is, the true exposure measurements of interest, while adjusting for covariates not involved in interactions (strand et al ., 2014). Here, we extend these forms to allow interaction between the unobservable predictors and the covariates . This extension allows us to account for situations where the health exposure relationship may be modified by subject characteristics or conditions . Regression calibration with instrumental variables algorithms employ unbiased but measured - with - error variables (unbiased surrogate variables) and variables that are linearly related to the true predictors of interest (instrumental variables) to obtain consistent and nearly unbiased estimators of the coefficients of the true predictors . Two particular algorithms are often used to carry out rciv, one that obtains the calibrated estimates as functions of estimates from the two separate model fits (rciv1) and another that obtains predicted values from one model fit, then plugs these predicted values into a second model in place of the unknown predictors (rciv2). (2006) and hardin and carroll (2003) describe methods to obtain asymptotic variances for estimated coefficients of interest in generalized linear models using stacked estimating equations for rciv1 and/or rciv2 estimators, but recommend the use of empirical (sandwich) variances due to model approximations with this approach . (2003) proved that rciv1 and rciv2 estimators have the same asymptotic variances when the outcome follows a generalized linear model of exponential family form and when there may be multiple covariates measured with error . In this article, we determine both model - based and empirical asymptotic variances of rciv1 estimators in linear mixed models with specific interaction terms by applying the delta method . This approach allows for true covariance structures to be retained (based on underlying models and assumptions), although in practice, we have found that approximations to covariance structures have worked adequately . Our motivating application involves a longitudinal air pollution and health study conducted on children with asthma at the national jewish health in denver and reported in rabinovitch et al ., we examined the relationship between a biomarker of inflammation, leukotriene e4 (lte4), and two instrumental variables related to pollutant exposures: urinary cotinine (related to shs exposure) and outdoor fine particulate matter (related to personal ambient fine particulate matter exposure), plus their interaction . Using these instrumental variables plus a small number of days of measured - with - error but unbiased exposure concentrations from personal monitors (the unbiased surrogate variables), we then used rciv in the second article to estimate the slopes of the two unobserved pollutant exposure variables, plus their interaction . Both reports demonstrated that while increases in exposures to ambient fine particulate matter and shs each had adverse effects on health, greater dose some preliminary analyses suggested that the health pollutant relationships for subjects might depend on whether or not children had upper respiratory infections (uris or colds). The extended models presented in this paper allowed us to examine this, and could be used to study other potential effect modifiers . In this paper, we first derive the form of regression calibration estimators for the extended rciv1 model, which includes interactions between unobservable predictors and observable covariates, and derive asymptotic standard errors for all estimators in this model using the delta method . We then revisit the air pollution and health data to better understand the role of uris in the health - exposure relationship . We consider linear mixed models, including random intercepts to account for subject heterogeneity in the responses, and a serial correlation structure for within - subject repeated measures . Although fitting models does involve approximation to the covariance structures as will be discussed later, including these two features in the approximated structures has allowed us to obtain reasonably accurate inferential results for parameters of interest, as verified through monte carlo simulation . The continuous outcome variable yij for subject i at time j, i = 1,,n and j = 1,,ri is expressed as a function of the unobservable predictors (x1ij, x2ij and their interaction), covariates measured without error that are involved in interactions with at least one of the unobservable predictors (denoted as), covariates without error that are not involved in interactions with the unobservable predictors (denoted as zij), and random terms to incorporate the correlation due to longitudinal measurement of subjects . If each z term is allowed to interact with all terms in xij=(1,x1ij, x2ij, x1ijx2ij) and we let zij=(z1ij, z2ij,,zkij),, then the model of interest is 1 where is a random intercept term for subjects and . We assume that the random intercept and error terms are independent of each other and that the subjects are independent . We use the spatial power structure for, which has the form, for days tij and in the study period associated with observations j and j, respectively, for subject i. this is a generalization of the ar(1) structure and is useful when data are collected intermittently, such as in our data application . (in our application, we do not consider days without data to be missing values because data collection was designed to be intermittent .) The fixed - effect parameter vectors are defined as,,, where, for p = 1,...,m . The symbol indicates the kronecker product; because xij and are both row vectors, the resulting product will also be a row vector with 4 m elements: (). Let denote the entire set of 4(m + 1) + k fixed - effect parameters in model (1). In our data application, y is natural log leukotriene e4, or ln(lte4), and x1 and x2 are shs and fine ambient particulate matter exposure concentrations, respectively . Both of the pollutant variables are considered in terms of fine particulate matter (technically, particulate matter less than 2.5 microns in diameter, or pm2.5), and measured in g per m. our primary models included an indicator for uri, or cold status (for presence, 0 for absence) as the covariate allowed to interact with x variables, and study year (z1,,z4; the last year was the reference year) as covariates not involved in interactions . If not all interactions between z and x variables are required, then the model can be reformulated as 2where is the (nontrivial) subset of xij to be included in interactions with, for p = 1,,m and is the corresponding subset of . Instead of observing the predictors of interest, x1 and x2, we observe instrumental variables m1 and m2 that are linearly related to them: 3 where are random intercepts for subjects, for p = 1,2, and 1ij and 2ij are error terms with . As with (1) [or (2)], we assume that the random intercept and error terms are independent (for each equation), subjects are independent, and assume the spatial power structure for . Instrumental variables in our application are cotinine (m1, measured in ng per mg of creatinine), a metabolite of nicotine that can be measured in the urine and used as a biomarker of exposure to shs, and ambient fine particulate matter concentrations measured by a fixed outdoor monitor (m2, measured in g per m). We can generalize (3) so that m1 and m2 are included in both equations . One of the difficulties of this model is that matrices associated with estimators have linear dependencies that need to be dealt with . It does not produce problems with estimators, but just complicates derivations . In this article, we will only consider (3) and not the expanded version primarily for the sake of simplifying notation, although results could be generalized to that case . In addition, the use of (3) seemed to be adequate for the data application, although the expanded version was used in the previous article to demonstrate the methods . The primary difficulty in measuring x1 and x2 is because they are mixed together when measured, along with pollution from other sources . Although we do not observe x1 and x2, in our application, there is a small amount of data for unbiased but measured - with - error versions of these variables, denoted as w1ij = x1ij+u1ij and w2ij = x2ij+u2ij, respectively, where upij has mean 0 and is assumed to be independent of, p = 1,2 . Incorporating w1 and w2 into (3) yields 4 where, for p = 1,2 . Portions of total measured fine particulate matter attributable to shs (w1) and ambient sources (w2) were estimated using personal monitors, chemical signatures, and methods as described in strand et al . This approach is expensive, cumbersome, and still results in measurements with error . But as long as we can assume that the w variables are unbiased for the x variables, we can apply rciv . In (4), let denote the error vector for subject i and p = 1,2 . We use the spatial power structure for, although it is likely to just be an approximation . Letting mij=(1,m1ij, m2ij, m1ijm2ij), the health outcome model fit for rciv1 is 5 this is essentially model (1) with mij used in place of xij, that is, unobservable predictors are replaced with their instrumental counterparts . Collectively, we define as the 4(m + 1) + k fixed - effect parameters in model (5) that correspond with the parameters in for model (1). Asterisks are included on the random terms in (5) to distinguish them from those in (1). A version of (5) that involves partial interactions can also be written [analogous to how (2) relates to (1)]. Models (4) and (5) are fitted to obtain estimates that are then combined via rciv1 to achieve the calibrated estimates, which are derived in the next section . The true covariance structure for response variables in models (4) and (5) as well as between responses in (4) and (5) based on underlying models (1) and (3) are displayed and/or discussed in appendix a. these covariance structures are quite complicated, but our work has shown that using the spatial power plus random intercept structures as approximations in models (4) and (5) works reasonably well . This is true for models here as well as those studied in strand et al ., asterisks are included on the covariance parameters to distinguish them from parameters in true underlying structures . While using these simpler covariance structures has appeared to be adequate, we also found that ignoring the repeated measures completely leads to very over - inflated variances . Let wij=(1,w1ij, w2ij, w1ijw2ij). Based on models (1) and (4), and assuming () and () are uncorrelated, we can write e(wij\|mij) = mijc, where 6 for our models and assumptions, in the aforementioned equations, we employ the following assumptions that are standard in deriving regression calibration estimators: wij and mij are surrogates for xij in predicting yij, such that (see carroll et al ., 2006 for a definition of surrogacy involving distributions);; random terms between models in (4) are uncorrelated and mij is uncorrelated with upij, p = 1,2, such that e(xij\|mij) = e(wij\|mij). For a given application, whether these assumptions are met should be considered carefully . Violations to these assumptions are a matter of degree; small violations may be inconsequential, but larger violations could be very problematic . To determine forms of estimators of, and, we first equate terms in the preceding mean derivation with those in the mean of (5), given mij and covariates: by properties of the kronecker product, we can re - express), and hence, the last aforementioned equation becomes where i m is an m m identity matrix . We also note that (imc)=(imc). By utilizing all of these equations and replacing parameters with their maximum likelihood estimators in the mixed model fits, we obtain where indicates that all parameters in c are replaced with their estimators; this matrix is invertible as long as estimates yield a nonsingular matrix, which is expected . A formulation of the model and estimators for partial interactions is given in appendix b. as in strand et al . (2014), we employed the delta method to obtain the approximate asymptotic variance of . We use the term approximate because of the approximations of covariance structures in the model; asymptotic methods are inherently approximate . Based on the delta method, where 11 is the covariance matrix for estimators of fixed - effect parameters in (4) and (5) and is a matrix of partial derivatives with (i, j) element, where [i] denotes the i element of and [j] denotes the j element of = (0,1,0,1,). The diagonal elements in (7) are the covariance matrices for fixed - effect estimators in model (4) (first two diagonal matrices) and model (5) (third diagonal matrix), and the off - diagonal matrices contain covariances between fixed - effect parameter estimators between models . Specific forms of and based on models (1), (4), and (5) and the defined estimators are given in appendix c. in (7), note that,, . Both model - based and empirical forms of were calculated (appendix c). When random terms between models in (4) as well as between those in (4) and (5) are uncorrelated, =0, although and are not necessarily 0 . For model - based variances, we set all of these submatrices to 0, primarily to avoid non - convergence or non - positive definite matrix issues . For the empirical - based estimates of, we did not set off - diagonal elements to 0 . One disadvantage of doing this is the same subject - day records must be used to fit different models in (4) and (5) so that off - diagonal covariances can be estimated (with our given approach). In the following are our steps to calculate model - based and empirical forms of used in the data application and simulations, followed by some important notes . Steps to calculate the model - based form of: set off - diagonal submatrices in (7) to 0.obtain diagonal submatrices in (7) from mixed model fits . Numerical forms of the diagonal covariance matrices are obtained easily with common statistical packages (for example, using the covb option in ods output in sas proc mixed or in the y.fit$varfix object in r, when using the lme function from the nlme package).compute using forms of as shown in appendix c, replacing parameters with their estimates . Numerical forms of the diagonal covariance matrices are obtained easily with common statistical packages (for example, using the covb option in ods output in sas proc mixed or in the y.fit$varfix object in r, when using the lme function from the nlme package). Compute using forms of as shown in appendix c, replacing parameters with their estimates . Steps to calculate the empirical form of: calculate empirical versions of submatrices in (7) using forms shown in appendix c, where the middle var and cov quantities are calculated based on squared residuals or residual cross products.compute using forms of as shown in appendix c, replacing parameters with their estimates . Calculate empirical versions of submatrices in (7) using forms shown in appendix c, where the middle var and cov quantities are calculated based on squared residuals or residual cross products . Compute using forms of as shown in appendix c, replacing parameters with their estimates . We consider linear mixed models, including random intercepts to account for subject heterogeneity in the responses, and a serial correlation structure for within - subject repeated measures . Although fitting models does involve approximation to the covariance structures as will be discussed later, including these two features in the approximated structures has allowed us to obtain reasonably accurate inferential results for parameters of interest, as verified through monte carlo simulation . The continuous outcome variable yij for subject i at time j, i = 1,,n and j = 1,,ri is expressed as a function of the unobservable predictors (x1ij, x2ij and their interaction), covariates measured without error that are involved in interactions with at least one of the unobservable predictors (denoted as), covariates without error that are not involved in interactions with the unobservable predictors (denoted as zij), and random terms to incorporate the correlation due to longitudinal measurement of subjects . If each z term is allowed to interact with all terms in xij=(1,x1ij, x2ij, x1ijx2ij) and we let zij=(z1ij, z2ij,,zkij),, then the model of interest is 1 where is a random intercept term for subjects and . We assume that the random intercept and error terms are independent of each other and that the subjects are independent . We use the spatial power structure for, which has the form, for days tij and in the study period associated with observations j and j, respectively, for subject i. this is a generalization of the ar(1) structure and is useful when data are collected intermittently, such as in our data application . (in our application, we do not consider days without data to be missing values because data collection was designed to be intermittent .) The fixed - effect parameter vectors are defined as,,, where, for p = 1,...,m . The symbol indicates the kronecker product; because xij and are both row vectors, the resulting product will also be a row vector with 4 m elements: (). Let denote the entire set of 4(m + 1) + k fixed - effect parameters in model (1). In our data application, y is natural log leukotriene e4, or ln(lte4), and x1 and x2 are shs and fine ambient particulate matter exposure concentrations, respectively . Both of the pollutant variables are considered in terms of fine particulate matter (technically, particulate matter less than 2.5 microns in diameter, or pm2.5), and measured in g per m. our primary models included an indicator for uri, or cold status (for presence, 0 for absence) as the covariate allowed to interact with x variables, and study year (z1,,z4; the last year was the reference year) as covariates not involved in interactions . If not all interactions between z and x variables are required, then the model can be reformulated as 2where is the (nontrivial) subset of xij to be included in interactions with, for p = 1,,m and is the corresponding subset of . Instead of observing the predictors of interest, x1 and x2, we observe instrumental variables m1 and m2 that are linearly related to them: 3 where are random intercepts for subjects, for p = 1,2, and 1ij and 2ij are error terms with . As with (1) [or (2)], we assume that the random intercept and error terms are independent (for each equation), subjects are independent, and assume the spatial power structure for . Instrumental variables in our application are cotinine (m1, measured in ng per mg of creatinine), a metabolite of nicotine that can be measured in the urine and used as a biomarker of exposure to shs, and ambient fine particulate matter concentrations measured by a fixed outdoor monitor (m2, measured in g per m). We can generalize (3) so that m1 and m2 are included in both equations . One of the difficulties of this model is that matrices associated with estimators have linear dependencies that need to be dealt with . It does not produce problems with estimators, but just complicates derivations . In this article, we will only consider (3) and not the expanded version primarily for the sake of simplifying notation, although results could be generalized to that case . In addition, the use of (3) seemed to be adequate for the data application, although the expanded version was used in the previous article to demonstrate the methods . The primary difficulty in measuring x1 and x2 is because they are mixed together when measured, along with pollution from other sources . Although we do not observe x1 and x2, in our application, there is a small amount of data for unbiased but measured - with - error versions of these variables, denoted as w1ij = x1ij+u1ij and w2ij = x2ij+u2ij, respectively, where upij has mean 0 and is assumed to be independent of, p = 1,2 . Incorporating w1 and w2 into (3) yields 4 where, for p = 1,2 . Portions of total measured fine particulate matter attributable to shs (w1) and ambient sources (w2) were estimated using personal monitors, chemical signatures, and methods as described in strand et al . This approach is expensive, cumbersome, and still results in measurements with error . But as long as we can assume that the w variables are unbiased for the x variables, we can apply rciv . In (4), let denote the error vector for subject i and p = 1,2 . We use the spatial power structure for, although it is likely to just be an approximation . Letting mij=(1,m1ij, m2ij, m1ijm2ij), the health outcome model fit for rciv1 is 5 this is essentially model (1) with mij used in place of xij, that is, unobservable predictors are replaced with their instrumental counterparts . Collectively, we define as the 4(m + 1) + k fixed - effect parameters in model (5) that correspond with the parameters in for model (1). Asterisks are included on the random terms in (5) to distinguish them from those in (1). A version of (5) that involves partial interactions can also be written [analogous to how (2) relates to (1)]. Models (4) and (5) are fitted to obtain estimates that are then combined via rciv1 to achieve the calibrated estimates, which are derived in the next section . The true covariance structure for response variables in models (4) and (5) as well as between responses in (4) and (5) based on underlying models (1) and (3) are displayed and/or discussed in appendix a. these covariance structures are quite complicated, but our work has shown that using the spatial power plus random intercept structures as approximations in models (4) and (5) works reasonably well . This is true for models here as well as those studied in strand et al ., asterisks are included on the covariance parameters to distinguish them from parameters in true underlying structures . While using these simpler covariance structures has appeared to be adequate, we also found that ignoring the repeated measures completely leads to very over - inflated variances . Let wij=(1,w1ij, w2ij, w1ijw2ij). Based on models (1) and (4), and assuming () and () are uncorrelated, we can write e(wij\|mij) = mijc, where 6 for our models and assumptions, in the aforementioned equations, we employ the following assumptions that are standard in deriving regression calibration estimators: wij and mij are surrogates for xij in predicting yij, such that (see carroll et al ., 2006 for a definition of surrogacy involving distributions);; random terms between models in (4) are uncorrelated and mij is uncorrelated with upij, p = 1,2, such that e(xij\|mij) = e(wij\|mij). For a given application, violations to these assumptions are a matter of degree; small violations may be inconsequential, but larger violations could be very problematic . To determine forms of estimators of, and, we first equate terms in the preceding mean derivation with those in the mean of (5), given mij and covariates: by properties of the kronecker product, we can re - express), and hence, the last aforementioned equation becomes where i m is an m m identity matrix . We also note that (imc)=(imc). By utilizing all of these equations and replacing parameters with their maximum likelihood estimators in the mixed model fits, we obtain where indicates that all parameters in c are replaced with their estimators; this matrix is invertible as long as estimates yield a nonsingular matrix, which is expected . (2014), we employed the delta method to obtain the approximate asymptotic variance of . We use the term approximate because of the approximations of covariance structures in the model; asymptotic methods are inherently approximate . Based on the delta method, where 11 is the covariance matrix for estimators of fixed - effect parameters in (4) and (5) and is a matrix of partial derivatives with (i, j) element, where [i] denotes the i element of and [j] denotes the j element of = (0,1,0,1,). The diagonal elements in (7) are the covariance matrices for fixed - effect estimators in model (4) (first two diagonal matrices) and model (5) (third diagonal matrix), and the off - diagonal matrices contain covariances between fixed - effect parameter estimators between models . Specific forms of and based on models (1), (4), and (5) and the defined estimators are given in appendix c. in (7), note that,, . Both model - based and empirical forms of were calculated (appendix c). When random terms between models in (4) as well as between those in (4) and (5) are uncorrelated, =0, although and are not necessarily 0 . For model - based variances, we set all of these submatrices to 0, primarily to avoid non - convergence or non - positive definite matrix issues . For the empirical - based estimates of, we did not set off - diagonal elements to 0 . One disadvantage of doing this is the same subject - day records must be used to fit different models in (4) and (5) so that off - diagonal covariances can be estimated (with our given approach). In the following are our steps to calculate model - based and empirical forms of used in the data application and simulations, followed by some important notes . Steps to calculate the model - based form of: set off - diagonal submatrices in (7) to 0.obtain diagonal submatrices in (7) from mixed model fits . Numerical forms of the diagonal covariance matrices are obtained easily with common statistical packages (for example, using the covb option in ods output in sas proc mixed or in the y.fit$varfix object in r, when using the lme function from the nlme package).compute using forms of as shown in appendix c, replacing parameters with their estimates . Numerical forms of the diagonal covariance matrices are obtained easily with common statistical packages (for example, using the covb option in ods output in sas proc mixed or in the y.fit$varfix object in r, when using the lme function from the nlme package). Compute using forms of as shown in appendix c, replacing parameters with their estimates . Steps to calculate the empirical form of: calculate empirical versions of submatrices in (7) using forms shown in appendix c, where the middle var and cov quantities are calculated based on squared residuals or residual cross products.compute using forms of as shown in appendix c, replacing parameters with their estimates . Calculate empirical versions of submatrices in (7) using forms shown in appendix c, where the middle var and cov quantities are calculated based on squared residuals or residual cross products . Compute using forms of as shown in appendix c, replacing parameters with their estimates . In our analyses, we consider the same data as described and analyzed in strand et al . (2014), with the addition of the time - varying cold indicator variable, obtained through surveys with the children . The variables are defined in section 2.1, with summary statistics presented in table1 . Within a study year data from the first 2 study years (20022004) were available to fit (4), while data from all 4years (20022006) were available to fit (5). For ease of interpretation of parameter estimates, we mean - corrected the w variables in the fit of (4). Instrumental data were much more abundant and easier to collect than data from personal monitors, making rciv a natural choice for analysis . Our assumed model for (1) was 12 descriptive statistics of variables involved in the analysis y = ln(lte4); w1=ln(shs exposure+1); w2=ln(ambient fine particulate matter exposure + 1); m1=ln(cotinine); m2=ln(ambient fine particulate matter from fixed monitor);=upper respiratory infection (1=present, 0=absent), that is, cold. Units for pollutant exposure variables and ambient fine particulate matter measured at the fixed monitor were g / m; units for lte4 were pg per mg of creatinine; units for cotinine were ng per mg of creatinine (all before transformation). Table2 shows results of model (4) and (5) fits, while table3 shows estimates of the key parameters in (8) via rciv1 . Our data suggested that the instrumental (m) variables were only weak to moderately associated with the personal exposure data . This was one of the reasons why more data were sought to fit (4), even though unbiased surrogate data did not exist in the last 2years of the study . Figure1 shows predicted values based on calibrated estimates; the figure demonstrates that while predicted values for lte4 generally increase as pollutants increase, there was noticeable negative interaction between pollutants for those without colds (p = 0.07); when one pollutant was lower, the dose response relationship between lte4 and the other pollutant was stronger . Such a pattern was not observed for those with colds (p = 0.88 for interaction). The quartiles used in figure1 were determined based on distributions of predicted values of w1 and w2 from fits of (4). (2014), for which quartiles from distributions of observed values of w1 and w2 were used . The distributions of predicted values are less variable, and so using quartiles from these distributions leads to estimates more on the interior of the distributions of exposures rather than on the extremes . Estimates of parameters in (4) and (5) for the data application (se in parentheses) w variables were mean - corrected before the fit of (4). There were 458, 560 and 1438 records for analysis in the regression models for w1, w2 and y, respectively; see the text for more detail about the variables and models . Estimates of parameters (or linear combinations of parameters) in model (8) for ln(lte4) based on the rciv1 estimation method model - based asymptotic standard errors were derived using the delta method as described in section 2.3, and p - values are based on wald z - tests assuming asymptotic normality . Because w variables were mean corrected before analysis, interpretations for shs and ambient pm2.5 slopes (rows 2 and 3) are relevant at the mean of the other pollutant, and the intercept is the estimate of mean ln(lte4) for the reference year (year 4) at the mean of both pollutants . The larger ses for cold are due to fewer subject - days for these conditions, relative to no cold . Relationships between lte4 and exposure to one pollutant, modified by exposure to a second pollutant, for children on (a) days without colds and (b) days with colds . Solid lines show estimated mean lte4 as a function of secondhand smoke (shs), by quartiles of ambient fine particulate matter . Dashed lines show estimated mean lte4 as a function of ambient fine particulate matter, by quartiles of shs . Quartiles were determined from distributions of predicted values from model fits of (4). Quartile symbols are q1=25th percentile, q2=50th percentile, q3=75th percentile . Predicted mean values were obtained using rciv1 methods, using estimates, as shown in table3, and scaled for the average across years . Because the response variable was analyzed on the natural log scale, the predicted values have the form on the original scale, where c = 1.18 for the average across years . The graph illustrates that for no - cold days, increases in lte4 per unit increase in one pollutant are steepest when the second pollutant is lower . However, when children have colds, lte4 is higher and there is less interaction between pollutants on health . Both outcome and predictors were analyzed on the natural log scale but inverted back for presentation, resulting in curves the estimated slope of the shs exposure variable in predicting log lte4fixing ambient fine particulate matter exposure at its mean level was approximately three times greater than for ambient fine particulate matter (fixing shs exposure at its mean level), both for those without and with colds (table3). Although this difference was not significant for either those with or without colds (p> 0.2), the consistency of estimates between sick and well children is notable . For those without colds, the shs slope at a fixed level c for ambient fine particulate matter was greater than the slope of ambient fine particulate matter slope at level c of shs with marginal significance (p = 0.047), while it was very insignificant for those with colds . (this is a different test than when comparing at the mean of the other pollutant because the means of the two pollutants differed .) The weaker significance of tests involving those with colds is at least in part due to fewer records under those conditions (approximately 20%). In the absence of shs, well children had a marginally significant relationship between ambient fine particulate matter and log(lte4), while there was no apparent relationship when children were sick (last row, table3). The comparison of shs slopes in absence of ambient fine particulate matter is less relevant and not included because children are generally exposed to ambient fine particulate matter, but not necessarily cigarette smoke . The increase in lte4 per iqr increase in one pollutant at the mean of the other pollutant ranged from approximately 3 to 15% both for those with and without colds . Fixing both shs and ambient fine particulate matter at mean values, those with colds had lte4 values that were approximately 8% higher than those without (p = 0.02; obtained as [exp(4.41) exp(4.33)]/exp(4.33), based on intercepts shown in table3). This suggests that having a cold has an acute effect on lte4 that is approximately the same as an iqr increase in a pollutant (fixing non - involved pollutants at mean values). The larger random subject variances for w1 and y models are expected; the first occurring due to differences in smoking behaviors of those living with the study subjects, and the second due to natural subject heterogeneity in lte4 levels . Both the random subject variance and within - subject correlation were relatively low for w2 models, but for consistency, we kept the terms in that model . Appendix d has a discussion of the predicted values for models without covariate interaction terms . Simulations were conducted in order to determine accuracy of the regression calibration estimators and to assess the coverage rates of confidence intervals . The models used were similar to those in the data application, without year indicators (i.e., no z variables in the model). One indicator covariate () was included and was allowed to interact with both x variables as well as their interaction (i.e., the full interaction model (1) was considered). Was included to mimic the variable, with the same approximate rate of event occurrence (for about 20% of the data). The simulations generally used parameter settings that lead to parameter estimates similar to those observed in the data application . The simulation conditions were as follows: sample sizes ranged from n = 50 to n = 500 subjects, with r = 10 to r = 20 repeated measures per subject on consecutive days . Complete records were simulated so that both model - based and empirical variance estimators could be determined . Additional conditions (e.g., different correlation parameter values or error distribution types) were considered in strand et al . Overall, the results here are similar to those presented in strand et al . (2014) for models without covariate interaction terms, but more complex pollutant models . Generally, the results indicate that the methods described in section 2 yield confidence intervals for parameters of interest that have appropriate (nominal) coverage rate, with some slight overcoverage when using model - based variances, for smaller sample sizes . Mse and bias simulation results for rciv1 one thousand replicates per condition, using normal errors; r= number of consecutive - day repeated measures per subject; n= subject sample size . Covariance and fixed - effect parameter settings were chosen so that fitted models had similar parameter estimates as those observed for the data application; for fixed - effect parameters, the following values were used:,,,,,,,(related to x variables that were not mean corrected) simulation confidence interval coverage rates for rciv1 one thousand replicates per condition; r= number of consecutive - day repeated measures per subject; n= subject sample size . Values are minimum (or maximum) confidence interval (ci) coverage rate among four parameters in set . For 1000 replicates, a correct method will have coverage within 1.4% of 95%, with 95% probability; ci coverages outside of this range are in bold . The properties of estimators and confidence intervals seemed to differ slightly between coefficients related to main effects () and interactions (). In particular, more precision was apparent for estimators of main effect coefficients, likely because of more data available to estimate them (subject - days with no colds); the interaction coefficients () involve comparisons between cold versus no - cold conditions . Mean bias was not significantly nonzero (for = 0.05 with t - tests) across simulation sets . There were slight trends of bias in some simulations, although it did not appear to adversely affect confidence interval coverage rates, and the bias tended to average out between sets of parameter estimates . For example, when and had negative bias, and were positive, and vice versa . Similar results were observed for although even more pronounced (likely because of greater standard errors for those estimates). These patterns were not consistent across simulations, and together with the nonsignificant observed bias, there is no indication of systematic problems with the estimation method . The results suggest that using empirical variances may be more reliable (when possible), although model - based variances did not perform too poorly . Because of the fact that the variances are asymptotic in nature, it is not recommended that sample sizes much less than 100 subjects be used . The simulations discussed earlier and presented in the tables generated data from random terms that were independent between as well as within models . A set of simulations was also conducted using correlated random terms between models in (4) in order to determine how violating the assumption affected results, as discussed in section 5 . In our analyses, we found that while pollutants appeared to have additive effects when children were sick, there was a marginally significant interaction between pollutants when they were not sick, such that the health pollutant relationship for one pollutant was stronger when the other pollutant was lower . This difference in interaction terms (i.e., the three - way interaction) suggests a complex relationship between exposure to pollutants and cold status of children with asthma, with respect to lte4 levels . One possible explanation for the observed results is that when they are not sick, children are more able to maintain homeostatis by limiting lte4 responses during co - exposures to pollutants, relative to when they are sick . However, more research is certainly warranted in order to not only test this hypothesis but also to verify the observed patterns here . The conditions and restrictions of the study should be kept in mind here, which include but are not limited to the following: fine particulate matter is only one type of air pollution that does not include toxic gases such as carbon monoxide; the study group here involves children with asthma and not the general population; relatively low levels of fine particulate matter and shs were observed, and results cannot necessarily be extrapolated to higher, unobserved levels ambient fine particulate matter concentrations in parts of china are often well over 10 times that observed in denver (e.g., see venners et al ., 2003); and the toxicity of ambient fine particulate matter in denver may differ from that in other cities because of different compositions . Specific forms of regression calibration estimators depend on exactly which terms are included in the models . Special cases for models with one predictor measured with error could be obtained easily from the results here . Methods could be extended to models with more than two predictors using the same approach given in this paper; however, notation would become burdensome . (2014) suggest that regression calibration for more complicated longitudinal models with interaction terms can yield fairly accurate estimators despite approximations made to covariance structures . In both of these studies, some over - coverage was apparent for relatively small sample sizes (no larger than 100) for model - based variances estimators . (2000) also found overestimated variances of regression calibration estimators associated with random effect models, for estimation without instrumental variable estimators; he also suggested modified approaches to reduce this overestimation . Whether or not these results are due to the same issue, the slight overestimation of variances of here did not appear to be large enough to consider adjustments, as long as adequate sample sizes are used . Certain assumptions were made that allowed us to derive the point estimators, and we discuss a few here with respect to our application . The non - differential error (or surragocy) assumption essentially states that the unbiased surrogate and instrumental variables do not provide any more information in predicting the health outcome beyond what the actual exposure variables and covariates do . Because cotinine is a metabolite specific to nicotine, we believe it is reasonable to assume that it will not provide more information in predicting lte4 than actual shs exposure . An exception to this would be the fact that cotinine may be affected by the gas portion of secondhand smoke, which could potentially influence lte4 and is not included in estimated or actual shs particulate exposure . Similarly, we believe it is reasonable to assume that outdoor fine particulate matter measured at a fixed location will not provide more information in predicting lte4 than the direct exposures to this pollutant . The assumption that w variables are surrogates for x variables seems even more logical, although the unbiased assumption is not easily verifiable . Further examination of our data indicated the assumption that random terms between pollutant models in (4) are uncorrelated was violated to a small degree . However, additional simulations that included correlation of approximately the same magnitude as was observed had a negligible effect on the point estimates . Another assumption in our models is that the instrumental variables are not measured with error . There is some potential for error in cotinine and creatinine measurements, although it is not expected to be greater than that of the w variables . One may wonder whether regressing the health outcome directly on unbiased surrogate variables is a reasonable alternative to using regression calibration as described in this article . While easier, this approach not only has the disadvantage that the measurement error in the predictors will attenuate the estimates by introducing classical measurement error, but also much less data are available to fit this model . This approach yielded effects that were not close to significant (results not presented). In our application, we used a binary covariate that interacted with the pollutant variables to illustrate the methods . Because it is just a binary variable, one could also perform stratified analyses for those with colds and those without, eliminating z variables from the models . However, doing such would not allow for statistical comparisons to be made between those with and those without colds . Using our presented methods, we could also add continuous covariates into the model such as subject height . In some analyses with other data, however, for these particular data, height was insignificant and did not contribute to the model and thus was not included.
Major conclusions are that there is a broad range of kinematics and that specific prostheses have specific advantages and disadvantages [2, 5, 27]. For example, posterior - stabilized knee prostheses were developed to prevent reversed anterior translations of the femoral condyles during flexion seen in cruciate sacrificing prostheses . The induced posterior displacement will avoid impingement and thereby improve the range of motion of the knee . However, it is no exception that the actual in vivo kinematics of knee prostheses is not in line with the desired kinematics as intended by the design . Understanding the effect of design choices on in vivo kinematics, stability and muscle activation has become more important because of the increasingly clear connection between knee prosthesis kinematics and clinical performance . Therefore, the aim of this study was to compare a broad range of total knee prostheses with different design parameters (multi - radius, single - radius, fixed - bearing, mobile - bearing, posterior - stabilized, cruciate retaining and cruciate sacrificing) to determine whether in vivo kinematics was consistently related to design . The hypothesis was that there are no clear recognizable differences in in vivo kinematics between different design parameters or prostheses . At two sites, data were collected by a single observer on 52 knees (49 subjects with rheumatoid arthritis or osteoarthritis). Total knee replacements were performed by five surgeons at three hospitals in two countries (the netherlands and united kingdom). All surgeons were specialized in total knee arthroplasty, and prostheses were implanted according to the operative techniques described by the manufacturer . Based on a previous fluoroscopy study, relative motions of 0.3 could be detected when ten patients were included in each group . Knee kinematics was recorded using fluoroscopy as the patients performed a step - up motion . Patients reported functional ability (knee score and function score) was quantified pre- and post - operatively for the prospective patients using the knee society score (kss). The study was approved by the respective local medical ethics committees, and all patients gave informed consent.table 1overview of the prostheses used, congruency of the insert and number of knees and patient characteristics (mean and standard deviation)prosthesisdesign parametersnumber of kneesfollow - up (months)male / femaleage (years)bmi (kg / m)pre - operativepost - operativefunction scoreknee scorefunction scoreknee scoreduraconmulti - radius1021 (8.9)3/768 (10.9)29 (3.7)xx88 (13)95 (3)fixed - bearingcruciate retainingtriathlon fbsingle - radius1113 (1.0)5/666 (9.1)30 (6.2)52 (18)43 (13)73 (24)92 (4)fixed - bearingposterior - stabilizedtriathlon mbsingle - radius912 (2.5)2/763 (9.6)31 (7.5)48 (13)49 (21)71 (26)90 (11)mobile - bearingposterior - stabilizedpfc - sigmamulti - radius85 (1.0)4/467 (7.6)31 (5.1)xxxxfixed - bearingposterior - stabilizednexgenmulti - radius743 (7.7)1/667 (8.2)30 (3.1)43 (16)44 (24)74 (30)84 (18)mobile - bearingposterior - stabilizedroccmulti - radius725 (0.8)3/463 (10.9)29 (5.6)50 (26)47 (12)79 (22)86 (11)mobile - bearingcruciate sacrificingmissing data are indicated with an xstryker, kalamazoo, mi, usadepuy orthopaedics inc ., warsaw, in, usazimmer inc ., warsaw, in, usabiomet, europe bv, dordrecht, the netherlands overview of the prostheses used, congruency of the insert and number of knees and patient characteristics (mean and standard deviation) missing data are indicated with an x stryker, kalamazoo, mi, usa depuy orthopaedics inc ., warsaw, in, usa zimmer inc ., warsaw, in, usa biomet, europe bv, dordrecht, the netherlands the patients were asked to perform a step - up motion (height 18 cm) with bare feet in front of a flat panel fluoroscope (15 frames / sec, resolution 1,024 1,024, pulse width <3.2 ms). Patients were instructed to keep their weight onto the leg of interest and to perform the motions in a controlled manner . Three - dimensional (3d) models (reverse engineered or computer aided design) of the tibial and femoral components were used to assess the position and orientation of the components in the fluoroscopic images . In case of a mobile - bearing prosthesis, during surgery 1 mm tantalum markers were inserted in predefined non - weight bearing areas of the mobile insert to visualize the polyethylene . Roentgen stereophotogrammetric analysis (rsa) was used to create accurate 3d models of the markers of the inserts to assess position and orientation of the mobile insert in the fluoroscopic images . This technique showed to have an axial rotation accuracy of 0.1 and 0.1 mm . The coordinate system was defined as the local coordinate system of the tibial component . At maximal extension the minimal distance between the femoral condyles and the tibial base plate was calculated independently for the medial and lateral condyle and projected on the tibial plane to show the anterior - posterior motions . This line was projected onto the transverse plane of the tibial plateau for each fluoroscopic frame . All images were processed using a commercially available software package (model - based rsa, medis specials b.v ., leiden, the netherlands). A chi - square test (cramer s v) was used to test whether the prosthesis groups were different on variables, such as age, gender, bmi and functional and knee scores . An anova was used to test for differences in outcome variables among the prosthetic groups . Levene s test was used to test for homogeneity of variances between prosthetic groups . For femoral axial rotation (p = 0.006) and insert axial rotation (p = 0.001), the variances were not equal . To correct for this unequal variance and to correct for the different group sizes, when a significant effect of prosthetic design on an outcome variable was found, post hoc tests were performed to test which groups were different . The patients were asked to perform a step - up motion (height 18 cm) with bare feet in front of a flat panel fluoroscope (15 frames / sec, resolution 1,024 1,024, pulse width <3.2 ms). Patients were instructed to keep their weight onto the leg of interest and to perform the motions in a controlled manner . Three - dimensional (3d) models (reverse engineered or computer aided design) of the tibial and femoral components were used to assess the position and orientation of the components in the fluoroscopic images . In case of a mobile - bearing prosthesis, during surgery 1 mm tantalum markers were inserted in predefined non - weight bearing areas of the mobile insert to visualize the polyethylene . Roentgen stereophotogrammetric analysis (rsa) was used to create accurate 3d models of the markers of the inserts to assess position and orientation of the mobile insert in the fluoroscopic images . This technique showed to have an axial rotation accuracy of 0.1 and 0.1 mm . The coordinate system was defined as the local coordinate system of the tibial component . At maximal extension, the minimal distance between the femoral condyles and the tibial base plate was calculated independently for the medial and lateral condyle and projected on the tibial plane to show the anterior - posterior motions . This line was projected onto the transverse plane of the tibial plateau for each fluoroscopic frame . All images were processed using a commercially available software package (model - based rsa, medis specials b.v ., leiden, the netherlands). A chi - square test (cramer s v) was used to test whether the prosthesis groups were different on variables, such as age, gender, bmi and functional and knee scores . An anova was used to test for differences in outcome variables among the prosthetic groups . Levene s test was used to test for homogeneity of variances between prosthetic groups . For femoral axial rotation (p = 0.006) and insert axial rotation (p = 0.001), the variances were not equal . To correct for this unequal variance and to correct for the different group sizes, brown - forsythe correction was used . When a significant effect of prosthetic design on an outcome variable was found, age at surgery, bmi, pre - operative kss knee score and function score did not differ significantly between groups (table 1). However, there was a small significant difference in post - operative kss knee score (p = 0.045). Post - operatively, the duracon patients (multi - radius fixed - bearing cruciate retaining) scored highest on both kss function score and knee score . In all groups, all patients were considered clinically successful without significant pain or measurable ligamentous instability . Also, no clinical deviations were reported, such as extension lags or flexion contractures . The nexgen group had significant smaller knee flexion angles compared to the other prosthetic groups (triathlon mb p = 0.005; triathlon fb p = 0.004; duracon p = 0.003; rocc p = 0.007; pfc - sigma p = 0.017). There were no significant differences between the other groups (table 2).table 2mean and standard deviation of the range of knee flexion (), axial rotation of the femoral component and the insert () and anterior - posterior (ap) translation (mm) of the lateral and medial condyle during the step - up motion for each prosthetic groupprosthesisknee flexion ()axial rotation ()ap translation (mm)femoral componentmobile insertmedial condylelateral condyleduracon59.7 (9.3)8.6 (2.3)9.0 (2.1)11.1 (3.4)triathlon fb60.3 (5.4)8.3 (2.7)6.6 (1.5)7.1 (1.8)triathlon mb62.0 (12.9)9.6 (4.3)8.7 (4.9)6.8 (2.0)6.0 (1.6)pfc - sigma56.5 (9.9)8.3 (4.5)5.3 (1.9)6.8 (2.5)nexgen34.5 (10.3)3.0 (0.5)2.0 (0.7)3.9 (2.1)4.8 (1.8)rocc59.0 (8.8)10.4 (5.4)7.3 (2.8)6.9 (2.0)7.0 (1.5)levene s test0.83 n.s.3.80 p = 0.0069.60 p = 0.0010.31 n.s.1.74 n.s.anova brown - forsythef(5, 36.7) = 8.38 p = 0.000f(5, 25.1) = 3.56 p = 0.014f(2, 13.2) = 9.11 p = 0.003f(5, 40.7) = 6.46 p = 0.000f(5, 34.6) = 8.55 p = 0.000also, the results of the levene s test and anova are presented . There was a significant effect of prosthetic design on all outcome variables: fixed - bearing prosthesis; therefore, no mobile insert datan.s . Not significant mean and standard deviation of the range of knee flexion (), axial rotation of the femoral component and the insert () and anterior - posterior (ap) translation (mm) of the lateral and medial condyle during the step - up motion for each prosthetic group also, the results of the levene s test and anova are presented . There was a significant effect of prosthetic design on all outcome variables: fixed - bearing prosthesis; therefore, no mobile insert data the nexgen group had significantly smaller femoral axial rotation compared to the duracon group (p = 0.000), the triathlon mb group (p = 0.024) and triathlon fb group (p = 0.001). The mean range of axial rotation of the insert of the nexgen patients was also significantly smaller (limited to 2.0) than the mean range of axial rotations of the inserts of the triathlon mb and rocc groups (p = 0.010 and p = 0.006, respectively). The mobile insert of the rocc followed the motion of the femoral component until approximately 60 of knee flexion . Beyond 60 of knee flexion, 3 of 7 rocc patients showed paradoxical axial rotations . The insert of the triathlon patients followed the femoral component during the complete motion (maximum knee flexion during step - up was 80), without showing paradoxical axial rotations . Under the assumption that the inserts will follow the femoral component, a centrally located pivot point of axial rotation of the femoral component was expected . In all groups, except for the rocc patients, the measured pivot point of axial rotation varied between a medial, central or lateral position . All the rocc patients had a central point of rotation, except for one subject having a medial pivot point of axial rotation (fig . 1).fig . 1example of a medial pivot point of axial rotation . The medial condyle moves to posterior and the lateral condyle to anterior during knee extension example of a medial pivot point of axial rotation . The medial condyle moves to posterior and the lateral condyle to anterior during knee extension the translations of the lateral condylar were essentially anterior throughout knee extension and translations of the medial condylar mainly posterior . The nexgen, duracon, pfc - sigma and triathlon fb patients showed no paradoxical anterior - posterior motions . The duracon group had larger translations of the medial condyle compared to the pfc - sigma group (p = 0.021) and the nexgen group (p = 0.005) and of the lateral condyle compared to the triathlon mb group (p = 0.015) and nexgen group (p = 0.003). Between the rest of the groups, there were no significant differences in anterior - posterior translation . The nexgen group had significant smaller knee flexion angles compared to the other prosthetic groups (triathlon mb p = 0.005; triathlon fb p = 0.004; duracon p = 0.003; rocc p = 0.007; pfc - sigma p = 0.017). There were no significant differences between the other groups (table 2).table 2mean and standard deviation of the range of knee flexion (), axial rotation of the femoral component and the insert () and anterior - posterior (ap) translation (mm) of the lateral and medial condyle during the step - up motion for each prosthetic groupprosthesisknee flexion ()axial rotation ()ap translation (mm)femoral componentmobile insertmedial condylelateral condyleduracon59.7 (9.3)8.6 (2.3)9.0 (2.1)11.1 (3.4)triathlon fb60.3 (5.4)8.3 (2.7)6.6 (1.5)7.1 (1.8)triathlon mb62.0 (12.9)9.6 (4.3)8.7 (4.9)6.8 (2.0)6.0 (1.6)pfc - sigma56.5 (9.9)8.3 (4.5)5.3 (1.9)6.8 (2.5)nexgen34.5 (10.3)3.0 (0.5)2.0 (0.7)3.9 (2.1)4.8 (1.8)rocc59.0 (8.8)10.4 (5.4)7.3 (2.8)6.9 (2.0)7.0 (1.5)levene s test0.83 n.s.3.80 p = 0.0069.60 p = 0.0010.31 n.s.1.74 n.s.anova brown - forsythef(5, 36.7) = 8.38 p = 0.000f(5, 25.1) = 3.56 p = 0.014f(2, 13.2) = 9.11 p = 0.003f(5, 40.7) = 6.46 p = 0.000f(5, 34.6) = 8.55 p = 0.000also, the results of the levene s test and anova are presented . There was a significant effect of prosthetic design on all outcome variables: fixed - bearing prosthesis; therefore, no mobile insert datan.s . Not significant mean and standard deviation of the range of knee flexion (), axial rotation of the femoral component and the insert () and anterior - posterior (ap) translation (mm) of the lateral and medial condyle during the step - up motion for each prosthetic group also, the results of the levene s test and anova are presented . There was a significant effect of prosthetic design on all outcome variables: fixed - bearing prosthesis; therefore, no mobile insert data the nexgen group had significantly smaller femoral axial rotation compared to the duracon group (p = 0.000), the triathlon mb group (p = 0.024) and triathlon fb group (p = 0.001). The mean range of axial rotation of the insert of the nexgen patients was also significantly smaller (limited to 2.0) than the mean range of axial rotations of the inserts of the triathlon mb and rocc groups (p = 0.010 and p = 0.006, respectively). The mobile insert of the rocc followed the motion of the femoral component until approximately 60 of knee flexion . Beyond 60 of knee flexion, 3 of 7 rocc patients showed paradoxical axial rotations . The insert of the triathlon patients followed the femoral component during the complete motion (maximum knee flexion during step - up was 80), without showing paradoxical axial rotations . Under the assumption that the inserts will follow the femoral component, a centrally located pivot point of axial rotation of the femoral component was expected . In all groups, except for the rocc patients, the measured pivot point of axial rotation varied between a medial, central or lateral position . All the rocc patients had a central point of rotation, except for one subject having a medial pivot point of axial rotation (fig . The medial condyle moves to posterior and the lateral condyle to anterior during knee extension example of a medial pivot point of axial rotation . The translations of the lateral condylar were essentially anterior throughout knee extension and translations of the medial condylar mainly posterior . The nexgen, duracon, pfc - sigma and triathlon fb patients showed no paradoxical anterior - posterior motions . The duracon group had larger translations of the medial condyle compared to the pfc - sigma group (p = 0.021) and the nexgen group (p = 0.005) and of the lateral condyle compared to the triathlon mb group (p = 0.015) and nexgen group (p = 0.003). Between the rest of the groups, there were no significant differences in anterior - posterior translation . The aim of this study was to compare different total knee prostheses (multi - radius, single - radius, fixed - bearing, mobile - bearing, posterior - stabilized, cruciate retaining and cruciate sacrificing) to determine whether in vivo kinematics is consistently related to kinematics intended by the knee prosthesis design . According to several authors, in vivo knee kinematics after total knee arthroplasty is directly related to the constraints of the design of the prosthesis [4, 5, 9]. On the other hand, several studies found aberrant and highly unpredictable kinematics, and there was no distinction in clinical results and kinematics between different types of prostheses [9, 14, 15, 20, 2224]. This study showed that despite kinematics being generally consistent with the kinematics intended by their design, there were no clear recognizable differences in in vivo kinematics between different design parameters or prostheses . Patients with a cruciate sacrificing prosthesis (rocc) cannot rely on the cruciate ligaments to provide stability . To compensate for this, the congruency of the insert is increased, providing more intrinsic stability between the insert and the femoral component . The increased congruency is also expected to lead to increased axial rotation of the mobile insert . This is supported by our fluoroscopic data, showing that the insert was following the femoral component until approximately 60 of knee flexion . Beyond 60 of knee flexion, diversion between the insert and the femoral component and reversed axial rotations occurred . Despite the lower congruency, the triathlon mb group showed equal motion of the insert and femoral component during the whole range of flexion, without occurrence of reversed axial rotations . A more uniform motion may reduce wear of the polyethylene, due to a reduction in shear forces at the liner interface [6, 19]. According to knee simulator studies, the reduction in sliding distance reduces the surface area of polyethylene being worn which in turn reduces wear [18, 19]. The cruciate retaining group (duracon) had the largest anterior - posterior motions, without revealing any reversed femoral tibial motion patterns . This is in accordance with the intended kinematics, keeping the posterior ligament to preserve normal rollback . The retained posterior ligament is assumed to increase joint stability compared to cruciate sacrificing total knees . This assumption is supported by the duracon group having the highest post - operative kss knee and function scores . Possibly, this patient group had also better function pre - operatively . Pre - operative scores and function are good indicators for post - operative scores and functions . All total knees showed comparable axial rotations of the femoral component with respect to the tibial component, except for the nexgen patients . The mobile inserts did not add additional mobility to the knee joint compared to the fixed - bearing groups . However, additional mobility was possibly not needed during the step - up motion performed . The inserts of two of the three mobile - bearing groups moved as predicted on theoretical grounds . The absence or reduced mobility in the nexgen patients makes this implant very similar to a fixed - bearing prosthesis . This absence or reduced mobility will also enhance wear of the polyethylene and could induce a higher incidence of loosening by transmitting larger forces to the bone - implant interface [1, 6, 7, 10, 12, 25, 26]. In all three mobile - bearing prostheses used, the centrally located trunnion imposed a centrally located pivot point of rotation of the insert on top of the tibial plateau . Under the assumption that the inserts will follow the femoral component, a centrally located pivot point of axial rotation of the femoral component only the rocc patients had a measured central pivot point of axial rotation of the femoral component with respect to the tibial component . In the other two mobile - bearing groups, these deviant pivot points might be caused by low congruency between the insert and femoral component and by laxity of the surrounding ligaments . However, no manifest laxity was seen in these patients . A possible limitation of this and other multicenter studies, which could explain the variability in kinematics, is patient diversity (osteoarthritis and rheumatoid arthritis), pre - operative deformities, muscle adaptations and the different surgeons . It is known that surgeons are still the biggest variable in outcome after total knee arthroplasty . Factors that play a major role in dysfunction of any knee and are determined by the surgeon are frontal plane malalignment, axial malrotation of the prosthesis, sagittal overstuffing of the knee, inappropriate level of joint space, inappropriate constraint or ligamentous imbalance and poor initial fixation of the implant [3, 8, 21]. Statistics showed that there was a significant effect of prosthetic design on all outcome parameters; however, post hoc tests showed that the nexgen group was responsible for 80% of the significant values . In this group, the range of knee flexion was much smaller, resulting in smaller anterior - posterior translations and rotations . It is not clear whether and why this patient group performed the step - up task differently . This study showed that the in vivo kinematics of most included total knee prostheses were consistent with the kinematics intended by their design . However, some prostheses showed reversed or paradoxical kinematics in some parts of their functional range of motion . If the theoretical kinematics is not in accordance with the in vivo kinematics, the manufacture should optimize the new prosthetic design to prevent large scale polyethylene wear with subsequent prosthesis loosening . This is of importance because of the growing population of younger patients who will require an implant to function for at least two decades . Because of the high accuracy, it is recommended that fluoroscopy is used for evaluating the kinematics of new total knee prostheses before introducing the new knee worldwide on the market . Despite kinematics being generally consistent with the kinematics intended by their design, there were no clear recognizable differences in in vivo kinematics between different design parameters or prostheses . Hence, the differences in design parameters or prostheses are not distinct enough to have an effect on clinical outcome of patients.
Wilson's disease is a rare autosomal recessive inherited disorder of copper metabolism causing excessive copper accumulation in the liver, brain, cornea and several other organs in the body, requiring specific treatment to remove or detoxify tissue copper and to prevent reaccumulation (1). Most patients with wilson's disease present with acute or chronic liver conditions, while some present with major extrahepatic manifestations such as neurological disturbances, hematologic abnormalities, and psychiatric disorders . Others may show relatively rare conditions such as renal diseases, arthritis, cardiomyopathy, pancreatitis, and endocrine manifestations including hypoparathyroidism, abnormal menstruation, infertility, and hypoglycemia (12). Hypopituitarism has been reported sporadically in wilson's disease, but cases we found clinically presented only as hypogonadotrophic hypogonadism or menstrual abnormalities . Here we report a case of a woman diagnosed with wilson's disease initially presenting with psychiatric symptoms, also accompanied by hypopituitarism in the form of hypothyroidism and adrenal insufficiency . A 40-year - old woman visited the department of psychiatry at our hospital on march 17, 2014 with depressive mood, general weakness, and loss of appetite . Her symptoms worsened over the past year as she refused to talk or eat . At admission, vital signs were stable and laboratory tests revealed the following results: hemoglobin 9.9 g / dl, hematocrit 30.5%, white blood cells 2,180/mm, platelets 115,000/l, total protein 5.4 g / dl, albumin 3.1 g / dl, bun / creatinine 10.5/0.6 mg / dl, total bilirubin 1.1 mg / dl, direct bilirubin 0.47 mg / dl, ast / alt 91/29 iu / l, prothrombin time 13.3 seconds (inr 1.22). An abdominal ultrasound was performed for further evaluation of low hemoglobin, thrombocytopenia and abnormal liver function test results, which showed liver cirrhosis and splenomegaly . As the patient had no history of alcohol consumption or recent use of hepatotoxic drugs, additional blood and urine tests were done to distinguish the cause of liver cirrhosis . The hepatitis b surface antigen, antibody, hepatitis c antibody, hiv and vdrl were all negative . Serum antinuclear antibody, anti - smooth muscle antibody, lkm-1 antibody and anti - mitochondrial antibody were all negative and igg was 824 mg / dl being in the normal range . Serum copper was decreased to 28.88 g / dl (normal range: 90 - 130 g / dl) and serum ceruloplasmin to less than 8 mg / dl (normal range: 20 - 40 mg / dl). The 24-hour urinary copper excretion was increased to 78.21 g / day (normal range: <40 g / day). Slit - lamp examination revealed a kayser - flesicher ring, a band of golden pigment around the cornea which led to the final diagnosis of wilson's disease (fig . Copper deposits in the descemet's membrane of the cornea (kayser - fleischer ring). Basal hormone levels were checked in order to exclude other causes of depression, appetite loss, and general weakness . Lh, fsh, estradiol, testosterone, igf-1, and prolactin were all within normal range while thyroid function test results showed tsh 0.28 iu / ml, ft4 0.72 ng / dl, and t3 50.85 ng / dl, all being below the lower limit . Acth and cortisol were checked at 8 am and 4 pm, being decreased to 1.00/1.72 g / dl and 1.00/2.84 g / dl, respectively . The thyrotropin releasing hormone (trh) stimulation test and insulin tolerance test tsh levels showed only a marginal increase from 0.227 iu / ml to 2.816 iu / ml which was an insignificant response to trh (table 1), indicating central hypothyroidism and not non - thyroidal illness syndrome . Acth remained as 1.00 pg / ml after provoking hypoglycemia with insulin injection while cortisol merely rose from 1.77 g / ml to 1.66, and gh from 1.39 mg / dl to 9.78 mg / dl (table 2). Acth, adrenocorticotropic hormone; gh, growth hormone; lh, luteinizing hormone; fsh, follicle stimulating hormone; igf-1, insulin - like growth factor 1; tsh, thyroid stimulating hormone . Baseline samples were taken after 8 hours of fasting; luteal phase . After being admitted, the patient showed trouble swallowing food and other neurological symptoms that could not be explained by depression such as rigidity, tremor and bradykinesia . Brain mri was done as an additional test for these symptoms as well as to determine the presence of any pituitary tumors . T2-weighted images showed high signal intensity in both basal ganglia and in the midbrain, whereas no abnormality was found in the pituitary (fig . Bilateral symmetric increased signals of the basal ganglia on t2 weighted mr image (a) while no focal lesion was found in the pituitary gland (b). Oral d - penicillamine (1.0 g / day) was given for copper chelation with pyridoxine (50 mg / day), while thyroid hormone and steroid replacement with levothyroxine (50 g / day) and prednisolone (7.5 mg / day) were additionally used . Increase in 24-hour urinary copper excretion was monitored which reached 922.8 g / day after initiating treatment and was maintained in the range of 200500 g / day after 6 months . The patient's general weakness, appetite loss and depressive mood improved as well . She is currently receiving treatment with the same regimen and has had no side effects from medication or other newly developed symptoms for the last 7 months . Hypopituitarism refers to decreased secretion of pituitary hormones as a result of abnormalities in the pituitary itself or the hypothalamus (3). It usually occurs due to a pituitary tumor or as a consequence of its treatment, and symptoms vary depending on which hormone is deficient (3). Other causes include pituitary apoplexy, sheehan's syndrome, stroke, traumatic brain injury, cerebral hemorrhage and infiltrative diseases such as sarcoidosis or hemochromatosis which are relatively rare and are often wrongfully labeled as idiopathic since imaging is usually normal (34). In our case, hormone studies were consistent with secondary adrenal insufficiency and hypothyroidism due to hypopituitarism which we concluded to be associated with wilson's disease, since no other definite cause or coexisting disease was found . The patient had no headaches, visual field defects or disturbances and brain mri was negative of any pituitary lesion . Among infiltrative diseases, hemochromatosis is known to cause pituitary dysfunction by depositing iron in the anterior pituitary, especially as hypogonadotrophic hypogonadism probably due to the preference for iron of gonadotrophic cells (3). As for our case, we suggest copper deposition or secondary neuronal damage in the pituitary to explain the association between hypopituitarism and wilson's disease, but the mechanism could not be proven in this report . Recent studies showed that wilson's disease may be complicated by high iron concentrations in the brain as low ceruloplasmin levels and reduced ferroxidase activity lead to iron overload along with copper in excessive amounts, causing oxidative damage in a synergistic cascade (5). Proposed dopamine - dependent neurotoxicity as another possible mechanism, decreasing survival of dopaminergic cells in the brain and contributing to the regional differences in copper accumulation or its consequent neuronal damage . In another report, a biomolecular basis of copper toxicity was presented, demonstrating that both copper and iron induce genome damage and inhibit its repair process (7). Whether these suggestions could be applied to the pituitary in wilson's disease t2 hyperintense signals in the basal ganglia is the most common finding on the brain mri in wilson's disease, but interpretation of this lesion is ambivalent since copper deposition may be undetectable without the summation of underlying iron accumulation . Secondary neuronal damage might occur without direct local copper accumulation (8). Also, it is not uncommon for pituitary imaging to be normal in hypopituitarism (4). Despite normal pituitary imaging in our case, we could not rule out the possibility of copper infiltration as the cause for hypopituitarism in wilson's disease . Symptoms such as delayed puberty or amenorrhea were sporadically reported, as well as glucose intolerance and parathyroid function disorders (910). (1) reported galactorrhea and menstrual abnormalities in a patient with wilson's disease, presuming that the symptoms may have been caused by local deposits of copper in the pituitary and mammary glands . (10) studied 16 patients with wilson's disease to detect potential endocrine dysfunctions . In their report, most patients had low or borderline lh levels and a dynamic gnrh test revealed blunted lh and fsh responses . Also regarding a possible association between pituitary dysfunction and wilson's disease, a case of delayed puberty in an 18-year - old male with wilson's disease was reported, suggesting copper accumulation as the potential cause of decreased synthesis or secretion of gnrh in the hypothalamus or pituitary . Like in our study, the brain mri was negative of any pituitary lesion (11)., our study is an unprecedented case of wilson's disease coexisting with hypopituitarism presenting as tsh or acth deficiency in korea, as other pituitary hormones were maintained within the normal range . Meanwhile, common psychiatric symptoms in wilson's disease are depression, incongruous behavior, and cognitive impairment (12). It may be challenging to differentiate these symptoms from hypothyroidism or adrenal insufficiency which can be easily misdiagnosed or neglected if not examined for other potential conditions (13). This study highlights the importance of screening for hormone deficiency in wilson's disease especially in patients presenting with psychiatric symptoms . We also suggest considering wilson's disease when evaluating possible causes for idiopathic hypopituitarism . Regardless of whether the two conditions are causal or coincidental, further confirmation is needed on the endpoint of hormone therapy and on whether or not copper - chelating treatment alone brings pituitary function recovery, as pituitary dysfunction was successfully reversed by liver transplantation or therapeutic phlebotomy in some cases of hemochromatosis . Also, it would be worthwhile to reevaluate the pituitary function in 6 to 12 months after discontinuing hormone replacement . In conclusion, this case indicates a possible link between wilson's disease and hypopituitarism presenting as altered thyroid homeostasis and pituitary - adrenal axis dysfunction.
Integral membrane proteins, such as ion channels, receptors, and transporters, perform their vital tasks through complex mechanisms that often involve major structural rearrangements triggered by stimuli such as ligand or substrate binding . The molecular response to the stimuli is propagated across the membrane, connecting the extracellular environment to the interior of the cell . That the membrane environment is involved in the allosteric mechanisms and their regulation is now well - established, but the mechanisms by which this environment participate in the observable and measurable activities of the embedded proteins are yielding only slowly to quantitative understanding . In particular, it has become evident that in order to carry out their tasks membrane proteins take advantage of many structural, thermodynamic, and mechanistic properties of the cell plasma membranes and that in turn the lipid membranes respond dynamically to conformational changes in proteins by locally adjusting their lipid composition, bilayer thickness, and/or curvature . Many consequences of such function - dependent cross - talk between proteins and lipids have been identified, including compartmentalization and oligomerization (often enhanced by specific plasma membrane domains termed rafts), which play central roles in the physiological mechanisms of the cell . One difficulty in connecting the physiological observations to the rapidly growing information about the detailed structure and dynamic properties of individual membrane protein molecules is that most structural and many functional assays are conducted in non - native environments in which the role of the plasma membrane and its components (i.e., cholesterol and charged lipids) in modulating structural and functional properties of the integral proteins (e.g., ref (15)) are not accounted for . Thus, most protein preparation methods for studies of integral membrane proteins involve their overexpression, followed by their detergent - mediated extraction from the cell and purification and reconstitution into proteoliposomes . When present in water above certain critical concentrations, the detergents, which have been selected to replace the native environment of these proteins (i.e. The membrane) and possess a relatively large polar headgroup and a short hydrophobic tail, self - assemble in aggregates of positive curvature, termed micelles . In such micelles, the polar headgroups of the detergent are exposed to the aqueous solvent whereas hydrophobic tails face each other and thus mimic the membrane hydrophobic environment for an encapsulated protein while excluding aqueous solvent from the micelle interior . Although micellar aggregates effectively shield hydrophobic trans - membrane (tm) segments of integral proteins from unfavorable polar exposure while bringing polar loop regions into contact with an aqueous phase, they cannot capture all of the complexities of the native plasma membranes . Indeed, several recent studies have provided dramatic demonstrations of changes in functional properties of membrane proteins due to detergent (e.g., refs (18) and (19)). One such class of membrane proteins studied in detergent environments is the family of neurotransmitter / sodium symporters (nss) that is responsible for the removal of extracellular solutes from the synaptic cleft into the presynaptic nerve terminal . The uptake mechanism is energized by the coupling the transmembrane na gradient to the uphill transport of the respective substrate . Several x - ray structures of a prokaryotic homologue of nss, the leucine transporter (leut), have identified the centrally located high - affinity substrate binding site, termed the s1 site, and two sodium binding sites termed na1 and na2 . Computational and experimental studies have identified a second high - affinity substrate binding site, the s2 site, located in an extracellular vestibule 11 above the s1 site that is essential for the transport mechanisms of leut . In particular, an allosteric mechanistic model of a na - coupled symport was proposed in which intracellular release of the s1-bound substrate is triggered by the binding of a second substrate molecule in the s2 site . Remarkably, follow - up studies suggested that experimental conditions and, in particular, a high concentration of n - dodecyl-,d - maltopyranoside (ddm) detergent, can obscure the functionally relevant s2 site and result in reduced substrate binding . But the reconstitution of leut, previously preincubated with high concentrations of ddm detergent, into e. coli membranes, showed a full recovery of functionality for the s2 site . Interestingly, the loss of the s2 site upon detergent treatment appeared to have a concentration threshold in the range of 0.150.175% ddm . These experimental results illustrate that the function of integral membrane proteins can be modulated by the experimental preparation and the environment in which the protein is studied . To extrapolate the knowledge gained from the experimental explorations conducted under non - native conditions to mechanisms in vivo, it is necessary to understand on the molecular level how detergent micelles form around proteins and what fundamental changes occur when the protein is surrounded by a detergent micelle . The literature discussing molecular models of detergent / protein interactions (e.g., refs (3039) and citations therein) has not addressed these fundamental questions in a systematic way . To point out the shortcomings associated with the interpretation of membrane protein structure and function in experimental environments, we provide here, to our knowledge for the first time, a detailed molecular view of the leut protein embedded in ddm detergent micelles formed at different detergent / water / protein ratios . This view is offered from extensive atomistic molecular dynamics (md) simulations carried out in order to (1) establish the aggregation number of ddm micelles surrounding leut, (2) explore the overall organization of the detergent micelle containing the transporter, and (3) obtain molecular - level insight into the nature and consequences of interactions between leut and ddm . Analyzing various protein - to - detergent (p / d) number ratios (i.e., from 1:160 to 1:300), we show that the aggregation number of ddm in the micelle that surrounds the transporter is strongly dependent on the p / d ratio . Moreover, the md simulations of the system at various p / d ratios suggest a mechanism for the dependence of leut substrate binding stoichiometry on detergent concentration . Thus, we found that the detergent can penetrate leut through two alternative pathways . As a consequence of such penetration, ddm molecules establish long - lasting contacts with several functionally critical residues located in the s2 site of leut . Remarkably, we find that the detergent penetration phenotype is determined by the aggregation number of ddm around leut so that nontransient ddm insertion is observed only in the high - detergent - concentration regime . These results, discussed here in the light of recent experimental findings suggesting the modulation of leut activity by detergent, can explain experimentally observed phenotypes caused by the occlusion of the s2 site in leut at high detergent concentration . For atomistic molecular dynamics (md) simulations, we used the x - ray structure of leut with the pdb accession code 3gjd . The transporter in this structure is in the occluded state with leucine (leu) at the s1 primary binding site and the two na ions bound at na1 and na2 sites, respectively . Thus, the structure also contains detergent n - octyl-,d - glucopyranoside (og) at the s2 binding site . The detergent molecule was removed prior to the simulations, leaving the s2 site empty at the beginning of the md runs . The leut residues that were missing from the 3gjd structure (first four residues on n terminus, last eight residues on c - terminus, and the p132n133a134 stretch in the el2 loop) were added with modeler . All crystallographic waters were retained in the simulations, and glu112, glu287, and glu419 were treated as protonated . The leut model was immersed in a box containing water and ddm detergent molecules . As described in figure 1 (starting configurations) and illustrated in figure 2, some ddm molecules were initially placed in a spherical micelle formation around leut (see below) whereas others were placed randomly as monomers outside this central micelle . The starting conditions were varied with respect to the number of ddms in the initial micelle and in the monomers . Thus, for the initial set of simulations, the micelle was composed of either 160 or 246 ddm molecules and was surrounded by different numbers of monomeric ddms (figure 1). Accordingly, throughout this work, various simulated constructs are given the designation a / b, where a denotes the initial number of ddms in the central micelle surrounding leut (figure 2) and b is the starting number of monomeric detergent molecules outside this micelle . Schematic representation of conditions probed in our all - atom md simulations of leut / detergent complexes: protein - to - detergent number ratios and initial spatial distribution of detergent around leut . The first stage of simulations (starting configurations) involved leut surrounded by a ddm micelle consisting of either 160 or 246 detergent molecules . In addition, different numbers of monomeric ddms (0, 54, or 115) were placed randomly outside the central protein / detergent micelle (figure 2a, b). Note that conditions with 160 total ddms were probed in two separate md simulations initiated from different random seeds . Because all starting configuration simulations resulted in similar numbers of shell ddms (detergent molecules within 4 of a protein, termed shell in methods), we initiated a second set of simulations (resulting configurations) in which leut with only its shell of detergents (120 ddms 4 from the protein) was retained (chosen from the representative snapshot from starting configuration trajectories). This protein / detergent complex was then surrounded by randomly placed monomeric ddms (41, 115, 174 detergents), and new md trajectories were accumulated . The time durations for each simulation conducted are given in the respective boxes . (a) snapshot of the initial configuration of the 160/115 system (figure 1, starting configurations). The cubic simulation unit box of 180 linear length contains leut protein (in cartoon), ddm detergent molecules (in licorice, 160 ddms depicted in gold are in the micelle surrounding leut and 115 ddms in white are in monomeric form outside), a water box (silver dots represent water oxygen atoms), and 0.15 m nacl (yellow and cyan spheres). The leucine ligand bound to the leut s1 site and the two na ions are omitted . (b) same as in panel a only with water and salt ions removed . (c) same as in panel b, only after 140 ns of md simulations . Different types of aggregates (definitions in methods) are highlighted with arrows and labeled . (d) final snapshot of the 160/115 system, after 242 ns of simulations, showing only leut (cartoon) and shell ddm molecules (within 4 of protein) (licorice). Notice strong intermixing of initially micellar (gold) and monomeric (white) ddms in panels c and d. to build a micelle containing a number a of detergent molecules around leut, we used a multistep algorithm described in ref (37). According to this procedure, in step 1 n pseudoparticles were randomly placed on an imaginary sphere surrounding the protein, excluding areas around intracellular and extracellular parts of leut (figure 2); in step 2, the pseudoparticles were replaced with explicit ddm molecules, oriented with their hydrophobic tails facing the center of leut; and in step 3, the imaginary sphere (containing leut and all of the ddm molecules) was incrementally shrunk subject to concomitant energy minimization to a final radius of 51 . With that, we ensured that in the starting configuration ddm tails were appropriately placed to cover the hydrophobic core of leut while leaving hydrophilic regions of the protein exposed to the solvent . To the box containing the leut - detergent micelle complex (proteomicelle) obtained with the procedure described above, we added the desired number of monomeric ddms (figures 1 and 2) positioned randomly outside the central micelle, and the system was then solvated with tip3 waters and ionized with na and cl to achieve an ionic concentration of 0.15 m. the final simulation box had nearly cubic geometry with a linear dimension of 180 and contained 500 000 atoms . Correspondingly, the detergent concentration was kept above the established critical micelle concentration (cmc) of 0.17 mm for ddm in all of the constructs (table s1 in the supporting information). The all - atom md simulations were done with the namd 2.7 package, and the all - atom charmm27 force field, with cmap corrections for proteins and a charmm - compatible force - field parameter set for detergents . Molecular constructs were initially equilibrated using a two - phase protocol: (i) short energy minimization was carried out during which protein, water, and ion atoms were fixed and the coordinates of only ddm molecules were allowed to evolve freely and (ii) 1.5-ns - long md simulations were conducted with the protein backbone harmonically constrained . The constraints were released gradually, in 0.5 ns steps, with decreasing force constants of 1, 0.5, and 0.01 kcal/(mol). The equilibration procedure was similar to that implemented by others for md simulations of protein / micelle complexes (e.g., ref (38)). The simulations implemented pme for electrostatics interactions and were carried out in an npt ensemble under isotropic pressure coupling conditions and at 310 k temperature . The nose - hoover langevin piston algorithm was used to control the target p = 1 atm pressure with the langevin piston period set to 100 fs and the langevin piston decay set to 50 fs . As detailed in the results, the md simulations led to the spontaneous formation of aggregates of different types (figure 2c). Accordingly, we distinguished ddm detergents in the following types of aggregates: detergent micelles (dms) ddms that are bound to the central micelle as monomers;free aggregates (fas) ddms that are monomeric in the solution.ddm shell (shell) ddms within 4 from leut . (see figure s1 in the supporting information for a comparison with the results using alternative definitions of the shell .) Ddms that are in the central micelle around leut; bound aggregates (bas) ddms in aggregates that bind to the central micelle; bound monomers (bms) ddms that are bound to the central micelle as monomers; free aggregates (fas) ddms that are part of aggregates in the solution; free monomers (fm) (see figure s1 in the supporting information for a comparison with the results using alternative definitions of the shell .) To identify the number of constituent detergent molecules in each type of aggregate defined above, we used an expansion algorithm . Thus, to calculate the number of ddms in dm, we first identified detergent molecules within 4 of leut (ddm shell, figure 2d). Next, we found all ddm molecules within 4 of this detergent shell, and such an expansion was repeated until the search failed to identify any new ddm molecules . In this procedure, only the atoms in the hydrophobic tails of detergent molecules were used to differentiate between ddms in the protein - surrounding micelle from those that are interacting with this micelle via headgroup atoms . After dm was defined, ddms in bas and bms were counted by selecting detergent molecules whose headgroup atoms were within 5 of the dm and building complexes using the expansion algorithm described above . After all bound aggregates were located, the algorithm identified the ddms in fas and fms by building the remaining aggregates in solution . Note that all types of aggregates defined above can, in principle, contain ddm molecules that were initially either part of the central micelle or were monomeric outside the micelle (figure 2b d). The micellar aggregation number is a description of the number of molecules present in a micelle once the cmc has been reached and is defined as the ratio of micelle concentration over the concentration of monomeric detergent . Here, the aggregation number of the leut / ddm proteomicelle was calculated as the dm + ba + bm sum . (see above .) To quantify the shape of the micelle around leut, we calculated the eccentricity e of the dm following the procedure implemented in ref (34). To this end, we determined the three principal moments of inertia (i) of an ellipsoid that encloses the non - hydrogen tail atoms of those ddm molecules that were identified as being part of the dm . Using the magnitudes of the smallest principal moment (imin) and of the average of all three moments (iav), we then obtained the eccentricity of the micelle as e=1 note that according to this definition, e = 0 for a perfectly spherical micelle . To quantify the extent of detergent penetration into leut in our md simulations, we monitored the time evolution of selected distance measures from different trajectories . Specifically, because we observed that ddm is inserted into the transporter and binds in the s2 site (see results), we first screened the residues that have been established from experimental and computational studies to comprise the s2 site in leut (see also discussion): leu25, gly26, leu29, arg30, gln34, tyr107, tyr108, ile111, w114, ala319, phe320, phe324, leu400, and asp404 . We then tracked the minimal distance from these residues to the nearest detergent molecule in different simulations . The penetration of the detergent molecule was observed in different simulations to follow two distinct pathways (see results), one resulting ultimately in interactions with arg30 and gln34 in transmembrane helix 1 (tmh1) of leut and the other resulting in interactions with phe320 in extracellular loop 4 (ecl4) and leu400 in tmh10 . Therefore, we chose to quantify ddm insertion by monitoring the time evolution of the minimal distance (dmin) of these four key residues arg30, gln34, phe320, and leu400to the nearest ddm molecule . In a certain md trajectory, therefore, a detergent molecule was considered to be fully inserted (complete insertion) into the leut s2 site if dmin between the detergent and any of the above - mentioned four residues was 3 or shorter during at least the last third of the trajectory . If the detergent molecule interacted with either arg30, gln34, phe320, or leu400 (dmin <3) for a shorter period of time, it was considered to be transiently inserted into the s2 site . In this scenario, the interactions between the detergent and s2 site residues were forming and breaking dynamically (e.g., figure 5). Finally, if dmin> 5 at all times during the trajectory, then the leut molecule was not considered to be penetrated by ddm . To determine the behavior of the ddm molecule number in a micellar system surrounding leut, we conducted md studies on a series of leut - ddm complexes constructed to explore systematically the effect of protein - to - detergent (p / d) number ratios on the nature and dynamics of the resulting complexes . The md simulations of the systems containing from 1:160 (low detergent content) to 1:300 (high detergent content) p / d ratios, starting from the constructs described as starting configurations in figure 1 in which various numbers of monomeric ddm molecules were added randomly outside the protein / micelle complex (figures 1 and 2a, b). The various constructs converged (at the simulation times indicated) to configurations with very similar ddm shells (defined in methods as the shell) surrounding the protein . Specifically, as shown in figure 3, we found that in all of the simulations the number of detergent molecules in the shell was 120 . We note that the convergence of the ddm numbers in the shell to similar values in the different simulations is independent of the range explored specifically, from 3 to 5 (figure s1 in the supporting information). Time evolution (after initial equilibration phase) of the number of ddm molecules within 4 of the protein (i.e., shell) in different simulations . The traces were smoothed by the running average algorithm . To investigate further the stability of the identified shell, a second set of md simulations the starting constructs were obtained by extracting leut with its shell ddm molecules (120 detergents) from a representative snapshot from the starting configurations trajectories and surrounding this complex with different numbers of monomeric ddms . Md trajectories that were 200350 ns long were collected in the environment defined in methods . As illustrated in figure 3, this second set of md simulations also converged to configurations in which the shell surrounding leut contained 120 ddm molecules . These results suggest that irrespective of the p / d ratio a converged shell of ddms forms around leut . Interestingly, however, quantitative analysis of various types of detergent aggregates forming in the md trajectories revealed that the detergent aggregation number, i.e. The number of ddm molecules comprising the entire micelle enclosing leut (denoted as dm in methods) strongly depends on the p / d ratio . Figure 4a shows the average number of ddm molecules in the entire micelle surrounding leut (dm) as well as in aggregates bound to dm (bmand ba) and in the entire complex of detergent associated with the central protein / micelle complex (i.e., dm, bm, and ba together, which is the proteomicelle aggregation number). The averages were obtained from the analysis of the last 50 ns of each trajectory . The time trace in figure 4b illustrates the evolution of these aggregates from one particular 160/115 simulation, and figure 4c shows, from the same simulation, the evolution of ddm numbers in the free aggregates compared to that in the central protein / micelle . (analogous plots for other constructs can be found in figures s2s3 in the supporting information .) (a) average number of ddm molecules in the detergent micelle around leut (dm), in the aggregates bound to dm (ba and bm), and in dm, ba, and bm taken together . For each simulated system, the averages were calculated using the last 50 ns segments of the respective trajectories . The dashed horizontal line indicates the average number of ddms (120) in the detergent shell around leut established from figure 3 . (b) time evolution (after initial equilibration phase) of the measures from panel a in the 160/115 simulation . (c) percentage of ddm detergents in dm, ba, and bm (blue line), in fa (red line), and in fm (black line) as a function of time in the 160/115 simulation . For definitions of the various types of aggregates, the results in figure 4c show that over 80% of 275 ddms present in the 160/115 system become part of the central micelle (blue line in figure 4c). The majority of the detergent that is still in the solution assembles spontaneously into free aggregates (fas, red line in figure 4c), whereas free monomers (fms) practically disappear (black line in figure 4c). The interconversion of the different bound aggregates (ba and bm) and their melting into the central dm that surrounds leut are illustrated in figure 4b . Thus, within the first 50 ns, the number of ddms in dm increases from 160 to 190 and remains at this level until the 100 ns time point (red trace in figure 4b). At the same time, the number of aggregates bound to dm remains more or less unchanged (green trace in figure 4b). Interestingly, in the subsequent 100150 ns time interval we observe a sudden increase in the number of detergent molecules in dm as a result of the fusion of the bound aggregates with the central micelle (green curve in figure 4b decreases to 0). After the fusion process is completed (150 ns), the detergent count in dm and in dm + ba + bm remains nearly identical for the remainder of the trajectory (red and blue traces in figure 4b), indicating that practically all bound aggregates (over 80% of the ddm, see figure 4c, blue curve) become part of the dm . Although the size of the ddm shell appears to be 120 ddm (as calculated at a 4 distance cutoff) regardless of the p / d ratio, we found the aggregation number to change significantly in the p / d interval of 1:246 to 1:300 interval, from 2045 (in the 120/174 simulation) to 2437 (for the 246/54 system), as seen in figure 4a . Moreover, these values are substantially higher than the 1453 to 1562 aggregation number range that we find for the three different simulations under low detergent condition (1:160 p / d ratio). Indeed, our results suggest that for the low ddm constructs all of the available detergent eventually aggregates with the central dm around the protein . Interestingly, at an intermediate p / d ratio of 1:235, the aggregation number (1865) is in between these two regimes . We note that the extensive simulation times that we have reached in the current studies still may not be sufficient for complete equilibration of the dynamic variables discussed in figure 4a (figures 4b, c and s2s5 in the supporting information). However, the examination of a number of control trajectories for the high detergent regime (figure 1 flowchart) allowed us to assess with confidence the convergence of the aggregation numbers from below (120/174, 120/115, and 160/115 simulations) as well as from above (246/0 and 246/54 simulations). On the basis of the average values from these simulations initiated from different starting conditions (figure 4a), we therefore conclude that for the high detergent regime the ddm aggregation number around leut is 22617 . In the low ddm regime, such as the p / d ratio of 1:160, our results suggest that all available detergents will constitute the dm . In the course of simulations with some of the constructs, we observed ddm molecules inserting into leut . However, when a stable complex was formed, the single ddm molecule penetrated the leut molecule until it ended up interacting with residues in the identified such complete ddm penetration occurred only in the constructs with relatively high detergent content, i.e. For p / d ratios in the 1:246 to 1:300 range (table 1). In contrast, only transient ddm insertion into leut was observed in simulations of systems with low (1:160 p / d ratio) or intermediate (1:235 p / d ratio) detergent content . We identified two alternative pathways for ddm insertion into leut, one termed from the side penetration and the other termed from the top penetration (table 1). For the definition of different modes of detergent penetration, see results . Different simulations are labeled as a / b, where a and b represent the initial number of ddm molecules in the micelle and in solution, respectively . Penetration was observed in several of the md simulations (table 1), and the path is illustrated in figure 5 for the 160/115 construct . The ddm molecule enters the transporter through an area between the extracellular (ec) ends of tmh11 and tmh6 . The first contacts established by the ddm molecule involve residues pro241, gly242, and ile245 in tmh6, trp467, and val466 in tmh11 (figure s4 in the supporting information). As it penetrates deeper into the transporter, the detergent molecule engages in additional strong interactions with phe405 in tmh10 (figure s3) and eventually with gln34 and arg30 in tmh1 (figure 5c, d). The participation of the latter two residues is especially intriguing because both gln34 and arg30 are situated in the functionally important s2 site of leut . As seen in figure 5a, b, the ddm is stabilized in the s2 site through both hydrophobic and polar interactions as the ddm headgroup engages with the arg30-gln34 pair whereas the tail interacts with hydrophobic residues lining the ec ends of tmh6 and tmh11 . Notably, this entry pathway and the identity of residues participating in interactions with the detergent were found to be remarkably consistent among these different simulations, although the extent of the detergent penetration in these trajectories (figure 5c, d and also methods for insertion criteria) ranged from completely inserted (in the 160/115 and 246/54 systems) to transiently bound (120/115, 246/0, 120/174, and 160/0 ii simulations). From the side entry pathway of a ddm molecule into leut . (a, b) representative snapshot from the 160/115 simulation showing ddm detergent tmh6 and tmh11 of the transporter are colored blue and red, respectively, and the key residues are shown in a space - filled representation and are labeled . (c, d) time traces of the minimal distance from the inserted detergent molecule to arg30 (panel d) and to gln34 (panel c) in 246/54, 160/115, and 120/115 trajectories . Penetration from the top of the transporter was observed in the 246/54, 120/174, and 160/0 i simulations (table 1). The initial contact was observed to involve the phe235-asp240 stretch of ec loop 3 (ecl3), and the insertion proceeded toward the gly307-ala317 segment in ecl4 (figure s5 in the supporting information). As the ddm molecule penetrated with its headgroup deeper into the transporter, the final set of stabilizing interactions included phe320 in ecl4 and leu400 in tmh10 (figure 6). The participation of these particular residues in interactions with the inserted detergent is important because they are again, as in the from the side penetration, situated in the functionally important s2 site and leu400 has been directly linked to the functional mechanisms of substrate transport through leut . From the top entry pathway of ddm molecule into leut . (a, b) representative snapshots from the 246/54 (a) and the 246/0 (b) simulations showing ddm detergent (in licorice) penetrating leut (in cartoon). Different segments of the transporter are highlighted as follows: tmh10 (blue), 235240 stretch in ecl3 (pink), 307316 in ecl4 (red), and 317336 (yellow). Glu236 and ile314 are shown by the white space fill, and phe320 and leu400 are depicted by the orange space fill . (c, d) time traces of the minimal distance from the inserted detergent molecule to phe320 (panel c) and to leu400 (panel d) in 246/0, 246/54, and 120/174 simulations . An interesting variation on the mode of binding is observed in the 246/0 trajectory where the inserted detergent molecule is engaged in stabilizing interactions with the same residues as identified in the simulations of the 246/54, 120/174, and 160/0 i constructs (figure 6c, d), but it is the hydrocarbon tail that interacts with phe320 and leu400 residues rather than its headgroup atoms (cf . The convergence of penetration paths and stabilization sites from the various simulations with different constructs indicates the robust nature of these conclusions . Moreover, we note that in two trajectories (246/54 and 120/174) in which the detergent entered the protein from the top, the specific ddm molecule that eventually ended in the s2 site of leut came from outside the protein - surrounding micelle, from among those in the monomeric form (data not shown). Similarly, the ddm molecule that entered s2 site from the side in the 120/115 simulation (figure 5c, d) inserted itself into the protein after diffusing toward leut from the solution . These trends suggest that the md simulations were sufficiently long to allow ddm molecules to diffuse over relatively long distances before reaching the binding sites in the transporter . The dependence of specific measurable properties of leut on the quantitative parameters of the micellar system offers the first detailed molecular perspective on the manner in which detergent solubilization can affect functional phenotypes of this type of membrane protein . The insights result from extensive atomistic md simulations of different regimes of protein - to - detergent number ratios that are commonly used to prepare protein for biochemical / biophysical studies and crystallography . This study followed our recent work that highlighted the effect of the experimental conditions on the activity of leut, in particular, how high ddm concentrations can obscure substrate binding to the functionally important s2 site . Taken together, our studies indicate that ddm at high concentrations can occupy the s2 site in leut, just like og . These studies emphasize the sensitivity of the s2 site, a site that is yet to be identified with crystallographic approaches in a substrate - occupied state . Because the crystallization of membrane proteins requires high protein concentrations that are routinely obtained with centrifugal filtration of purified material in protein detergent mixtures, our studies emphasize a common problem that is associated with the use of protein in detergent - solubilized form . A key observation of the present study is the formation of a converged shell around leut established irrespective of detergent concentration (within the wide range explored in our studies). This nucleus of detergent molecules effectively protects the transporter tm segments from unfavorable hydrophobic / hydrophilic exposure and therefore is key to the stability of the protein . Indeed, in control simulations initiated with a nucleus of 100 ddm molecules surrounding leut, the system exhibited instabilities during the trajectory, which resulted in substantial water penetration of the hydrophobic core of the transporter (data not shown). Taken together, our findings establish the importance of considering ddm - to - protein number ratios at or above 120 in md simulations of leut proteomicelles (i.e., the number required for the formation of a converged shell at 4 radius). It is not surprising given the shape of the protein that our simulations show that the proteomicelles forming around leut are nonspherical in shape (figure s6 in the supporting information). The aggregation number of ddm in the micelle that surrounds the transporter depends strongly on the p / d ratio (figure 4a). Specifically, in the high ddm concentration regime we identified 22617 detergent molecules in the protein - solubilizing micelle, which is significantly larger than the usual aggregation number for ddm in micelles (140); for low detergent content (p / d ratio of 1:160), our results indicate the aggregation of all of the available detergent into the leut - binding micelle . We note that the calculated proteomicelle aggregation number (22617) is not dependent on the simulation box size because there always will exist, irrespective of the available volume, partially formed detergent micelles in the solution together with protemicelles, as observed in our simulations . Remarkably, the penetration of a ddm molecule that inserts fully into the transporter, whether it follows the from the top or from the side path described in the results of the md simulations, is found to occur only in the constructs with high detergent content (p / d ratio range of 1:246 to 1:300); any penetration observed in systems with low or intermediate ddm fraction is at most transient . Because the shell surrounding leut does not depend on the detergent concentration and it is the ddm aggregation number for the system (and thus the protein - to - detergent number ratio) that determines penetration, it becomes clear that ddm insertion into leut is not simply related to the interaction with the detergent immediately surrounding the transporter . Specifically, we reason that at low detergent concentrations (at or below 22617 aggregation number established by our studies) all of the ddm molecules in the system are expected to participate in the stabilization of the proteomicelle . But at higher concentrations, excess ddms, not associated with the proteomicelle, will be present in the solution and will diffuse freely in monomeric and/or in aggregate forms . Such dynamics of free detergent increases the probability of random encounters with the protein regions exposed to the solvent and specifically with the large extracellular vestibule of leut, which eventually leads to the observed detergent penetration . The final position of any completely penetrating ddm was found to be the s2 site in the extracellular vestibule of leut and to involve arg30, gln34, phe320, and leu400 in strong interactions with the inserted ddm . Specifically, in crystal structures of leut these residues are among those implicated in stabilizing interactions with tricyclic antidepressants (tcas) in the extracellular vestibule as well as with og detergent and to bind inhibitors (such as trp). Furthermore, highly conserved ionic interactions between arg30 and asp404 in tmh10 have been established as one of the structural / functional hallmarks in nss family proteins that regulate the access of the substrate from the ec vestibule down to the s1 site during the transport cycle . In addition, the importance of leu400 residue in the leut function is highlighted by the phenotypes of leu400-to - ser or leu400-to - cys mutations that impair leu substrate binding in the s2 site . Notably, detergent penetration events observed in the simulations occur at p / d ratios that are expected to be realized in the local environments of the solubilized leut proteins during in vitro experiments . Thus, the results from the simulations presented here correspond to the experimental observation that the binding of the leu substrate at the s2 site is impaired if leut is preincubated in the presence of high ddm (0.3%), yielding a protein - to - detergent ratio of 1:225, but this does not occur at low ddm concentrations (0.1%, with a protein - to - detergent ratio of 1:170). A detailed scan through the detergent concentration range revealed that the loss of leu binding occurs abruptly in the interval between 0.15 and 0.175% ddm concentration . In qualitative agreement with these experimental observations, our data indicate the existence of two distinct regimes for ddm concentration, separated by a narrow range of p / d ratios (from 1:235 to 1:246), that determine detergent penetration . This suggests a mechanistic explanation for the experimentally observed impairment of the s2 site under high detergent conditions that involves the steric hindrance of the s2 site by a penetrating ddm molecule, much like the proposed effect of og on leut when bound in the s2 site . In the low detergent concentration regime, our results suggest that ddm molecules penetrate leut only transiently, therefore leaving the s2 site more accessible for substrate binding . Importantly, we stress that because even at the lowest p / d ratios studied here (1:160) the detergent can still penetrate leut (albeit transiently) and given the narrow p / d ratio range that determines the extent of detergent insertion, the data and hypothesis presented above provide a plausible explanation for the findings from the recent functional experiments on leut that were interpreted to suggest the existence of only a single high - affinity s1 site even at low detergent concentrations . Furthermore, the results presented here illustrate, on the molecular level, how even small variations in protein preparation with detergent can lead to the differential behavior of the proteomicelle and to differences in processes such as the penetration of detergent into the transporter . We note that to establish unambiguously the conditions concerning ddm penetration behavior and protection by lipids it becomes essential to expand the current md simulations to more complex environments that involve mixed phospholipid - detergent micelles . This is because the treatment of the protein with detergent generally retains a relatively small annulus of lipid molecules that are being extracted together with the protein during the solubilization process . This annulus of lipids can be expected to create a new set of local interactions with the protein and with the detergent, and these could affect the detergent penetration (especially from the side) of leut . To establish the specific conditions regarding the lipid core, we are currently extending the studies of leut in mixed micelles by probing different simulation conditions, and the results will be presented in a subsequent publication.
Recently, the detection of cystic lesions in the pancreas has greatly increased because of the wide use of high - resolution diagnostic imaging techniques.1 in contrast to solid tumors which are usually ductal adenocarcinomas, which have a very poor prognosis, most cystic lesions of the pancreas are either benign or low - grade malignancies; thus, it is relatively easy to perform curative resection for them.2 therefore, cystic and intraductal tumors of the pancreas consist of an essential category with a challenging differential diagnosis.3 pancreatic squamoid cyst is defined as a lesion with cyst - like dilated ducts that are lined by non - keratinized squamous epithelium, and it is a recently recognized type of cystic lesion of the pancreas.2 we herein present a case of a patient with a pancreatic squamoid cyst who underwent laparoscopic resection . A 60-year - old woman who underwent abdominal computed tomography (ct) for a routine health check - up was found to have a 1.8-cm cystic lesion in the tail of the pancreas . Her abdomen was soft and flat, and no tenderness or rebound tenderness was observed . There was no evidence of pancreatitis . Both the serum carcinoembryonic antigen (cea) and carbohydrate antigen 19 - 9 (ca19 - 9) levels were normal . A dynamic ct of the pancreas showed multiple cystic lesions in the tail of the pancreas and the largest cystic lesion showed a lobulated contour without communication with the pancreatic duct . There were no indications of invasion into the surrounding tissues or dilatation of the main pancreatic duct (fig . Magnetic resonance cholangiopancreatography revealed a 2.2-cm round cystic lesion in the tail of the pancreas which had no communication with the main pancreatic duct . Endoscopic ultrasonography revealed a 2.21.3 cm - sized cystic lesion and a 52.5 mm - sized mural nodule; the septation within the cyst and the cyst wall was thin; and the cyst was well - demarcated from the surrounding tissues . The pancreas showed normal parenchymal echogenicity and the main pancreatic duct had a normal caliber (fig . We diagnosed the cystic lesion as an intraductal papillary mucinous neoplasm . To treat this lesion, we performed laparoscopic spleen - saving distal pancreatectomy . During the operation, the cystic lesion, approximately 3 cm in diameter, was found arising from the border between the body and tail of the pancreas without any infiltration into the surrounding tissues . Most of the lesion was located within the pancreatic tissue, and its border was well - defined . The patient showed the usual routine postoperative course and was discharged 10 days after surgery . The patient quickly returned to her normal activities . On examination of the resected specimen, the cystic lesion of the pancreas (21.81.5 cm) was multi - locular with thin septa (fig . A well - defined, oligo - locular cystic mass was in the pancreatic tail without a solid portion . Inner surface of the cyst was smooth and glistening and the cyst contained serous fluid . Histologic examination revealed that the cysts had linings ranging from flat squamoid cells to transitional cells with non - keratinization (fig . The epithelia of the cysts reacted negatively with muc 1 and muc 6 (a marker for intercalated duct centroacinar cells), and positively with ck7 and p63, and negatively with inhibin . In 2007, othman et al reported on 6 cases of cystic lesions with distinctive morphologic, immunophenotypic, and clinical characteristics, and proposed the term squamoid cyst of the pancreatic ducts to refer to these types of lesions.2 they mentioned that this type of cystic lesion typically resulted from uni - locular cystic dilatation of the ducts and had variable linings ranging from attenuated, flat, non - stratified squamous to transitional and stratified squamous epithelium, without a cornified layer or parakeratosis.2 however, no tall - columnar mucinous cells or acinar cells are evident in a squamoid cyst.1 the squamoid cyst of the pancreas is characterized by a flat, transitional, or squamous epithelium, and a p63 expressing basal and parabasal cell component . Kurahara et al mentioned that the microscopic version of this lesion is a relatively common incidental finding in the pancreas, and these, along with the expression of muc 1 and muc 6, suggest that the process originates from the entroacinar / intercalated ductal system.1 however, the surface cells were negative for muc1 and muc6 in our case . So we suggest that the immunophenotyping of the squamoid cyst should be studied further, especially from the point of view of the expression of muc1 and muc6 . The expression of p63 in the cells forming the basal region is essential because p63 is a transitional / squamous cell marker and is not detected in the normal pancreas or in non - squamous neoplasms . The surface cells were negative for muc1 and muc6 (markers present in intercalated duct cells) in this case . Thus, we were able to determine that squamoid cysts can be diagnosed without muc1 or muc6 being expressed . There are other types of squamous - lined cysts of the pancreas, such as lymphoepithelial cysts, epidermoid cysts within an intrapancreatic accessory spleen, and dermoid cysts.4 lymphoepithelial cysts are lined by well - differentiated stratified squamous epithelium surrounded by a band of dense lymphoid tissue.4 epidermoid cysts within the intrapancreatic accessory spleen are lined by attenuated squamous cells, usually non - stratified, surrounded by normal - appearing splenic tissue.5 dermoid cysts are morphologically similar to teratomas seen in other sites . The presence of adnexa - type elements (sebaceous glands, hair, etc) is more typical of dermoid cysts.6,7 these cystic lesions of the pancreas are thought to be all benign . Clinically, it is important to distinguish squamoid cysts of the pancreatic ducts from mucinous cyst - forming neoplasias, especially mucinous cystic neoplasms and intraductal papillary mucinous neoplasms.2 in contrast to squamoid cyst of the pancreatic ducts, which appears to be a benign lesion, these mucinous lesions have the risk of malignant transformation and have the potential to progress to invasive adenocarcinoma . However, the differential diagnosis of pancreatic cystic lesions is not easy because the radiologic and clinical findings are not specific . Kurahara et al mentioned that tumor markers (cea and ca19 - 9) may be high in squamoid cysts of the pancreas.1 we investigated 5588 patients who underwent a pancreatic biopsy or pancreatic resection from 2009 to 2012 in our institution, and found 7 cases of squamoid cysts of the pancreas . There were no connections to the main pancreatic duct and no solid portion in any of the cases . The levels of cea, ca19 - 9 and ca72 - 4 were 1.860.91 ng / ml, 19.5526.9 u / ml, 2.570.23, respectively . There was one patient whose ca 19 - 9 was 76.6 u / ml, but this was not a specific finding because a pancreatic intraepithelial neoplasia was also found in her pancreas biopsy report.8 thus, we suggest that tumor markers can be an important method to use in the differential diagnosis . Cea is a very accurate tumor marker for diagnosing mucinous pancreatic cystic neoplasms, although the accuracy of this marker and the cut - off level vary among laboratories.9,10 in conclusion, squamoid cyst of the pancreas is a very rare disease, and it has been proposed only recently as a distinct pathologic lesion . However, the diagnosis of squamoid cyst will increase due to improvements in imaging techniques such as ct scanning and magnetic resonance imaging . This lesion is regarded as a benign entity, thus an extended operation should be avoided and resection of the lesion can be performed minimally.
Children with cerebral palsy experience restrictions on handwriting due to difficulties of motor coordination and balance due to damage to the central nervous system and involuntary movements . One study found that 81.3% of children with cerebral palsy were found to write incorrect, uneven and unrecognizable letters1 . Previous studies1,2,3,4,5 of handwriting related factors of children with cerebral palsy have been carried out, but these studies had limitations regarding the identification of comprehensive relevant factors and the selection of efficient priorities . In the last ten years, perspectives on outcome assessments of cerebral palsy have been influenced and shaped by the world health organization s international classification of functioning, disability, and health (icf)6, as a guiding principle . Icf provides descriptions of three major domains of body function, body structure, and activities and participation (execution of tasks and activities and involvement in a life situation). These domains are further clarified with contextual factors, either personal or environmental, and by applying the icf s conceptual framework, vocational evaluators can systematically gather, organize, synthesize and interpret health related assessment information7 . Also, icf domains have been used to understand and describe the many impacts of cp on individuals and allow the categorization of various cp outcome measures by the domain that is being assessed8 . We investigated factors affecting handwriting articulation of children with cerebral palsy based on the icf model to provide comprehensive data for the assessment and design of interventions for effective handwriting of children with cerebral palsy . Among the subjects who participated in the research, there were 54 male (56.3%) and 42 female subjects (43.8%). Their average age was 11.16 (2.06), average education year was 3.75 (1.97). There were 78 right - handers (81.3%) and 18 left handers (18.8%). There were 54 subjects with diplegia (56.2%), 30 with hemiplegia (31.3%) and 12 with quadriplegia (12.6%) (table 1table 1.general characteristics of the subjectscomponents of icfn (%) gendermale54 (56.3)female42 (43.8)age8 years14 (14.6)9 years13 (13.5)10 years13 (13.5)11 years7 (7.3)12 years14 (14.6)13 years23 (24.0)14 years12 (12.5)(msd)11.162.06hand dominanceright78 (81.3)left18 (18.8)education (elementary school)first grade19 (19.8)second grade13 (13.5)third grade11 (11.5)fourth grade8 (8.3)fifth grade18 (18.8)sixth grade27 (28.1)(msd)3.751.97type of cerebral palsyspastic74 (77.1)athetoid5 (5.2)athetoid with spasticity4 (4.2)ataxic11 (11.5)flaccid2 (2.1)affected sidediplegia54 (56.2)hemiplegia30 (31.3)quadriplegia12 (12.5)). The subjects did not have any diseases related to the hands, fractures, or past histories of diseases . Before the study, the principal investigator explained all procedures to the subjects in detail . All subjects signed an informed consent form, which was approved by the cheonnam university college of human health science studies committee . Based on the icf, factors related to handwriting were classified as personal factors (gender, age, education, hand dominance, type of cerebral palsy), environmental factors (writing aids, sitting aids, type of desk), body structure(affected side), body functions (cognitive function: orientation, visual perception, spatial perception, motor praxis, visuomotor organization, thinking operation; sensory function: proprioception, tactile; strength function: upper extremity, grasp, prehension; motor function: bilateral coordination, upper extremity speed), and activity and participation (sitting balance, pencil grasp posture). To identify the factors related to handwriting articulation, assessment tools were classified and used based on the icf model (table 2table 2.assessment based on the icfcomponents of icf handwriting factorsassessment personal factorsgenderinterviewageeducationhand dominancetype of cerebral palsyenvironmental factorswriting aidsinterview sitting aidstype of deskbody structureaffected sideinterviewbody functionscognitive functionorientationdotcavisual perceptionspatial perceptionpraxissensory functionvisuomotor constructionproprioception testthinking operationtactile testproprioceptionmanual muscle teststrength functiontactiledynamometerupper extremitypinch metergraspb - o test (subtest 3)motor functionprehensionb - o test (subtest 8)bilateral coordinationupper extremity speedactivity & participationsitting balancesitting balance testpencil grasp posturesurveyhealthwriting articulationkorean alphabet writing assessment). Some personal factors (age, education) and body functions (cognitive function: visual perception, visuomotor organization; strength function: upper extremity elbow flexion and extension, wrist extension, lateral deviation and medial deviation, grasp; and motor function: bilateral coordination, upper - extremity speed, factors were related to handwriting articulation . According to the backward regression analysis, wrist lateral deviation, upper - extremity speed and education were significantly associated with handwriting articulation (table 3table 3.factors related to handwriting articulationcomponent of icfhandwriting factorsunstandardized coefficientsstandardized coefficientsvifbstandard errorbetabody functionstrength functionupper extremity - wrist lateral deviation1.0460.2990.3071.001motor functionupper extremity speed0.0790.0180.3931.024personal factoreducation0.3640.1760.1831.023p<0.05, * p<0.001). Cerebral palsy may involve problems in the neuromuscular system such as spasticity, contracture, muscle weakness, and loss of selective movement9 . This decreased functioning limits the ability of children with cp to participate in various activities . So, the identification of factors that lead to functional impairment is of fundamental importance in clinical decision making and the evaluation of the effect of therapeutic strategies11 . This study investigated handwriting articulation among school - aged child with cerebral palsy and factors related to it . Lower body function cognitive function: visual perception, visuomotor organization; strength function: upper extremity (elbow flexion and extension, wrist extension, lateral deviation, and medial deviation, grasp; and motor function: bilateral coordination, upper - extremity speed) contributed towards poor handwriting articulation, and also occurred in individuals with lower ages and education . Several studies have described the influence of visual perception, visual motor organization1, strength function of the upper extremity12, and motor function4 . Visual perception is the ability to recognize forms, notice likenesses and differences, infer the movements necessary for the production of the form . Visuomotor organization is the ability to copy or transpose from printing material to cursive or manuscript writing1 . Bilateral coordination impairment negatively affects handwriting skills4 . Crossing the midline is an integral skill related to bilateral coordination . A child who avoid midline crossing may have difficulty coordinating both sides of the body, has difficulty establishing hand dominance and tends to use alternate hands when writing . Backward regression analyses shows that wrist lateral deviation of muscle strength, upper - extremity speed and education were important predictors of handwriting articulation . A previous study reported that the muscles of the wrist stabilize and prevent unwanted wrist movements, which allow the finger muscles to maintain an adequate length that is favorable for producing tension and prehension13 . Upper extremity mobility is a coordinated effort of the upper extremity structure and function, and poor upper extremity speed can lead to functional limitation . This study had several limitations, and one of them was the small sample size, therefore these results cannot necessarily be generalized to all children with cerebral palsy . It also exclusively investigated physical body function and structure and personal factors but not environmental factors . In conclusion, this study comprehensively handled overall health - related factors on the basis of the icf health model for intervention approaches for children with cerebral palsy, therefore, it can be used as basic data in setting systematic intervention goals and plans for articulation of handwriting . Comprehensive personal function and body function assessments that involve cognitive function (visual perception, visuomotor organization), strength function (upper extremity: elbow flexion, extension, wrist extension, lateral deviation, medial deviation, grasp) and motor function (bilateral coordination, upper - extremity speed) must be performed . Efforts to manage and improve the handwriting articulation of children with cerebral palsy should focus on wrist lateral deviation and upper - extremity speed.
Patients of sarcomatoid carcinomas of adrenal can present with non - functional adrenal swellings with pressure symptoms by themselves or the result of their metastatic damage to other organs . Diagnostic modalities include endocrine as well as radiological evaluation of the patient, but the definitive diagnosis is by histopathological and immunohistochemical examination postoperatively after excision . We present a case of sarcomatoid carcinoma of right adrenal gland for which open adrenalectomy was done . However, she succumbed after 4 months as she had developed similar swelling in left adrenal gland along with metastases to para aortic nodes . A 62-year old female presented with complaints of on and off episodes of pain in the right upper quadrant of abdomen since 4 months . Computed tomography (ct) scan of abdomen and pelvis suggested 4.32.5 cm well defined moderately enhancing right adrenal mass with central necrosis (figure 1). Histopathology suggested it to be tumor composed of spindle cells and permeated by intense inflammatory infiltrate chiefly neutrophils . The tumor cells showed strong immunopositivity for vimentin and focal immunopositivity for epithelial membrane antigen . Ct scan of pelvis and thorax was done to search for the primary focus of malignancy but both suggested no obvious focus except patient having a 60% thrombosis of distal arch of aorta . The surgery was completed uneventfully . On gross examination, specimen was brown and nodular measuring 6.55.53.5 cm and weighed 55 g. its cut surface showed an unencapsulated firm greyish white tumor (figure 2). On microscopy the tumor showed pleomorphic tumor cells with epithelial and spindle cell morphology with evidence of foci of necrosis and intense inflammatory infiltrate with neutrophils and lymphocytes (figure 3). Focal immunopositivity for pancytokeratin (figure 5) and bcl-2 . The tumor cells were immunonegative for synaptophysin, chromogranin, melan - a and inhibin and described as sarcomatoid carcinoma of right adrenal gland . Follow up after 3 months showed recurrence with involvement of left adrenal and para - aortic nodes on ct scan . It is broadly classified into: i) conventional adrenocortical carcinomas, and ii) adrenocortical carcinoma variants . Variants of adrenocortical carcinomas include: i) oncocytic adrenocortical neoplasms; ii) myxoid adrenocortical neoplasms; iii) adrenocortical carcinomas with a sarcomatous or sarcoma - like component; iv) adrenocortical blastoma . The tumor has been reported in relatively common organs such as the respiratory tract, gastrointestinal tract, and mammary gland . Sarcomatoid carcinomas of the adrenal glands are extremely rare and extremely malignant tumors known with 1 year mortality of 100% . Patients usually present with complaints of vague abdominal pain, abdominal lump in the flanks, loss of weight and generalized weakness . If the tumor is functional patients may present with the corresponding endocrine abnormalities like hypercortisolism causing cushing s syndrome, hyperaldosteronism causing severe hypertension, muscle cramps, testosterone secretion in women causing deepening of voice, acne, balding, hyperestrogenemia in males causing gynaecomastia, impotence and in females causing irregular menses, menorrhagia . In the review of literature, six out of 9 patients diagnosed with sarcomatoid carcinoma of adrenal, presented with flank or abdominal pain or discomfort as in present study . Tumours tend to be very large at the time of presentation (mean size 13 cm, weight 1113 g) (table 1). These tumors are notoriously known for their metastasis at the time of presentation and the routes of metastasis include hematogenous, lymphatic or transcoelomic spread . The tumor cells on microscopy show dual line of differentiation into mesenchymal (sarcomatous) and epithelial (carcinomatous) elements . The mesenchymal differentiation may be towards muscle (rhabdomyosarcoma), bone (osteosarcoma), fibrosarcoma etc . Or just undifferentiated spindle cell type . There are many theories proposed to explain the dual line of differentiation of these tumors . They are: i) the composition tumor theory (paradoxically requiring a non - malignant non - epithelial component as a reactive proliferative response induced by the epithelial component via paracrine secretion); ii) the collision or biclonal tumor theory (a collision between two synchronous, histogenetically independent, biclonal tumors); iii) the conversion tumor theory (neoplastic transformation within a monoclonal tumor recapitulating the naturally occurring event of conversion of epithelial to mesenchymal cells during embryogenesis); and iv) the combination or divergent tumor theory (deriving from a common monoclonal stem cell precursor), molecular genetic evidence of monoclonality supports the single pluripotential stem - cell - divergence hypothesis and the epithelial - to - mesenchymal transition as well . Adrenal cortical carcinosarcomas tend to be aggressive tumours, with locoregional recurrence and rapid metastases from sarcomatoid component . Endocrine assays and ct are the modalities available to diagnose as well as to rule out primary focus from elsewhere . Dynamic enhanced scan with multi - detector row ct may show the compositions and the blood supply of a tumor . Ct scan shows heterogenous enhancement which may raise the suspicion of sarcoma, however further studies are required to confirm . The best available treatment option is surgical excision of the tumor with clearance of metastasis . In radical resection adrenal sarcomatoid carcinoma seems to be a highly malignant tumour and has very poor prognosis . Adrenal sarcomatoid carcinomas even though are operable on early presentation, may present with metastases quite early and be fatal.
Heart rate variability (hrv) assesses the differences of the periods between consecutive heartbeats, which vary under autonomic control . Hrv can be measured by the time domain method, the frequency domain method and others . In the time domain method, mean heart rate (mhr), the square root of variance of rr intervals (sdnn) and square root of the mean squared differences of successive rr intervals (rmssd) can be calculated . On the other hand, frequency domain parameters include total power (tp), very low frequency (vlf), low frequency (lf), high frequency (hf), normalized low frequency (lf norm), normalized high frequency (hf norm) and lf / hf ratio . Sdnn reflects all cyclic components of the variability in recorded series of rr intervals. [13] rmssd is an estimate of high - frequency variations in short - term rr recordings and therefore reflects parasympathetic regulation of the heart. [13] tp reflects overall autonomic activity . The lf power is modulated by both sympathetic and parasympathetic outflows as well as by other factors, including baroreceptor activity . The advantage of the normalized low frequency (lf norm) and the normalized high frequency (hf norm) is that they minimize the effect of changes in very low frequency power and emphasizes changes in sympathetic and parasympathetic regulation respectively . Lf norm is the ratio between absolute value of the low frequency and difference between total power and very low frequency . Hf norm is the ratio between absolute value of the high frequency and difference between total power and very low frequency . Lf / hf ratio signifies the overall balance between sympathetic and parasympathetic systems. [13] although enhanced cholinergic activity of asthmatics has been established at least four decades ago, little hrv studies were done on asthma patients. [811] moreover, the results of these studies are not reproducible, probably due to inter - individual differences of autonomic balance in test groups or inadequacy of methods . Almost all previous researches on hrv of asthmatics considered asthma severity at the time of the studies without special concern for the disease control . Although long - term hrv looks more attractive, it may be less effective in determining the relation between autonomic modulations and asthma activity . Other determinants of heart rhythm are similarly not constant during the day, e.g., respiratory rate and blood pressures. [1517] therefore, it seems more logical to study short - term hrv under similar conditions where these parameters are less likely to change significantly . Several studies confirm that short - term correlate very well with long - term hrv . Demonstrate that fifteen to thirty second heartbeat measurements were long enough to produce reliable long - term patterns of hrv features . Thus, short and intermittent recordings of heartbeats could be used to detect long - term hrv patterns . This is especially true for power spectral measures of rr variability calculated from short (2 to 15 minutes) ecg recordings . Another advantage of short - term hrv is that it is more practical and less expensive when needed for the purpose of follow - up in outpatient clinics . This study aims to evaluate the pattern of autonomic modulations in asthmatic patients based on short - term hrv studies . The present study has considered asthma activity over the last month prior to patients evaluations using internationally valid and reliable test, namely asthma control test (act). [2123] the relation between autonomic modulation and the duration of bronchial asthma was also appraised . The study involved one hundred patients with past medical history of bronchial asthma (at least for two years) selected from chest clinics of teaching hospitals in khartoum state patients with past medical history suggestive of other chronic respiratory diseases, diabetes mellitus, hypertension, heart diseases or any illness that may alter heart rate were excluded from the study . Asthma history and drug therapy was recorded to assess asthma activity at the time of examination as well as over the last month prior to patients evaluation using asthma control test (act). Responses from the act are summated to yield a score that ranges from 5 (no control) to 25 (complete control). A score of 20 or higher was used as discriminating cut - off to define totally from poorly controlled patients while a score of 15 or lower was used as discriminating cut - off to define asthma that was not controlled at all. [2125] allflow spirometer (version 5.18 - clement clarke international limited u. k) was used for assessing pulmonary function according to ats / ers standards . Statistical evaluation was performed using the microsoft office excel (microsoft office excel for windows; 2003) and spss (version 19). Screening studied variables for significant correlation between measurements of asthma severity and hrv indices were performed using bivariate correlation . In physiological studies comparing hrv in different well - defined groups, the differences between underlying heart rate should also be properly noted . Therefore, heart rate (mhr) was introduced as a covariate in the statistical analysis of hrv using partial correlations . According to act, seventeen subjects (17%) of the studied patient were suffering from well controlled asthma, twenty - seven (27%) were poorly controlled, and fifty - six (56%) were uncontrolled . The means (m) and standard deviations (sd) of age and other physical characteristics of studied asthmatic patients are summarized in table 1 . Table 2 demonstrates the m sd of the time and the frequency domains hrv indices of studied asthmatic patients when classified according to act . Figures 1 and 2 compare lf norm, hf norm, and lf / hf between different classes of asthma patients . The significances of the correlations between act state and hrv indices are given in table 3 . Characteristics of studied asthmatic patients m sd of hrv indices in studied asthmatic patients lf norm and hf norm of studied asthmatic patients lf / hf norm of studied asthmatic patients the correlations between act state and hrv indices following adjustment for mhr and anti - asthma medications, act score correlates positively with both lf norm (cc = 0.302, p = 0.002) and lf / hf (cc = 0.212, p = 0.036) and negatively with hf norm (cc = -0.317, p = 0.001). All studied asthma classes, namely well controlled, poorly controlled, and uncontrolled asthmatic patients, had long - standing history of asthma (m sd = 17.88 10.55, 8.93 6.97 and 10.93 8.14 years, respectively). However, asthma duration was significantly higher in well controlled compared with both poorly controlled and uncontrolled asthmatic patients (p = 0.000 for both). Duration of asthma correlates positively with hf norm (cc = 0.235, p = 0.020) and negatively with lf norm (cc= -0.250, p = 0.013). High fev1 is associated with higher hf component of the power spectral density (cc = 0.200, p = 0.049). High fvc is associated with improved hrv as indicated by sdnn (cc = 0.275, p = 0.006), rmssd (cc = 0.294, p = 0.003), tp (cc = 0.271, p = 0.007), lf (cc = 0.248, p = 0.014), and hf (cc = 0.271, p = 0.07). It is evident from the results that higher level of asthma control is associated with improved sympathetic (lf norm and lf / hf) and depressed parasympathetic (hf norm) modulations . Moreover, asthmatic patients with better ventilatory functions have the benefit of enhanced hrv as indicated by the significant positive correlations between fvc and sdnn, rmssd, tp, lf, hf . These results are comparable with the earlier hrv researches in asthmatic patients, although previous studies were mostly in children and based on smaller sample size. [811] almost all previous studies were designed to compare healthy controls with different classes of asthma severity and none of them was able to demonstrate significant correlations between parameters of asthma severity and hrv indices . For example, garrard et al . Evaluated ten healthy controls, nine asymptomatic, untreated asthmatic subjects, and ten asthmatic patients during treatment for acute asthma, by measurement of the variation in resting heart rate using frequency spectrum analysis . Spectral density of the beat - to - beat heart rate was measured with the lf and hf . Sympathetically mediated heart rate variability (lf norm) however, the study failed to prove dominance of parasympathetic modulations (hf norm) in asthmatic groups . In contrast, du et al . Were able to demonstrate enhanced vagal tone when comparing 23 healthy volunteers and 69 asthmatic young adults . The autonomic nervous function (anf) of asthma subjects was lower in comparison to the normal group . One year later, kazuma and his group examined the circadian rhythm of parasympathetic nervous function in asthmatic children . Circadian rhythm disappeared in 11.25% of asthmatic children and was observed in all the individuals in the healthy children . The current results also showed that asthma duration correlates positively with hf norm and negatively with lf norm . One major difference between mild and severe asthma is the duration of contact of small airways to inflammatory mediators . This is especially true if one consider inadequate clearance of inflammatory mediators secondary to arterial luminal narrowing observed in bronchial circulation of uncontrolled / persistent asthma patients . The attenuated sympathetic activity (as indicated by lf norm) in those with longer asthma duration offers a good choice for vasodilatation of the already narrowed blood vessels by the effect of remodeling and hence better washout of the inflammatory mediators . Alternatively, the enhanced parasympathetic activity (as indicated by hf norm) in those with longer asthma duration will further aggravate bronchoconstriction and worsen asthma symptoms . The current results also showed that all studied asthma classes, namely well controlled, poorly controlled, and uncontrolled asthmatic patients, had long - standing history of asthma . However, asthma duration was significantly higher in well controlled compared with both poorly controlled and uncontrolled asthmatic patients . Although asthma duration was significantly higher in well controlled compared with both poorly controlled and uncontrolled asthmatic patients, it seems that there was enough time for inflammatory mediators to act in both controlled and uncontrolled asthmatic patients (m sd of asthma duration = 17.88 10.55 and 10.93 8.14 year, respectively). Moreover, longer asthma duration in well controlled patients could be explained by the fact that asthmatics with longer history of the disease became more knowledgeable in dealing with asthma, e.g., avoidance of triggers, compliance with treatment protocols, and therefore, release of less quantity of inflammatory mediators . In conclusion, previous studies examining the pattern of autonomic modulations in asthmatic patients report mixed findings possibly due to inadequacy of methods . In the present study, all diseases that may affect hrv were excluded in studied subjects; statistical analysis has considered variations due the differences in mhr and anti - asthma medications among asthmatic patients; and hrv was studied over a short duration to safeguard against the labile activity of asthma . All these considerations make the effects of possible confounding factors on autonomic modulation unlikely in this study . Results were interesting and revealed that poor asthma control is associated with depressed sympathetic and enhanced parasympathetic modulations especially in those with longer asthma duration.
Drug induced safety issues, to include renal impairment, continue to be a significant reason that drugs fail the development process 1 . However, the ability to identify drug induced renal function changes during the development process continues to be a challenge . The traditional renal biomarkers of urea and creatinine are frequently obtained in all phases of clinical trials as these markers are accessible, analytically stable 3, and cost effective, but viewed as insensitive and non - specific monitors of renal function 4 . The non - specific nature of urea and creatinine as renal markers may be advantageous in clinical trials if the ability to identify drug induce renal impairment can be improved . Therefore, a sensitive statistical tool to identify drug induced changes in renal function, using these traditional biomarkers, will be valuable in the drug development process . The key element to identify a drug induced change in renal function from serum urea and creatinine is understanding that small changes are significant for these biomarkers . Multiple definitions of a toxicity signal exist such as published toxicity tables, identifying values outside the reference interval, using multiples of the reference interval, evaluating multiples of the baseline value (2 fold increase, 3 fold increase), and% change from baseline . These definitions of a drug induced signal often fail because important drug induced changes for serum urea or creatinine will be small relative to the reference interval . Alternatively stated, the index of individuality (ioi = cvi / cvg where cvi is the intra - individual biological variability and cvg is the inter - individual biological variability) is 0.67 for urea and 0.56 for creatinine 5 . A z - score can be defined as the number of standard deviations between a visit value and a baseline mean: where cva is the coefficient of analytical variation and n is the number of baseline readings 5 . Assuming a cva of 5%, the 99% confidence interval (z=2.58) for a change from two baseline values, and given cvi's of 5%, 10%, and 20%, the important change for an analyte value is approximately 22%, 35%, and 65% increases from the baseline mean respectively . These% changes could easily be masked by the definition of the renal impairment signal . The cvi for creatinine is typically about 5% while urea's cvi is typically in the range of 10% 5 . Consequently looking at z=2.58 shift in the patient's values, a 22% change in a serum creatinine value or a 35% change in a urea value, should be identified as a potential drug effect in a subject in a clinical trial . In comparison, if the mean value of the reference interval is used, 75 mol / l for creatinine and 5.0 mmol / l for urea, the absolute changes would be 16.5 mol / l (final value 91.5 mol / l) for creatinine and 1.8 mmol / l for urea (final value of 6.8 these changes are well within the reference intervals (see methods for reference intervals) and all traditional definitions of renal impairment for urea and creatinine would be negative for more than half of the trial population if all subjects had a significant increase in biomarker value based on the estimated% cvi . The solution for improving the sensitivity of urea and creatinine as renal toxicity markers is to move from a traditional fixed limit definition of a toxic signal to a statistical evaluation of the signal . A z - score approach of defining an important change from the biological and analytical variation is partial solution to this issue 5 . Recently, it has been shown that both traditional reference intervals and z - score formulas are two particular cases of a more general adaptive bayesian model 6 . Population cvi and cvg can be used as prior information in a bayesian network (bn) to move from population- to individual - based intervals as the number of individual readings increases . Heterogeneous factors leading to stratified reference intervals (such as age and gender) can be integrated in the bn to further remove between - subject variation . An assumption in the z - score formula is that the population% cvi is constant across the population . By moving to an adaptive bayesian approach, the detection algorithm is customized for biomarker and subject thereby minimizing assumptions and maximizing the sensitivity of signal identification . The adaptive bayesian approach has been successfully applied for the evaluation of biomarkers as formalized in the so - called biological passport 7 - 8 . Biomarker signal defined statistically and detected through a very sensitive statistical technique may find an unimportant signal . However, in phase i and phase ii trials where populations are small, identifying a signal permits one to watch the signal in the development process to see if the signal becomes medically important 9 . The lack of a toxicity signal viewed through a very sensitive statistical tool early in the development process minimizes the risk of a toxicity being found later in development . Two phase iii clinical trial datasets, with known drug induced renal impairment, are analyzed for a renal impairment signal by traditional methods and an adaptive bayesian approach in this report . Interestingly, the knowledge of renal impairment makes it possible to estimate the specificity (control group) and sensitivity (treated group) of the applied methods for the first visits post - administration when the signal is most probably weak . Two phase iii clinical trial datasets, one for urea, one for creatinine, were analyzed retrospectively . The clinical trial database for urea was composed of 590 patients (aged 26 - 92, median 65) distributed among the placebo (200 patients) and two drug concentrations (group 1: 194 patients, group 2: 196 patients). The creatinine clinical trial database had 532 patients (aged 26 - 97, median 65) distributed among the placebo (178 patients) and two drug concentrations (group 1: 174 patients, group 2: 180 patients). The patients had between 1 and 3 pre - treatment visits and data was analyzed for the first 3 visits after drug administration . The blood for the urea and creatinine measurements was collected in bd vacutainers (becton dickinson, franklin lakes, nj), serum separated by the site, and transported to a central laboratory (covance central laboratory). Samples were analyzed on roche chemistry analyzers (roche, basel, switzerland) using roche urea and rate blanked compensated alkaline picrate creatinine reagents . All statistical simulations were performed on matlab version 7.7.0 with statistics toolbox version 7.0 (mathworks, natick, ma). The following stratified reference intervals were used for creatinine: males 18 - 50 years, 40 - 110 mol / l; females 18 - 70 years, 31 - 101 mol / l; males 50 - 70 years, 40 - 119 mol / l; males 70 - 80 years, 40 - 137 mol / l; females 70 - 80 years, 31 - 110 mol / l; males> 80 years, 40 - 145 mol / l; females> 80 years, 31 - 128 mol / l (covance central laboratory) and for urea: males and females 18 - 70 years, 1.4 - 8.6 mmol / l; males and females 70 - 80 years, 1.4 - 10.4 mmol / l; males and females> 80 years, 1.4 - 12.1 mmol / l (covance central laboratory). Toxicity of grade 1 were computed for all data following the 2009 toxicity table of the national institute of allergy and infectious disease (niaid). This table lists a grade 1 creatinine toxic value as 1.1 to 1.5 times the upper limit of normal (uln) and a urea toxic value as 1.25 to 2.5 uln . The adaptive bayesian approach was implemented in a hierarchical bn (see figure 1) with biomarker signal decomposed into within - subject and between - subject components . It is assumed that a series of urea or creatinine values is normally distributed with mean and variance specific to each subject . Prior distributions of within - subject variation (denoted in figure 1 by the variable cvi) and of the mean have been modeled as follows . The cvi is assumed to be log - normally distributed with geometric mean (gm) equal to -1.97 and -2.6 and geometric standard deviation (gsd) equal to 0.14 and 0.095, for urea and creatinine respectively . With these parameters, the 99%-interval of prior distribution of cvi is [8 - 22]% for urea and [5.6 - 9.2]% for creatinine . The mean is assumed to be log - normally distributed with gm=0.89 and gsd of 0.15 for urea, gm=3.92 and gsd=0.13 for creatinine . The latter gsd gives the amplitude of the between - subject variations post - stratification . The stratification according to gender and age was modeled so that to obtain the stratified reference intervals given above (assuming 99%-intervals). A bn was associated to each subject, with age and gender integrated as evidence in the bn before the start of the trial . Then, bayesian inference techniques were used to iteratively and adaptively integrate pre - treatment visit values . Posterior distributions of expected values were generated by the bn to analyze the values obtained in treatment . Finally, 99%-intervals were computed as the 0.5 and 99.5 percentiles of the posterior distributions . These intervals can be viewed as biomarker and subject and pre - treatment data specific reference intervals . In addition to testing single visit values, the bn was also used to test sequences of visit values obtained after treatment 6 . For example, if the first two visit values fall both at the 95 percentile of the distribution of expected values, the sequence composed of these two values falls approximately at the 99.75 percentile of the distribution of expected sequences of length 2 . It has been shown that the latter approach has a significantly better sensitivity in the analysis of longitudinal biomarker data in the presence of a weak, but consistent over time, signal 9 - 11 . On pre - treatment data, an analysis of variance returned a cvi of 14% for urea and 6% for creatinine . These cvi were used in the z - score formula of equation (1). The magnitude of the z - score is a measure in standard deviations of how far the value has moved from the baseline values . It has been shown that the z - score formula of equation (1) is a particular case of the bn described above 10 . The bn gives the same results as the z - score formula when (1) between - subject variations are assumed to be infinite, (2) the cvi is assumed to be universal (i.e. No subject - based cvi) and (3) no heterogeneous factors are taken into account 10 . Similarly, by design, the reference intervals returned by the bn before the integration of any individual biomarker data correspond to the traditional reference intervals stratified according to age and gender 11 . The greatest false - positive signal for both urea and creatinine was% values above the reference intervals . The adaptive bayesian approach applied to single visit values or to sequences returned false - positive signals between 0.5 and 3.2% . These results are in agreement with a theoretical rate of 1% induced by the use of 99%-intervals . The mean and sd baseline values for urea and creatinine values across the cohorts are very similar (control, group 1, group 2 respectively, urea mean, 5.7, 5.8, 5.6 mmol / l, sd, 1.8, 1.9, 1.6 mmol / l; creatinine, 75.6, 76.6, 76.4 mol / l, sd 20.5, 17.9, 22.7 mol / l) prior to the administration of the drug indicating a well - executed randomization of the patients . The number of subjects per visit ranged from 187 to 173 for the urea data and ranged from 178 to 77 for the creatinine data . The number of patients for the creatinine measurement decreased by approximately 50%, by design, in moving across the visits . The creatinine mean values increased with time and drug dose while the urea mean values increased most directly with drug dose . A small signal was found with the traditional signal definitions of% high flags, niaid toxicity grade 1, and 2 fold increases from baseline values . The adaptive bayesian analysis of sequences was still more sensitive than the bayesian approach based on a single visit value . No values were observed with a toxicity grade greater than 1, no values exceeded twice the upper limit of the reference interval, and no values were observed with a 3 fold increase or greater . At the population level, the mean values by group and visit show that the mean values increased after drug administration . Although the increases in mean values are small relative to the reference intervals, all increases were significant since the first visit in - treatment (p<0.01). In figure 2, the graphs of% change from baseline to the adaptive bayesian z - value demonstrate the same% change can have different z values and that the placebo distributions are different from the drug treated patient distributions . Placebo patients have only one z greater than 3 for urea and 2 z greater than three for creatinine . The adaptive bayesian methodology generated a prominent drug induced signal for renal impairment on both analytes at the first visit after drug administration . All traditional definition of renal toxicity produced none or significantly weaker signals than the adaptive bayesian approach . A dose dependent increase in the adaptive bayesian sequential signal indicates a drug effect is present and is increasing over time . The data shows that a drug effect can be identified at the first time point after drug administration . This urea change, although significant from a statistical point of view, is difficult to interpret from a biological perspective as this change remains in the reference interval and the renal handling of urea is complex and dependent on patient hydration . Mol / l is more easily interpreted relative to the modification of diet in renal disease (mdrd) equation 12 . Subsequently, an increase in the group 2 creatinine results of 21% by visit 3 results in a decrease in the population gfr by 16% . The signal definition for renal toxicity needs to reliably identify this magnitude of population serum creatinine increase . The out - of - reference range high values and toxicity grade 1 values do provide an indication of a drug effect and as the results are different between the placebo and treated groups . The weak signal could be due to noise in the system, due to expected disease related changes, or a function of the patient's baseline value being close to the upper limit of the reference interval . The comparison of the out of range high results to the toxicity grade 1 results would imply that the high flagged results were false - positives as the% high results are 2 to 3 times greater that the tox grade 1 results . The use of multiples of the reference interval (2 or 3 uln) to define renal toxicity is less sensitive that the toxicity grade 1 limits . For instance calculating a z - value using literature cvi values, at the boundary of 2 uln from the low and high ends of the reference interval, the patient z - values are very different . The z - value change in moving from the lower limit of normal for urea and a male creatinine results to 2 times the z - value change in moving from the upper limit of normal to 2 times the upper limit of normal is z=152 for a urea result and z=17 for a male creatinine result . This calculation demonstrates that the probability of generating a toxicity signal varies greatly with the baseline value and reference interval using traditional definitions of the biomarker signal . Also multiples of the reference interval require a substantial drug effect on the kidney to define a toxic signal as the z - values are very high . The drug effect is concentration and time dependent using the adaptive bayesian methodology to calculate the signal . The false - positive reference interval high results were significantly greater for both biomarkers than the% mean false - positive adaptive bayesian results . The false - positive adaptive bayesian signal is in the range of one tenth of the true signal . On the contrary, the false - positive rate for the% above the reference interval signal was approximately 50% of the treated group's signal . The toxicity grading had a low false - positive rate equal to the adaptive bayesian approach . However, the toxicity grading had a very low rate of true positive signal identification also . The adaptive sequential bayesian analysis rapidly produced a signal that exceeded the signal from the traditional definitions . The adaptive bayesian approach identified a drug effect on both analytes at the first visit after drug was administered on a significant number of patients . The rapid identification of a drug induced effect on renal function permits the rapid medical evaluation of this observed effect and, if appropriate, generate requests for additional renal biomarker data to define the mechanism and magnitude of the effect on key kidney function parameters . The same approach has been successfully applied for the evaluation of creatinine data in early phase clinical trials 9 . The data in figure 2 shows a marginal correlation of the% change from baseline to the adaptive sequential bayesian z - value results . The uncertain part of looking at% change from baseline is that the significance of the same% change from baseline will be different for different biomarkers and subjects . The% change from baseline methodology creates difficulties in scoring individual patients as having moved significantly from the baseline value . The challenge of a development protocol is to move from population data to individual patient data and be able to identify the individual patient who needs to be medically evaluated . This dataset provides insight as to the magnitude of an adaptive bayesian signal with a change in the population mean value . An approximate 20% change in the urea mean biomarker value generated an adaptive bayesian signal for 31% of the treated subjects while the toxicity grade 1 signal was 3.4% . An approximate 20% change in the creatinine mean value generated an adaptive bayesian signal for 29% of the treated subjects while the tox grade 1 signal was 6.5% . Change from baseline analysis makes defining the point of significant change more difficult than the adaptive bayesian z value approach as the same% change has a different statistical meaning across biomarkers and across subjects . The adaptive bayesian approach will identify the specific patients that have changed from the baseline value and minimize the dilution of true positives with false - positive observations . Both the creatinine and urea concentrations were rapidly effected by the drug even though the mechanism of clearance of creatinine and urea are significantly different . If renal impairment using non - specific markers such as creatinine and urea can reliably identify important changes from baseline with the adaptive bayesian approach, the non - specific nature of these biomarkers may be very appropriate for first line screening of renal effects in clinical trial populations . Although much has been done by the predictive safety testing consortium (ptsc) to find new clinically relevant biomarkers of renal function 13, our study suggests that conventional biomarkers like creatinine and urea are able to detect early kidney dysfunction when using individual reference ranges . Also, the personalization of a biomarker signal makes possible the evaluation of drug efficacy and safety on an individual basis and in turn to tailor drug therapy at a dosage that is most appropriate for an individual patient . Correlations with genotyping data on metabolic pathways in drug metabolism may be searched for still improved personalization 14 . A further advantage of the adaptive bayesian approach is that as one looks at historical biomarker data, the approach is independent of the analytical method and the reference interval and therefore is very appropriate for data - mining historical data sets . Data mining using the adaptive bayesian approach should permit one to take clinical issues identified in large datasets and follow the biomarker data back through the phases of development . If a signal is found in early small populations, the long - term implications of small signals in early phases of development can be more thoroughly understood . Two phase iii clinical trial datasets were used to evaluate traditional methods of renal impairment and an adaptive bayesian approach . These two datasets were appropriate for this analysis because they present a weak increase in mean urea and creatinine values after drug administration as well as a known drug induced renal impairment . Traditional definition of toxicity limits such as greater than the upper limit of normal, toxicity table ranges, multiples of the reference interval, and greater than 2 or 3 fold changes from the baseline value all suffer from low sensitivity of signal generation and varying probabilities that the threshold will be exceed dependent on the baseline value . The% change from baseline is better than the fixed limit methods however there is not a single% change from baseline to define a significant signal for all biomarkers and for the same biomarker across subjects . The significance of a fixed value for the% change from baseline varies depending on the subjects cvi of the biomarker . The adaptive bayesian approach using all known factors provided superior sensitivity and specificity in these two large clinical trials for drug dependent renal impairment signal generation.
Stroke is a kind of acute cerebrovascular disease characterized by high morbidity, disability and death rates . It is reported that stroke has become one of the most frequently - occurring neurological injuries in usa and china, about 800,000 and 2,000,000 people every year respectively (roger et al ., 2011). Stroke includes ischemic stroke and hemorrhagic stroke, and a large number of clinical studies and animal experiments have shown that the cerebral injury and ischemia - reperfusion injury caused by stroke are complex physiopathologic processes, involving toxic effects of excitatory amino acids, inflammatory reaction, oxidative stress, the generation of free radical, brain edema, neuronal apoptosis / death (zhu et al ., 2015). Although the mechanisms of the injury are diverse, our treatment theory of neuroprotective strategies mainly focus on two aspects: on the one hand, we can activate the endogenous protective mechanisms of patients bodies to protect the brain from injury; on the other hand, it is advisable to try our best to minimize secondary brain injury and promote the recovery of the damaged tissues . For many years, people have been trying to understand and search effective treatments for stroke from every conceivable angle . For now, for ischemic stroke, thrombolytic and anticoagulant therapy has become regular treatments (mandava et al ., 2015), and for hemorrhagic stroke, hematomas cleaning operation and the stable control of intracranial pressure are conductive to improve the prognosis of patients (siler et al ., 2014). In addition, hypothermia has been considered to be a valuable clinical treatment for centuries (thome et al ., 2005), and mild hypothermia (33 - 36c) has been demonstrated to play neuroprotective role (antonic et al . Its mechanisms mainly involve decreasing the metabolism rates of brain tissues and alleviating brain edema (hobbs et al ., 2008). Besides, a variety of methods are used for the treatment of stroke, including stroke unit, neuronal electrophysiological treatment, and even the possibility of psychological therapy (pohjasvaara et al ., 1998; patel et al ., 2004; sheffler and chae, 2007). Our previous studies suggested that pramipexole - induced hypothermia could effectively inhibit subarachnoid hemorrhage - induced early brain injury in rats via phosphoinositide 3-kinase / protein kinase b / glycogen synthase kinase-3 signaling pathway (ma et al ., 2016). However, the present theories for stroke are still unsatisfactory and needed to be further improved, although the development of evidence - based medicine is ongoing . Specifically, in recent years, medical gas, including oxygen and hydrogen, have become issues of concern . No matter ischemic stroke or hemorrhagic stroke, the final outcome is hypoxic - ischemic damage of brain tissues, hyperbaric oxygen therapy can significantly increase the oxygen pressure and blood oxygenation content, accelerate collateral circulation to protect neurons, repair the damaged microvessels, stimulate angiogenesis and neurogenesis, and accordingly, play neuroprotective role (gill and bell, 2004; sanchez, 2013). Hydrogen has also been found play roles of anti - inflammation, anti - oxygenation and anti - apoptosis when a stroke occurs . Hydrogen sulfide (h2s) has always been considered as a toxic gas with its odor of rotten eggs, because it could play the cytotoxic role through inhibiting cytochrome c oxidase and mitochondrial respiration (furne et al ., 2001; have suggesting that h2s is an another important gaseous signaling molecule in biological systems following nitric oxide (no) and carbon monoxide and in central nervous system (cns) (wang, 2002; li and moore, 2008), a larger number of researches have shown that it has neuroprotective effects as neurotransmitter (kimura and kimura, 2004; whiteman et al ., 2004), and researchers hope to provide a new direction for the therapy of stroke through studying h2s . After the endogenous h2s was first found in the brain in the end of 20 century (warenycia et al ., 1989), researchers gradually found that the production of h2s in cns predominantly involve three particular enzymes: cystathionine--synthase (cbs), cystathionine--lyase (cse) and 3-mercaptopyruvate sulfurtransferase (3-mst), which could convert to h2s from l - cysteine (cys) in combination with cysteine aminotransferase (cat) (kolluru et al ., 2013; jung and jeong, 2014). That is to say, cse primarily induce the production of h2s in cardiovascular system, while cbs mainly is responsible for producing h2s in the cns (qu et al ., 2008; kimura et al ., 2012), and cbs uses cys and homocysteine (hcy) that have been known the risk factors for stroke and heart disease as substrates (abe and kimura, 1996; chen et al . Interestingly, although there are amounting evidences showing that h2s participates in the regulation of neuronal function and signaling pathway, concerning the neuroprotective or neurotoxic effects of h2s still remains controversial . In our review, we will discuss the main mechanism and the possible role of h2s in stroke . Because of its toxicity, people have always thought that the concentration of h2s in animal tissues is very low for a long time . However, as what we have known, h2s could be produced endogenously, and further studies indicate that h2s could exist in mammalian tissues at very high concentration, up to 50160 mm (goodwin et al ., 1989; warenycia et al ., 1989), which is dramatically higher than that in the peripheral blood (046 mm) (zhao et al ., 2003), suggesting that h2s may have some effects and show potential therapeutic value in some cns diseases, including stroke, traumatic brain injury, alzheimer's disease and parkinson's disease . Here, we give a brief description on the mechanisms of h2s in the cns as follows (figure 1): (1) anti - oxidation: facing the oxidative stress injury after stroke occurring, h2s could promote the activation of cystine / glutamate antiporter and increase the intracellular concentrations of cys that it is a kind of substrate for the generation of glutathione . In addition, in this process, -glutamyl - cysteine synthetase, a kind of rate - limiting enzyme, could also be activated by h2s and regulates the production of glutathione (kimura and kimura, 2004; kimura et al ., 2010), which is an important intracellular antioxidant . Thus, the intracellular levels of glutathione increases and could clear the reactive oxygen species (ros) existing in the mitochondria to achieve the goal of protecting the neurons from oxidative stress injury . (2) anti - inflammatory effects: the major sites that h2s plays the role of fighting inflammatory response are astrocytes and microglia (seifert and pennypacker, 2014), where h2s could inhibit the release of no, tumor necrosis factor- and the proinflammatory cytokine interleukin-1, and decrease the activation of p38/c - jun n - terminal kinase, conversely, increase the release of anti - inflammatory cytokines, for instance, interleukin-4/10 . It is reported that sb 203580, a kind of p38 mitogen - activated protein kinase (mapk) inhibitor, could mimic the anti - inflammatory effects, indicating that h2s may regulate the immune function by mapk signaling pathway (hu et al ., 2007), but concrete mechanisms still need to be investigated . (3) anti - apoptosis: a growing body of evidence points that h2s exerts its role of anti - apoptosis probably via mitochondrial apoptotic pathway, h2s could inhibit the caspase-3 signaling pathway mediated by ros and calcium pathway activated by hydrogen peroxide (liu et al ., 2014), accordingly, prevent the apoptosis induced by oxygen glucose deprivation / reoxygenation (luo et al ., 2012, 2013). In addition, h2s could promote the nuclear translocation of nuclear factor kappa b (nf-b), which activates the anti - apoptotic gene (sen et al ., 2012). (4) facilitating long - term potentiation: it has always been demonstrated that h2s can facilitate hippocampal long - term potentiation via activating n - methyl - d - aspartic acid (nmda) receptor (kimura, 2002). Subsequently, researchers found that unlike carbon monoxide or no, h2s promotes the accumulation of cyclic adenosine monophosphate rather than cyclic guanosine monophosphate (kimura, 2000), and nmda receptor activation induced by h2s could be inhibited by adenylate cyclase inhibitor (kimura, 2002). (5) regulating calcium concentration: h2s can activate calcium channel to increase calcium influx, whereas decrease the release of calcium stored in the cells, thus induce the production of calcium waves in primary cultures of astrocytes, and this process can be inhibited by calcium channel antagonist (nagai et al . In addition, h2s has been known as an atp sensitive potassium (katp) channel activator in secondary brain injury after stroke or other diseases, h2s could play its neuroprotection through mimicking katp channel (jiang et al ., however, the problem of calcium overload cannot be ignored (obryant et al ., 2014). There are accumulating evidence that the neuroprotection or neurotoxicity of h2s depends on the concentration of it, which needs further investigations, including animals and clinical trials . Note: h2s: hydrogen sulfide; cns: central nervous system; cbs: cystathionine--synthase; cse: cystathionine--lyase; 3-mst: 3-mercaptopyruvate sulfurtransferase; -gcs: -glutamyl - cysteine synthetase; ros: reactive oxygen species; no: nitric oxide; tnf-: tumor necrosis factor-; il-4/10: interleukin-4/10; jnk: c - jun n - terminal kinase; nf-b: nuclear factor kappa b; ltp: long - term potentiation . Nmda: n - methyl - d - aspartic acid; katp: atp sensitive potassium . As we have known, a large number of animal studies must be tested before clinical application for anything, h2s is no exception . In view of the toxicity and high solubility of h2s, it is even uncommon to inhale it directly; therefore, sodium hydrosulfide (nahs, a h2s donor) is widely applied in animal studies concerning cerebral injury after stroke . At present, researchers gradually reach a consensus that the neuroprotection of h2s in stroke is dose - dependent, that is, the relatively low - dose of h2s in brain may play neuroprotective effect in stroke, whereas high concentration of h2s may cause neurotoxicity, suggesting that we must view h2s from a comprehensive perspective, and enhance its advantage, while avoid its disadvantage, to apply h2s in clinical treatment more effectually . In our review, we will show our analysis for animal studies about the role h2s in stroke (table 1). The neuroprotective and neurotoxic effects of hydrogen sulfide (h2s) in stroke the neuroprotection of h2s is exhibited as follows: (1) li et al . (2011) reported that h2s, whose concentration is 0.1 mm, has neuroprotective effects by protecting hippocampal neurons, improving the deficiency of learning and memory, and increasing synaptic plasticity, decreasing the brain edema around pyramidal neurons and their nuclear shrink in a rat brain model of stroke . (2) in a rat model of reversible occlusion of the right middle cerebral artery, h2s was found to reduce infarct volume through long - term hypothermia and the decrease of the expression of nf-b, thus protecting the brain against hypoxic injury due to stroke (florian et al ., 2008). (3) after stroke, the endogenous concentration of h2s increases at 12 hours and decreases at 24 hours . In addition, if pretreated by nahs at a relatively low dose (25 mmol / kg), the brain injury could be attenuated at 7 days after stroke (ren et al ., 2010). Nahs is also reported to protect brain against oxygen glucose deprivation injury (tay et al ., (4) by stimulating the phosphorylation of protein kinase b and extracellular signal regulated kinases, up - regulating vascular endothelial growth factor and angiopoietin-1 expression, h2s could promote angiogenesis around the infarct area, and show better functional outcomes in a rat model of middle cerebral artery occlusion (mcao) (jang et al ., 2014). (5) h2s has anti - oxidative, anti - inflammatory, and anti - apoptotic effects in stroke; and in cerebral ischemia / reperfusion injury, it is also reported h2s could increase the superoxide dismutase activity, anti - inflammatory interlukin-10 and anti - apoptotic protein bcl-2, accordingly, play the role of neuroprotection (yin et al ., 2013). In addition, h2s, or its donor, can reduce the infarct size and decrease the blood brain barrier damage in a model of cerebral ischemia - reperfusion injury (gheibi et al ., 2014). However, different models, administration paradigms and concentration of h2s may cause different results, even get the opposite conclusion: (1) using the same model of mcao, qu et al . (2006) found that if the h2s concentration is raised to 0.18 mm, the infarct volume will increase, instead of decrease, and this process can be ameliorated by h2s synthesis inhibitors or nmda receptor channel inhibitors, suggesting that h2s has different effects on brain in different dose - concentration . (2) the concentration of h2s increases in the cortex, and reach the peak at 12 hours after cerebral ischemia, then it will decrease (ren et al ., 2010). A recent research showed that h2s, at a high dose level, can cause cell death in cerebral cortex based on the model of permanent mcao, and in this process, the infarct volume can be reduced by the inhibitors of h2s synthesis enzymes, on the contrary, decreased by the administration of nahs (qu et al ., 2006). (3) in addition, when the concentration of nahs is below 200 m, the apoptosis can be induced, while the concentration is higher than 200 m, nahs will induce necrosis and cause brain damage (cheung et al ., 2007). In general, the neuroprotective effect of h2s in stroke is concentration - dependent, only a relatively low dose of h2s can yield beneficial effect . However, there is a point we have to say, the most of research on h2s at present mainly focus on ischemic stroke, while study concerning the hemorrhagic stroke is very few, which need us to do more research to reveal the role of h2s in entire stroke . At present, there is no direct clinical studies about neuroprotection of h2s being reported . However, the plasma h2s level has been reported to be related to long - term clinical outcome in stroke patients . A clinical study involving 36 patients showed that high plasma cys levels on admission and before treatment tend to poor clinical outcome when assessed at 3 months after stroke, and the patients who have early stroke deterioration are usually correlated significantly with high plasma level of cys (wong et al ., 2006), suggesting that h2s may be indirectly responsible for this effect . Furthermore, as accumulating evidence shows that hyper hcy is a risk factor for stroke (abbate et al ., 2003; kim et al ., 2003; hassan et al ., (2004) found that the concentration of hcy increased in all stroke subtypes in a case - control study including 313 participants (161 patients and 152 controls), indicating that hcy may be used as a marker for the clinical outcome of stroke as well . However, the definite relationship between hyper hcy and stroke has not been found (fallon et al ., 2001), and several meta - analyses have not reached an agreement (homocysteine studies collaboration, 2002; fallon and ben - shlomo, 2003). Above all, although there is no direct evidence indicating that h2s has neuroprotective effect in clinical trials, the close touch with brain injury caused by stroke is no doubt, and needs us to make great efforts to study . As what we have mentioned above, the high dose - concentration of h2s can cause systemic toxicity reacts (nakata et al ., 2015; wu et al ., 2015), especially neurotoxic effect, because it inhibits cytochrome c oxidase and mitochondrial respiration . The cns is sensitive to hypoxia, h2s poisoning can lead to respiratory paralysis rapidly and cause a series of nerve dysfunction, including brain edema, disturbance of consciousness, and others . Therefore, the following strategies are extremely important, including oxygen uptake, keeping the airway open, and symptomatic and supportive treatments . Although h2s is known as a toxic substance, increasing evidences have indicated that h2s plays an important role of neuroprotection in stroke, while its neurotoxicity cannot be neglected . So, we need to view h2s in the point of dialectics, and we believe that further research on h2s, including animal studies and clinical studies, may provide a new insight into the treatment of stroke and other cns diseases, such as traumatic brain injury and neurodegenerative diseases.
The jhs recruited 5,301 african americans from the jackson, mississippi, metropolitan area between september 2000 and march 2004 . The cohort was composed of four components: 1) 31% of the cohort members were participants from the atherosclerosis risk in communities (aric) study recruited to the jhs; 2) 30% were representative community volunteers who met census - derived age, sex, and socioeconomic status eligibility criteria from the jackson, mississippi, metropolitan area; 3) 17% were randomly ascertained from the jackson, mississippi, metropolitan area through methods described previously (10,11); and 4) 22% were in the jhs family study . The sampling frame for the family study was participants in any one of the aric, random, or volunteer samples whose family size met eligibility requirements as detailed previously (10,11). The cohort consisted of 5,035 adults aged 3584 years old, and an additional 266 participants (251 participants aged 2134 and 15 participants aged> 85) who were added as a part of the jhs family study . This resulted in a final age range of 21 to 94 years (10,11). The present study included participants who underwent multidetector ct scanning from 2007 to 2009 as a part of the second jhs examination . Of these, 1,414 (35% men) underwent multidetector ct assessment for vat and pat . Of these 1,414 participants, 1,402 had a complete covariate profile, 1,378 had an available measure of vat, and 1,298 were free of cvd . The study protocol was approved by the institutional review boards of the participating institutions: the university of mississippi medical center, jackson state university, and tougaloo college . The research ct protocol included the heart and lower abdomen, using a 16-channel multidetector ct system equipped with cardiac gating (lightspeed 16 pro; ge healthcare, milwaukee, wi). Quality control and image analysis was performed at a core reading center (wake forest university school of medicine, winston - salem, nc). The protocol included scout images, one electrocardiogram gated series of the entire heart, and a series through the lower abdomen from l3 to s1 used for assessing vat . The estimated average whole - body effective dose for the entire protocol was 4 msv . The scanning procedure for cardiac gated ct scans of the coronary arteries was based on the standard protocols developed as a part of the national heart, lung, and blood institute's multi - ethnic study of atherosclerosis (mesa) and coronary artery risk development in young adults (cardia) studies . Pat measurement was performed on the ct images after segmentation of the heart and surrounding adipose tissue from the remainder of the thorax using specific anatomic landmarks . Because it is difficult to distinguish pericardial fat from epicardial fat by the ct images, the pat in our study was measured by combination of pericardial fat and epicardial fat . For pericardial fat volume, 18 slices, 2.5 mm thick, starting 1.5 cm above and ending 3.0 cm below the superior extent of the left main coronary artery were included for coverage of 4.5 cm along the head - foot (z - axis) of the individual . The anterior border of the volume was defined by the chest wall and the posterior border by the aorta and the bronchus . This region of the heart was selected because it includes the pericardial adipose tissue located around the major epicardial coronary arteries (left main coronary, left anterior descending, right coronary, and circumflex arteries). Volume analysis software (advantage windows; ge healthcare, waukesha, wi) was used to segment and characterize each individual voxel as a tissue attenuation of fat using a threshold range of 190 to 30 hounsfield units . The pat volume was the sum of all pericardial fat voxels over the 4.5-cm volume (cubic centimeters/4.5 cm). Twenty 2.5-mm - thick slices centered on the lumbar disk space at l4l5 were analyzed . In this study, a randomly selected sample of 60 participants, the correlation coefficient between two different readers was 0.95 for vat and subcutaneous adipose tissue and 0.96 for pat, indicating excellent reproducibility of ct imaging measurements . Ct images were read by experienced and trained technologists for quantity of coronary artery calcium (cac) and abdominal aortic calcium (aac). The agatston score, modified to account for slice thickness, was used to quantify the amount of calcified artery plaque, which was computed by multiplying each lesion (area) by a weighted attenuation score (in hounsfield units) on a terarecon aquarius workstation (terarecon, san mateo, ca). Bmi was defined as weight in kilograms divided by the square of height in meters . Two measures of the waist (at the level of the umbilicus, in the upright position) were averaged to determine baseline waist circumference for each participant . Fasting blood samples were collected according to standardized procedures, and the assessments of plasma glucose, lipids, and c - reactive protein (crp) were processed at the central laboratory (university of minnesota) as described previously (10,11). Sitting blood pressure was measured twice at 5-min intervals, and the average of two measurements was used for analysis . Participants were considered to have dyslipidemia if their hdl cholesterol levels were <40 mg / dl in men and 50 mg / dl in women and/or if they had ldl cholesterol> 160 mg / dl or used lipid - lowering medication . Participants were considered to be hypertensive if they were taking antihypertensive medications, if they self - reported a diagnosis of hypertension, and/or if their systolic pressure was 140 mmhg or diastolic pressure was 90 mmhg . Diabetes was defined as a fasting plasma glucose level 126 mg / dl or treatment with insulin or a hypoglycemic agent . Modified national cholesterol education program adult treatment panel iii criteria were used to define the metabolic syndrome (8,12). The jhs physical activity cohort (jpac) survey, which was derived from the modified aric physical activity survey was administered by trained interviewers at a home visit preceding the jhs clinical examination . The total physical activity score was calculated as the sum of the four different domains of physical activity (active living, work, home and garden, and sport and exercise indexes) (13). Pat was normally distributed, and crp was normalized by logarithmic transformation and the central tendency and spread represented by the median and interquartile ranges . Age - adjusted pearson correlations of pat and vat were performed with each of the metabolic risk factors including systolic and diastolic blood pressure, fasting plasma glucose and insulin, triglycerides, hdl cholesterol, and crp, hypertension, diabetes, and dyslipidemia . To estimate the standardized effect size of pat on continuous risk factors or the odds ratio (or) on dichotomized risk factor prevalence, pat was standardized to a mean of 0 and a sd of 1 . Next, a multivariable regression model was constructed with pat as the independent variable and each of the metabolic risk factors as the dependent variable . Three models were generated in stages: 1) the multivariable - adjusted models, for which covariates in all models included age, sex, smoking and alcohol, treatment for hypertension, diabetes, and dyslipidemia; 2) a second model in which the first model was additionally adjusted for bmi; and 3) a final model, in which the first model was additionally adjusted for vat . Sex interactions were examined in the first model . Sex interactions were not significant in this study (all p> 0.05), and the results presented are for women and men combined . The association between pat and the presence of cac and aac was assessed using logistic regression analysis to estimate the or of the presence of cac and aac per 1-sd increase in pat after adjustment for 1) age and sex; 2) age, sex, and vat; 3) age, sex, smoking, alcohol, total and hdl cholesterol, systolic blood pressure, diabetes, hypertension treatment, and lipid treatment; and 4) model 3 plus vat . All computations were performed by sas software (version 9.2, sas institute, cary, nc). The research ct protocol included the heart and lower abdomen, using a 16-channel multidetector ct system equipped with cardiac gating (lightspeed 16 pro; ge healthcare, milwaukee, wi). Quality control and image analysis was performed at a core reading center (wake forest university school of medicine, winston - salem, nc). The protocol included scout images, one electrocardiogram gated series of the entire heart, and a series through the lower abdomen from l3 to s1 used for assessing vat . The estimated average whole - body effective dose for the entire protocol was 4 msv . The scanning procedure for cardiac gated ct scans of the coronary arteries was based on the standard protocols developed as a part of the national heart, lung, and blood institute's multi - ethnic study of atherosclerosis (mesa) and coronary artery risk development in young adults (cardia) studies . Pat measurement was performed on the ct images after segmentation of the heart and surrounding adipose tissue from the remainder of the thorax using specific anatomic landmarks . Because it is difficult to distinguish pericardial fat from epicardial fat by the ct images, the pat in our study was measured by combination of pericardial fat and epicardial fat . For pericardial fat volume, 18 slices, 2.5 mm thick, starting 1.5 cm above and ending 3.0 cm below the superior extent of the left main coronary artery were included for coverage of 4.5 cm along the head - foot (z - axis) of the individual . The anterior border of the volume was defined by the chest wall and the posterior border by the aorta and the bronchus . This region of the heart was selected because it includes the pericardial adipose tissue located around the major epicardial coronary arteries (left main coronary, left anterior descending, right coronary, and circumflex arteries). Volume analysis software (advantage windows; ge healthcare, waukesha, wi) was used to segment and characterize each individual voxel as a tissue attenuation of fat using a threshold range of 190 to 30 hounsfield units . The pat volume was the sum of all pericardial fat voxels over the 4.5-cm volume (cubic centimeters/4.5 cm). Twenty 2.5-mm - thick slices centered on the lumbar disk space at l4l5 were analyzed . In this study, a randomly selected sample of 60 participants, the correlation coefficient between two different readers was 0.95 for vat and subcutaneous adipose tissue and 0.96 for pat, indicating excellent reproducibility of ct imaging measurements . Ct images were read by experienced and trained technologists for quantity of coronary artery calcium (cac) and abdominal aortic calcium (aac). The agatston score, modified to account for slice thickness, was used to quantify the amount of calcified artery plaque, which was computed by multiplying each lesion (area) by a weighted attenuation score (in hounsfield units) on a terarecon aquarius workstation (terarecon, san mateo, ca). Bmi was defined as weight in kilograms divided by the square of height in meters . Two measures of the waist (at the level of the umbilicus, in the upright position) were averaged to determine baseline waist circumference for each participant . Fasting blood samples were collected according to standardized procedures, and the assessments of plasma glucose, lipids, and c - reactive protein (crp) were processed at the central laboratory (university of minnesota) as described previously (10,11). Sitting blood pressure was measured twice at 5-min intervals, and the average of two measurements was used for analysis . Participants were considered to have dyslipidemia if their hdl cholesterol levels were <40 mg / dl in men and 50 mg / dl in women and/or if they had ldl cholesterol> 160 mg / dl or used lipid - lowering medication . Participants were considered to be hypertensive if they were taking antihypertensive medications, if they self - reported a diagnosis of hypertension, and/or if their systolic pressure was 140 mmhg or diastolic pressure was 90 mmhg . Diabetes was defined as a fasting plasma glucose level 126 mg / dl or treatment with insulin or a hypoglycemic agent . Modified national cholesterol education program adult treatment panel iii criteria were used to define the metabolic syndrome (8,12). The jhs physical activity cohort (jpac) survey, which was derived from the modified aric physical activity survey was administered by trained interviewers at a home visit preceding the jhs clinical examination . The total physical activity score was calculated as the sum of the four different domains of physical activity (active living, work, home and garden, and sport and exercise indexes) (13). Pat was normally distributed, and crp was normalized by logarithmic transformation and the central tendency and spread represented by the median and interquartile ranges . Age - adjusted pearson correlations of pat and vat were performed with each of the metabolic risk factors including systolic and diastolic blood pressure, fasting plasma glucose and insulin, triglycerides, hdl cholesterol, and crp, hypertension, diabetes, and dyslipidemia . To estimate the standardized effect size of pat on continuous risk factors or the odds ratio (or) on dichotomized risk factor prevalence, pat was standardized to a mean of 0 and a sd of 1 . Next, a multivariable regression model was constructed with pat as the independent variable and each of the metabolic risk factors as the dependent variable . Three models were generated in stages: 1) the multivariable - adjusted models, for which covariates in all models included age, sex, smoking and alcohol, treatment for hypertension, diabetes, and dyslipidemia; 2) a second model in which the first model was additionally adjusted for bmi; and 3) a final model, in which the first model was additionally adjusted for vat . Sex interactions were examined in the first model . Sex interactions were not significant in this study (all p> 0.05), and the results presented are for women and men combined . The association between pat and the presence of cac and aac was assessed using logistic regression analysis to estimate the or of the presence of cac and aac per 1-sd increase in pat after adjustment for 1) age and sex; 2) age, sex, and vat; 3) age, sex, smoking, alcohol, total and hdl cholesterol, systolic blood pressure, diabetes, hypertension treatment, and lipid treatment; and 4) model 3 plus vat . All computations were performed by sas software (version 9.2, sas institute, cary, nc). The mean sd pat volume was 67.1 29 cm in women and 79.8 37.1 cm in men (table 1). Approximately 52% of participants were obese, 61% had hypertension, 39% had metabolic syndrome, and 15% had diabetes . Clinical characteristics of study participants data are means sd,% (n), or median (25th, 75th percentile) unless indicated otherwise . Age- and vat - adjusted correlation coefficients of pat with metabolic risk factors are displayed in table 2 . Pat was correlated to all of the cardiometabolic risk factors tested, with the notable exception of total cholesterol and diastolic blood pressure . However, the partial correlation coefficients of pat with cardiometabolic risk factors did not remain significant after additional adjustment for vat . Partial pearson correlation coefficients between pericardial fat volumes and metabolic risk factors p <0.0001 . Na, not available . To investigate the strength of the association of pat with both continuous and dichotomous metabolic risk factors, we performed multivariable regressions as summarized in table 3 . We observed significant associations per 1-sd increment of pat and continuous measures of systolic blood pressure, fasting glucose, triglycerides, hdl cholesterol, and log crp . However, the significant effect of pat on these risk factors became weaker or diminished when bmi or vat appeared in the same model (table 3). The ors of metabolic syndrome (1.89), hypertension (1.48), and diabetes (1.40) per 1-sd increase in pat were significant (all p <0.05). These relations became weaker or diminished after additional adjustment for bmi or vat (table 4). Multivariable adjusted regression coefficients between pericardial fat (per 1 sd) and metabolic risk factors data are multivariable (mv) regression coefficients (means se) adjusted for age, sex, smoking, alcohol and treatment of hypertension, diabetes, or dyslipidemia . Multivariable ors adjusted for hypertension, diabetes, and metabolic syndrome with an increase in pericardial fat (per 1 sd) data are multivariable (mv) ors (95% ci) adjusted for age, sex, smoking, alcohol, and treatment of hypertension, diabetes, or dyslipidemia . Generally, men had a slightly higher percentage for the presence of cac than women (49 vs. 41%) with a similar value for aac (65 vs. 61%). The relationship between pat and the presence of cac was found to be significant in the minimally adjusted model (or 1.37 [95% ci 1.201.56]; p <0.0001). This association was not materially different with additional adjustment for vat (or 1.37 [1.151.64]; p <0.0004) and persisted after additional adjustment for vat and cvd risk factors (1.34 [1.101.64]; p = 0.004), including smoking, alcohol, total and hdl cholesterol, systolic blood pressure, diabetes, hypertension treatment, and lipid treatment . Conversely, there was no association between pat and aac in the multivariable - adjusted model (1.03 [0.821.29]; p = 0.80). Age- and vat - adjusted correlation coefficients of pat with metabolic risk factors are displayed in table 2 . Pat was correlated to all of the cardiometabolic risk factors tested, with the notable exception of total cholesterol and diastolic blood pressure . However, the partial correlation coefficients of pat with cardiometabolic risk factors did not remain significant after additional adjustment for vat . Partial pearson correlation coefficients between pericardial fat volumes and metabolic risk factors p <0.0001 . To investigate the strength of the association of pat with both continuous and dichotomous metabolic risk factors, we performed multivariable regressions as summarized in table 3 . We observed significant associations per 1-sd increment of pat and continuous measures of systolic blood pressure, fasting glucose, triglycerides, hdl cholesterol, and log crp . However, the significant effect of pat on these risk factors became weaker or diminished when bmi or vat appeared in the same model (table 3). The ors of metabolic syndrome (1.89), hypertension (1.48), and diabetes (1.40) per 1-sd increase in pat were significant (all p <0.05). These relations became weaker or diminished after additional adjustment for bmi or vat (table 4). Multivariable adjusted regression coefficients between pericardial fat (per 1 sd) and metabolic risk factors data are multivariable (mv) regression coefficients (means se) adjusted for age, sex, smoking, alcohol and treatment of hypertension, diabetes, or dyslipidemia . Multivariable ors adjusted for hypertension, diabetes, and metabolic syndrome with an increase in pericardial fat (per 1 sd) data are multivariable (mv) ors (95% ci) adjusted for age, sex, smoking, alcohol, and treatment of hypertension, diabetes, or dyslipidemia . Generally, men had a slightly higher percentage for the presence of cac than women (49 vs. 41%) with a similar value for aac (65 vs. 61%). The relationship between pat and the presence of cac was found to be significant in the minimally adjusted model (or 1.37 [95% ci 1.201.56]; p <0.0001). This association was not materially different with additional adjustment for vat (or 1.37 [1.151.64]; p <0.0004) and persisted after additional adjustment for vat and cvd risk factors (1.34 [1.101.64]; p = 0.004), including smoking, alcohol, total and hdl cholesterol, systolic blood pressure, diabetes, hypertension treatment, and lipid treatment . Conversely, there was no association between pat and aac in the multivariable - adjusted model (1.03 [0.821.29]; p = 0.80). Among the jhs cohort of 1,414 participants undergoing multidetector ct scanning, pat was found to be significantly associated with cardiometabolic risk factors . However, most of the observed associations were attenuated or diminished with additional adjustment for bmi or vat, suggesting that pat is not more strongly correlated to cardiometabolic risk factors than vat or bmi . In contrast, commensurate with its proposed primary role as a locally acting fat depot, pat was predominantly associated with cac but not with aac even after vat and other relevant cvd risk factors were accounted for . Our findings have important implications for the role of pat in relation to cardiometabolic risk factors and vascular calcification . Anatomically, the coronary arteries and the myocardium of both the right and left ventricles are surrounded by or in direct contract with this small fat depot . Consequently, this close proximity of pat to the coronary artery and the underlying myocardium may exert a local toxic effect on the nearby organs rather than a systemic effect on cardiometabolic risk factors . Our data support the hypotheses that pat is associated with cardiometabolic risk factors because of shared risk factors with vat and that the independent association of pat with cac is probably due to its close anatomic relationship with the coronary vasculature . More importantly, despite lower amounts of vat at similar levels of bmi in african americans (14), the associations of pat with cardiometabolic risk factors and cac found in this african american cohort is consistent with the findings from the framingham heart study (15). Pat has been found in several clinical studies to be significantly associated with clinical key components of metabolic syndrome and insulin resistance including bmi, waist circumference, elevated levels of blood pressure, glucose, ldl cholesterol, hdl cholesterol, triglycerides, and inflammatory cytokines including high - sensitivity crp (3,4,7,15), and it has been proposed as a new indicator of cardiovascular risk (4). However, such an association may result from the systemic effects of the abdominal visceral fat depot because pat is highly correlated with abdominal visceral fat . Furthermore, a large sample from the framingham heart study demonstrated that the association of pat with these cardiometabolic risk factors was weaker or diminished when vat was taken into account (1,3). These data suggest that pat may be a residual marker of visceral adiposity . As a part of the metabolically active visceral fat depot, pat is an important local source of free fatty acids and a number of proatherogenic, proinflammatory, and prothrombotic hormones, and cytokines, including leptin, monocyte chemotactic protein-1, interleukin-6, and tumor necrosis factor- (6,1619). In addition, pat is usually located in close proximity to the coronary vasculature compared with other fat depots . Such distinguishing biochemical properties and the unique anatomic location of pat are hypothesized to enhance its paracrine role in the development of coronary atherosclerosis (1,20,21). Therefore, a local interaction between pat and the nearby coronary vasculature may explain the association between pat with cac observed in our data . Strengths of this study include the large sample size from a community - based cohort of african americans, simultaneous volumetric quantification of pat and vat, and multiple adjustments for potential confounders . Limitations include 1) the cross - sectional study design, which limits our ability to infer causality; 2) the primarily african american sample (although this may be seen as a limitation of the current study, it is important to note that our results are consistent with the findings from the framingham heart study, suggesting that our findings may apply across the ethnic groups); and 3) the potential misclassification of pat due to combined measurement of pericardial and epicardial fat inherent in our methodology . In addition, because the ct - measured pat and vat were collected several years after clinical data were collected, this time gap between clinical data collected and ct measurements could result in misclassification of risk factors . However, this limitation should not affect the relative association among pat, vat, and cac, which were measured contemporaneously on the same ct scans . In summary, pat is correlated with most cardiometabolic risk factors but not more strongly than vat . However, pat is significantly associated with cac, suggesting that pat may exert a local toxic effect on the coronary vasculature in the jhs cohort . Among the jhs cohort of 1,414 participants undergoing multidetector ct scanning, pat was found to be significantly associated with cardiometabolic risk factors . However, most of the observed associations were attenuated or diminished with additional adjustment for bmi or vat, suggesting that pat is not more strongly correlated to cardiometabolic risk factors than vat or bmi . In contrast, commensurate with its proposed primary role as a locally acting fat depot, pat was predominantly associated with cac but not with aac even after vat and other relevant cvd risk factors were accounted for . Our findings have important implications for the role of pat in relation to cardiometabolic risk factors and vascular calcification . Anatomically, the coronary arteries and the myocardium of both the right and left ventricles are surrounded by or in direct contract with this small fat depot . Consequently, this close proximity of pat to the coronary artery and the underlying myocardium may exert a local toxic effect on the nearby organs rather than a systemic effect on cardiometabolic risk factors . Our data support the hypotheses that pat is associated with cardiometabolic risk factors because of shared risk factors with vat and that the independent association of pat with cac is probably due to its close anatomic relationship with the coronary vasculature . More importantly, despite lower amounts of vat at similar levels of bmi in african americans (14), the associations of pat with cardiometabolic risk factors and cac found in this african american cohort is consistent with the findings from the framingham heart study (15). Pat has been found in several clinical studies to be significantly associated with clinical key components of metabolic syndrome and insulin resistance including bmi, waist circumference, elevated levels of blood pressure, glucose, ldl cholesterol, hdl cholesterol, triglycerides, and inflammatory cytokines including high - sensitivity crp (3,4,7,15), and it has been proposed as a new indicator of cardiovascular risk (4). However, such an association may result from the systemic effects of the abdominal visceral fat depot because pat is highly correlated with abdominal visceral fat . Furthermore, a large sample from the framingham heart study demonstrated that the association of pat with these cardiometabolic risk factors was weaker or diminished when vat was taken into account (1,3). These data suggest that pat may be a residual marker of visceral adiposity . As a part of the metabolically active visceral fat depot, pat is an important local source of free fatty acids and a number of proatherogenic, proinflammatory, and prothrombotic hormones, and cytokines, including leptin, monocyte chemotactic protein-1, interleukin-6, and tumor necrosis factor- (6,1619). In addition, pat is usually located in close proximity to the coronary vasculature compared with other fat depots . Such distinguishing biochemical properties and the unique anatomic location of pat are hypothesized to enhance its paracrine role in the development of coronary atherosclerosis (1,20,21). Therefore, a local interaction between pat and the nearby coronary vasculature may explain the association between pat with cac observed in our data . Strengths of this study include the large sample size from a community - based cohort of african americans, simultaneous volumetric quantification of pat and vat, and multiple adjustments for potential confounders . Limitations include 1) the cross - sectional study design, which limits our ability to infer causality; 2) the primarily african american sample (although this may be seen as a limitation of the current study, it is important to note that our results are consistent with the findings from the framingham heart study, suggesting that our findings may apply across the ethnic groups); and 3) the potential misclassification of pat due to combined measurement of pericardial and epicardial fat inherent in our methodology . In addition, because the ct - measured pat and vat were collected several years after clinical data were collected, this time gap between clinical data collected and ct measurements could result in misclassification of risk factors . However, this limitation should not affect the relative association among pat, vat, and cac, which were measured contemporaneously on the same ct scans . In summary, pat is correlated with most cardiometabolic risk factors but not more strongly than vat . However, pat is significantly associated with cac, suggesting that pat may exert a local toxic effect on the coronary vasculature in the jhs cohort.
Annually, 80,000 anterior cruciate ligament (acl) injuries occur in the united states with an estimated cost of almost a billion dollars . The most common mechanisms of acl injury are noncontact in nature, characterized by sudden deceleration prior to a landing motion or change of direction [2, 3]. These noncontact injuries may be due to coordination failure involving a complete and momentary loss of normal protective muscle support . People at high risk of acl injury frequently demonstrate high dynamic knee valgus (i.e., knee abduction moment) during landing from jumping, which may be due to decreased neuromuscular control [3, 5]. Current literature has proposed several laboratory - based tools to identify risk factors for acl injury . However, these screening tools require expensive 3d motion capture equipment, highly trained staff, and significant amount of time to administer and analyze rendering these tools inefficient and impractical for a clinical setting . Several jumping and landing tests are used in the clinical setting, including the landing error scoring system (less), the drop jump video screening test, and the tuck jump assessment (tja) [69]. For example, the tja protocol, unlike the other two tests, starts and stops from ground level instead of jumping from a box; this better represents techniques encountered in normal jumping activities [6, 8]. The tja protocol also requires participants to jump for 10 seconds, while the less and drop jump video screening test require only 1 to 2 jumps [69]. Therefore, the tja evaluates a measure of performance endurance, introducing a potential fatigue effect that might highlight landing flaws not observable in 1 to 2 jumps [69]. Plyometric assessment, identifying jumping and landing technique flaws pertaining to risk of acl injury [8, 10]. Although there are no published reports of the tja being widely used, the availability of test protocol and minimal equipment required may make this a favored tool to use in varied clinical settings with diverse personnel . The tja includes 10 technique flaws related to jumping and landing that are scored qualitatively as either having the flaw or not [6, 8]. Empirical evidence suggests a participant who demonstrates greater than or equal to 6 out of 10 technique flaws during the tja should be targeted for intervention to address flaws, such as correcting lower extremity valgus at landing [6, 8, 10]. The tja requires minimal equipment (e.g., video cameras and tape markers) and takes only several minutes to administer . Scoring follows standard criteria for each technique flaw and can be completed relatively quickly by watching video playback . These features make the tja a practical screening tool for injury risk assessment in a clinical setting for people of different educational backgrounds and levels of experience, as it is currently being used . Previous literature reported the interrater reliability of the tja as high with percentage exact agreement (pea) of two testers across all scoring criteria of 93% (range 80%100%) when scoring 10 participants . The same study also reported intrarater reliability to be high (pea of 96%100%). Although reliability was reported high, several limitations in study design necessitate further study of reliability of the tja . Specific information concerning training and background of tja scoring for the raters was not included, limiting generalizability to clinicians unfamiliar with jumping assessment . Additionally, the small sample size may have allowed raters to remember previous scores, introducing bias, resulting in higher intrarater reliability . Reliability has not been tested with raters of different educational backgrounds or levels of experience . Demonstrating that scoring is consistent between raters of different educational and experiential backgrounds would allow implementation of the tja in the clinical and performance settings with improved understanding of accuracy . The purpose of this study was to investigate intrarater and interrater reliability of the tja with raters of different educational backgrounds and levels of clinical experience with healthy injury - free men and women . The hypothesis for this study was that the raters would demonstrate good intra- and interrater reliability for the tja and that there would be no difference in tja total score between raters of different educational and/or experiential backgrounds . A sample of 108, both undergraduate and graduate, recreationally active students who were not currently involved in college athletics, were recruited for participation . All participants were healthy, injury - free men and women, 18 to 24 years old, without prior tuck jump training . From this cohort, the videos of 40 participants (n = 13 men and n = 27 women) were randomly selected for this reliability study . Participants received a full explanation of the nature, purpose, and risks of the study and were given the opportunity to ask questions . The physical activity readiness questionnaire (par - q) was administered to screen for contraindications for testing . All participants signed an informed consent document approved by the institutional review board at northern arizona university before participating in the study . Prior to participants completing the tja, height and weight were measured with a wall - mounted stadiometer and digital scale (cardinal scale, webb city, mo, usa). The tja was performed using instructions from a previously published tja study by the developers of the test . Initial set up for the tja required 2 two - dimensional video cameras (sony handycam, sony corporation, san diego, ca and jvc camcorder jvc americas corporation, wayne nj) on tripods to provide sagittal and frontal views of the participants . Two pieces of masking tape were placed on the ground, parallel to each other 8 inches apart . Participants were instructed to stand with one foot on each tape strip to ensure proper positioning for the cameras (figure 1). The participants were instructed in purpose and protocol of tja test which included: jumping repeatedly for 10 seconds with high effort level, bringing knees up as high as possible so both thighs were parallel with the ground, landing softly in the same footprint (2 pieces of tape) with each jump, and then immediately begin the next jump . No feedback was given to participants while performing the assessment . Technique flaws included: (1) lower extremity valgus at landing (figure 2), (2) thighs do not reach parallel (peak of jump), (3) thighs not equal side - to - side (during flight), (4) foot placement not shoulder width apart, (5) foot placement not parallel (front to back), (6) foot contact timing not equal, (7) excessive landing contact noise, (8) pause between jumps, (9) technique declines prior to 10 seconds, and (10) does not land in same footprint (excessive in - flight motion). Additional figures depicting these technique flaws can be found in previously published tja studies [6, 8, 10]. The participants were rated as either demonstrating a technique flaw or not . Per previously published literature, the flaws were then summed for the tja total score [6, 8]. Five raters of varying educational backgrounds and clinical experiences were chosen to analyze video and score the tja of the 40 participants . Raters included a physical therapist with a doctor of physical therapy degree and 4 years clinical experience (rater 1); the head strength and conditioning coach at a division 1 university with a masters of science in exercise science and strength and conditioning specialist certification and 7 years of clinical experience (rater 2); the head athletic trainer as a certified athletic trainer at a division 1 university with 17 years of clinical experience (rater 3); a third - year doctor of physical therapy student (rater 4); and a first - year doctor of physical therapy student (rater 5). All raters were given identical flash drives with instructions, a microsoft access database to input scores, a copy of myer et al . That described the tja and scoring in detail, 80 video files for 40 participants (frontal and sagittal plane view for each participant), and 2 video files for 1 example participant (frontal and sagittal plane views) previously scored independently by 3 of the authors of this paper (l. a. dudley, c. a. smith, and m. warren). The sample video was chosen from the larger sample of 100 to assist with consistency training of the raters and was not included in the 40 participants evaluated by raters . Raters were instructed to read an excerpt from myer et al . To create consistency between raters regarding the scoring procedures of the tja . This included looking at the tja scoring tool created with pictures of the first 6 technique flaws to ensure consistency with previously established tja scoring procedures . Raters were then instructed to watch and score the example video, which had the purpose of ensuring consistency in procedures between all raters and establishing the same volume setting to accurately analyze technique flaw number 7 (excessive contact landing noise). Once raters accurately scored the example video, participants' videos were scored independently, with no discussion amongst raters . Raters were instructed to use frontal plane views to score technique flaws (1), (3), (4), (6), and (7); sagittal plane views for (2) and (5); and frontal and sagittal views for (8), (9), and (10). Raters were given instructions on how to change playback speed on the computer program to allow viewing of the video in slow motion . Raters were encouraged to watch the videos as many times as necessary to give an accurate score . Approximately 1 month later, 3 of the 5 raters scored the videos again to determine intrarater reliability . Descriptive statistics were calculated as means with standard deviation for interval data and percents for categorical data . Analysis of variance with tja total score as the dependent variable and rater as the independent variable was completed to assess differences in mean values between raters . Intraclass correlation coefficients (icc, 2,1) and associated 95% confidence intervals (ci) were calculated to determine intrarater (3 different raters at 2 timepoints) and interrater (5 raters for first session and 3 raters for second session) reliability of the total tja score . The clinical significance was defined as poor for an icc below 0.50, moderate for 0.500.75, and good for 0.75 or higher . Icc was calculated as (1)icc=2b2b+2w, where b is the between person variance and w in the within person variance . Maximum likelihood estimates of b and w were obtained from linear mixed models with rater as a fixed effect for interrater reliability . Least square means were calculated for the mean tja scores for each rater, with p values calculated to determine significant differences in tja scores . All analyses were completed using sas, version 9.2 (sas institute, inc ., cary, nc) and an a priori alpha level of 0.05 was used to denote statistical significance . Participants in this study were 40 university students, 18 to 24 years of age (mean age standard deviation (sd) 21.0 1.6 years). There were 13 males and 27 females with an average height and weight of 170.8 8.9 cm and 67.4 14.7 kg, respectively . The average number of flaws identified on the tja (tja total score) were 6.30 1.76; and technique flaw (2) (thighs do not reach parallel) was the most frequently identified (87.5% of participants), using scoring from rater 5 who was randomly chosen . Additionally, technique flaw (2) was the most consistently agreed upon by all raters . Technique flaws (5) (foot placement not parallel front to back) and (9) (technique declines prior to 10 seconds) were the least agreed upon by all raters . Analysis of variance showed differences between raters for mean tja total score (f = 11.82; p <0.001). Post hoc comparisons showed no consistent pattern in scoring by educational and/or clinical experience (table 1). Interrater reliability between the 5 raters was poor (icc = 0.47; 95% ci: 0.330.62). However, interrater reliability between raters 1, 2, and 5 (raters used for intrarater reliability) improved in the second scoring session (icc = 0.52, 95% ci: 0.350.68 versus icc = 0.69, 95% ci: 0.550.81). Intrarater reliability testing was completed by raters 1, 2, and 5 who scored the tja videos at two different timepoints . The purpose of this study was to investigate intrarater and interrater reliability of the tja with raters of different educational backgrounds and levels of clinical experience with healthy injury - free men and women . This study showed intrarater reliability to be poor to moderate for the 3 raters scoring videos of 40 participants . It is noteworthy that those with more education and experience were not more consistent in scoring the tja than those raters with minimal education and experience . Rater 5 had highest intrarater reliability of the 3 raters and also had the most experience administering the tja . These results suggest the tja may not be used reliably, following published protocol, by a single clinician regardless of level of education or experience [6, 8, 11]. This study also showed poor interrater reliability between 5 raters when assessing tja performance of the same 40 participants . This suggests the tja may not be used reliably, following published protocol, when being scored by different raters [6, 8, 11]. However, interrater reliability improved the second session between the 3 raters that scored the videos in 2 separate sessions . Additional training beyond what was done in the study, as well as practice scoring to achieve consensus between raters, may be required . These results are in contrast to herrington et al . Who reported the tja had good intrarater and interrater reliability (greater than 0.75) when 2 raters scored videos of 10 recreationally active university students at 2 different scoring sessions . Did not specify raters' educational background, level of experience, or previous training in the tja . However, as this study was written by a developer of the tja, the two raters likely had more experience in scoring the tja and were more familiar with the flaws than the 5 raters of this study . The improved interrater reliability in the current study from session one to session two suggested a possible learning effect . Further, the small sample size used by herrington et al . Could promote bias due to raters remembering the scores of only 10 participants . The authors of the current study took measures to ensure continuity with previously established tja procedures and scoring criteria . However, current tja instructions do not specify whether a technique flaw should be scored only if it is seen consistently throughout the test or if the technique flaw may be scored if seen only once during the test . Due to this exclusion in the published protocol, it is likely that raters may inherently interpret this differently, thus creating scoring inconsistencies between raters . For example, during a 10 second tja a participant may demonstrate lower extremity valgus at landing only once throughout several jumps . One rater may score this as a technique flaw because it was demonstrated in the assessment; however, another rater may not score this as a technique flaw because the majority of jumps did not demonstrate lower extremity valgus at landing . More training and specific tja instruction prior to administration may correct this issue and promote consistency between raters . The current study includes the most diverse set of raters to date, representing 3 professions that may administer the tja . This allows for greater external validity in the clinical and performance setting than other published reliability studies to date . However, there are limitations of the current study . Participants in the current study were not competitive athletes, though were recreationally active university students . The results of this study suggest the tja may not be consistently scored, following the previously published protocol, although modifications to instructions and rater training may correct this . Since, further research is necessary to determine whether more specific scoring guidelines or training in tja administration would improve reliability . Using a published protocol and training of raters, the tja has poor to moderate interrater and intrarater reliability . There may be a learned effect with the tja since interrater reliability improved with repetition . Enhanced training or scoring instructions may be required to improve reliability of the tja among professionals of varying education backgrounds and experience.
Currently available genotyping methods, based on reverse hybridization with subtype - specific primers and probes targeting the 5-untranslated region (5-utr) and core regions (versant hcv genotype 2.0 system; siemens healthcare diagnostics, milan, italy), as well as real - time pcr assays based on 5-utr and ns5b sequencing (abbott hcv genotype ii assay; abbott diagnostics, lake forest, il, usa), accurately differentiate major hepatitis c virus (hcv) genotypes in the majority of cases and are widely used because of their technical simplicity and lower costs.1 routine genotyping methods, however, may cause wrong, inaccurate, or incomplete assignment in up to 10% of cases, due to indeterminate results, mixed infections, wrong subtyping, and even wrong genotyping, as we recently reported.2 misclassifications of hcv genotype were described in characterization of genotypes 5 and 6 and in subtyping of genotypes.26 versant hcv genotype 2.0 fails to identify genotype 1 subtype in 2.2%7.4% of the cases.5 in particular, cause of misclassification was due to variability of 5-utr of hcv . This region is not appropriate for discriminating hcv strains at the subtype level and for distinguishing many genotype 6 samples from genotype 1 as well as for distinguishing subtypes within genotypes 1, 2, and 3.6 incorrect assignments of genotype were associated with nucleotide polymorphisms in the 5-utr of hcv that may alter the ability of probe / primer of commercial assays to recognize viral strains . Indeterminate genotype results were reported for changes at positions 166 and 119 in genotype 2b and 138, 108, and 99 in genotype 3h of 5-utr of hcv.7 as most of the currently available therapeutic options target hcv genotype 1 strains, treatment of other genotypes, in particular genotype 3, is presently less efficient and more costly when directly acting antivirals (daas) are used.8 reassessment of hcv genotype and subtype by sequencing prior to interferon - free regimens may reduce the risk of treatment failure and consequent undue pharmacological costs driven by inappropriate genotype characterization and may, therefore, turn out quite cost beneficial.2,9 we investigated the cost - effectiveness of re - evaluating hcv genotype by population sequencing, prior to choosing a daa regimen among currently available options, in a mono - centric italian cohort . Between march, 2015, and september, 2015, at the liver and infectious diseases units of pescara general hospital, italy, hcv sequence analysis was performed in order to confirm previous commercial assignment by a second - generation lipa - hcv genotype assay (versant hcv genotype 2.0; siemens healthcare diagnostics) in all consecutive candidates to treatment with daas . The local health district authority was requested to evaluate the possible benefits of the experimental procedure, and found it likely cost - effective and funded 200 consecutive assays for the purpose of this evaluation . Direct population sequencing was, therefore, performed for both ns5a and ns3 hcv genes at the university of tor vergata (rome) through highly efficient home - made protocols as previously reported.2 expert interpretation of mutations and sequence variants detected was also provided by the same source (university of rome tor vergata) for each patient, to help in choosing the most appropriate and individualized regimen . The turnaround time allowance from sample dispatching to availability of assay results was set at a maximum of 3 weeks, to avoid undue delaying of treatments . For the purpose of the present study, in each case of resolved or discordant hcv genotype and/or subtype after sequencing, we calculated the difference in treatment costs between the most likely prescription that would have been possible on the basis of previously available commercial hcv genotype and subtype only, and the most likely prescription made possible by the availability of sequence results with expert interpretation . Interferon - free regimens were prescribed by attending specialists in accordance with current european association for the study of the liver guidelines and italian regulatory agency (aifa) reimbursement requirements.10,11 sustained virologic response 12 (svr-12) was defined as undetectable viral load at 12 weeks after treatment . Expected efficacy for each prescribed regimen was derived by currently available published svr-12 data for selected reference treatments . The costs considered in the analysis are direct medical costs (referred to 2015) related to each prescribed drug and/or regimen, retrieved from the pharmacy unit of pescara general hospital, and the genotypic test reimbursement received by the hospital from the regional health service (ie, 60 for lipa - hcv genotype assay and 500 for sequence analysis of hcv genotype). As a consequence, in the analyses of actual costs, reimbursements from the pharmaceutical companies were not considered, as the local pharmacy did not receive any refund sum as of date (february 8, 2016). The cost - effectiveness analysis was conducted considering the aforementioned direct medical costs and efficacy values for each patient in two scenarios: with the use of sequence analysis of hcv genotype vs use of lipa - hcv genotype assay . The time horizon considered was up to 12 weeks after hcv antiviral therapy conclusion . To test the robustness of the results, a resampling bootstrapping analysis was performed, simulating 500 cohorts of 134 patients, randomly extracting patients from the base case scenario . The local health administrative board in chieti - pescara reviewed in detail the study design, set up by the infectious diseases staff at pescara general hospital . The study was thoroughly discussed by the direzione generale strategica della azienda unit sanitaria locale di pescara / comitato etico di chieti / pescara . A final authorization was granted by direzione generale strategica della azienda unit sanitaria locale di pescara early in 2015, as documented in issue deliberazione del direttore generale number 319 march 16, 2015 . Written informed consent the local health administrative board in chieti - pescara reviewed in detail the study design, set up by the infectious diseases staff at pescara general hospital . The study was thoroughly discussed by the direzione generale strategica della azienda unit sanitaria locale di pescara / comitato etico di chieti / pescara . A final authorization was granted by direzione generale strategica della azienda unit sanitaria locale di pescara early in 2015, as documented in issue deliberazione del direttore generale number 319 march 16, 2015 . Written informed consent was obtained from all participants . During the study period, 134 consecutive patients were included . Starting from january 2008, routine genotype and subtype assays, we found three (2.3%) discordant genotypes, nine (6.7%) discordant g1 subtypes, four (3%) mixed infections, and five (3.7%) indeterminate g1 subtypes that were then correctly attributed . Suboptimal treatment choice due to wrong genotype assessment in the three individual cases would have caused a reduction in efficacy which we gauged at our best, based on literature data, as ranging between 17% and 40% (table 1). G1a was assigned by sequencing instead of g1b by commercial assays in eight out of nine cases with incorrect g1 subtype . This would have caused suboptimal treatment courses, with an expected decrease in efficacy from 5% to 7% . Conversely, in the single opposite case (g1b assignment instead of g1a), the consequence would have been a slight increase in costs and the requirement of ribavirin use (table 2). For patients with g1 and indeterminate subtype, the choice of regimens would have targeted the subtype 1a option, with a potential sensible increase in costs (table 2). Finally, the possible consequences of inappropriate prescription due to mixed hcv infections are shown in table 3 . In these cases, pan - genotypic regimens would be mandatory, with an increase in costs up to 32,000 per treatment . The use of sequence analysis of hcv genotype, compared with lipa - hcv genotype assay, would lead to a mean increase of efficacy (+ 1.42%) and per capita costs (+ 1,280 per patient). The 2.4% of the sensitivity analysis simulations led to a dominance of sequence analysis of hcv genotype compared with lipa - hcv genotype assay, reducing costs and increasing efficacy, while the remaining 97.6% of the results showed an increase in costs and efficacy (between 1,269 and 4,617, and 1.43% and 3.88%) with incremental cost - effectiveness ratios between 845/efficacy unit and 2,009/efficacy unit . Although some studies found uninterpretable or indeterminate genotype assignations in varying percentages of assayed samples, especially in non - g1 hcv infections,4,1216 the possible consequences of hcv genotype misclassification on treatments with daas were not yet evaluated and may have been perceived as a minor problem so far . However, in the new scenario of hcv therapy, the costs of genotyping by sequencing are definitely lower than those of daa regimens, so that even a few treatment failures averted may make reassessing hcv genotype before daas very convenient.2 as a consequence, studies aimed at gauging the improvement of treatment outcomes by the systematic deployment of sequence genotype should be encouraged . We introduced mandatory sequencing assays of hcv prior to hcv therapy with daas at our site, and compared the results of all consecutive assays performed with previous routine genotype assessments, in an attempt to pinpoint all possible differences in treatment efficacy and on possible parallel cost savings obtained in this way . Although based at a single italian site among the many authorized for the prescription of new treatments for hcv, one major strength of our experimental design was the consecutive enrolment to sequence genotyping of all daa eligible patients . This allowed a close estimate of the benefits of this investigational procedure in a relatively small sample of treated patients . We found percentages of hcv genotype / subtype misclassification approximately in line with those reported by others,3,13,1618 identifying four main groups of failures in routine genotyping . These would have had the potential of causing remarkable reductions in efficacy up to 40% of their respective daa treatments, which means a high likelihood of inducing at least one treatment failure . This first group alone justified our effort and covered all study expenses . In the second and third groups these would not have caused a significant reduction in efficacy; however, significant increases in costs would have occurred, especially for patients receiving a diagnosis of genotype 1a instead of 1b, and unduly longer and more toxic treatments . In these patients, the reduction in potential toxicity was an added and precious value on the top of averted expenses . In the last group of patients, mixed infections would have invariably induced the prescription of pan - genotypic regimens, causing a remarkable increase in costs, at least 50% greater than necessary . The cost - effectiveness results show an overall increase in terms of efficacy due to the use of sequence analysis of hcv genotype along with a slight increase in terms of costs . The cost per unit of efficacy gained is difficult to interpret due to the lack of threshold values associated with svr at 12 weeks for hcv - infected patients . The analysis performed does not consider the savings due to the mean efficacy increase and the need of further antiviral treatments and monitoring for patients not reaching svr in the lipa - hcv genotype assay scenario . Moreover, adverse events related costs were not considered in the analysis due to the absence of interferon in the antiviral treatments considered (assuming similar safety profiles), which would, however, increase the direct medical costs due to retreatments . Further cost savings not considered in the analysis due to higher efficacy of treatments are those related to the lack of progression of hcv infection . Future analyses should include a measurement of monitoring activities, adverse events management, and follow - up . The main limit of the cost - effectiveness analysis performed is related to the short - term time horizon and the lack of use of effectiveness parameters that allow to interpret incremental cost - effectiveness ratio of technology use based on national recognized thresholds (ie, quality adjusted life years). Overall, our experience highlights the importance and convenience of accurate hcv genotyping before current daa regimens . If wrong or incomplete, hcv genotype information would have caused a preventable risk of treatment failure . The relatively low incremental costs of hcv sequence analysis compared with the current costs of daas suggests that precise assignation of hcv genotype and subtype by sequencing may be beneficial until new and more potent pan - genotypic regimens will be available for all patients . Therefore, for a personalized and tailored therapy, genotype by sequencing should be performed in advance of first - line treatment with daas . Further analyses on the cohort, considering a longer time horizon, should investigate if, as potentially suggested by the results of the analysis presented, the use of sequence analysis of hcv genotype would lead to a decrease in direct medical costs for the treatment of hcv - infected patients, due to savings for the lower number of retreated patients and lack of disease progression.
Advancing age is related to considerable changes in mental and physical health, including loss of muscle mass (sarcopenia) and muscle function . This can lead to impaired physical ability and reduced quality of life.13 after the age of 70 years, about 1.5% of muscle mass is lost per year.4 denervation, metabolic, hormonal, or immunological reasons and the reduction of physical activity, in particular, contribute to sarcopenia.5 however, muscles can maintain a high degree of plasticity in advanced age, whereas tendons lose their plasticity predisposing them to injuries.6 resistance training may reverse tendon stiffness and reduce the risk of strain injuries . Furthermore, it can increase muscle strength and improve mobility as well as physical functioning in the elderly.7 several studies have investigated exercise programs for the very elderly and found moderate to very high improvements in muscle strength, balance, gait speed, and other outcomes that are indispensable for an independent life.810 however, most studies examined healthy and community dwelling elderly,1113 whereas persons with impaired physical ability as well as nursing - home residents or persons in long - term care facilities were underrepresented.14 a recent systematic review on the effects of progressive resistance training in elderly nursing - home residents showed that the intensity of the training should be vigorous and the duration at least 2 months.15 most authors describe a frequency of three training sessions per week, but the optimal frequency has not been defined.1619 therefore, the aim of this study was to investigate how much progressive strength training twice a week over a period of 8 weeks improves mobility, muscle strength, and quality of life in nursing - home residents with impaired mobility aged 75 years and older . Between may and july 2009, 34 nursing homes in berlin were contacted by phone . Out of the twelve that agreed to have a personal meeting with more detailed information, seven homes were excluded due to lack of interest in participation, residents without impaired mobility, or residents were looked after by a legal guardian . Nursing - home residents were included if they fulfilled all of the following criteria: (1) 75 years of age and older; (2) mild to severely impaired mobility defined as an elderly mobility scale (ems) score between six and 18 points; (3) sufficient language skills; (4) written informed consent; and (5) written consent by their general practitioner . Exclusion criteria were: (1) moderate to severe dementia (mini - mental state examination <18 points;20 (2) rheumatologic, orthopedic, or any other condition that could be aggravated by sport; (3) elevated systolic / diastolic blood pressure during exercise (> 230/110 mmhg); (4) cardiac arrhythmia; (5) epilepsy; or (6) legal care . The ethical review committee of the charit university medical center (berlin, germany) approved the study . Participants underwent a progressive resistance training program twice a week for 8 weeks using the following six gym machines: chest press, rowing machine, and butterfly reverse for the upper limb, leg press and leg extension for the lower limb, and a crunch trainer for the abdominals (gym80 international gmbh, gelsenkirchen, germany). Training was performed in three sets of eight repetitions of the individual lifted weight . Between the sets, participants had to pause for at least 1 minute ., all participants received an individual detailed introduction at each gym machine by qualified fitness trainers . The 15 participants were divided into two groups, which trained separately and were supervised by the trainers and the study investigator throughout the program . At each machine, height and angle of the seat participants who needed help to get on the seat were supported by the training staff . Sitting height and position as well as velocity and range of movements at each machine the lifted weight was set by the training staff, documented, and regularly adapted to the augmented muscle strength of participants . As soon as a participant could lift the weight more than eight times in a row in all of the three sets, the weight was increased to the next level . Primary outcome of the study was the change in mobility determined by the standardized and validated ems, a seven - item instrument with a good sensitivity and practicability, assessed by the study physician . The score ranges from zero (minimal mobility) to 20 (maximal mobility).2123 secondary outcomes were changes in muscle strength and quality of life . Muscle strength was measured indirectly by documenting the respective lifted weight . For the assessment of quality of life, the short form-36 health survey (sf-36) was used.24 all participants underwent a short medical exam including a resting electrocardiogram in the department of sports medicine at the charit university medical center . In the gym, the trainers determined the individual eight - repetition maximum, defined as the highest weight that a participant can lift a maximum of eight times in a row . It is comparable to 80% of the one - repetition maximum (the highest weight a person can lift only once). The latter was not used due to a higher risk of causing blood pressure peaks in comparison to the eight - repetition maximum . During this introductory exercise, blood pressure was measured after every weightlifting series at each machine to detect potential blood pressure peaks . All participants received a free 3-monthly membership for the gym including insurance, which covered possible accidents occurring during the stay at the gym . During transportation from the nursing home to the gym, they were insured by a private transportation company . A pre post comparison of assessed outcomes (mean standard deviation) was carried out by the wilcoxon signed - rank test for paired samples . The alpha - type error was set at 0.05, with a power of 80% . Data analysis was conducted with spss 12.0.0 (spss inc, chicago, il, usa). The outcome variables mobility and muscle strength were assessed as continuous variables from zero (minimum) to 20 (maximum ems score) and lifted weight in kilograms, respectively . Between may and july 2009, 34 nursing homes in berlin were contacted by phone . Out of the twelve that agreed to have a personal meeting with more detailed information, seven homes were excluded due to lack of interest in participation, residents without impaired mobility, or residents were looked after by a legal guardian . Nursing - home residents were included if they fulfilled all of the following criteria: (1) 75 years of age and older; (2) mild to severely impaired mobility defined as an elderly mobility scale (ems) score between six and 18 points; (3) sufficient language skills; (4) written informed consent; and (5) written consent by their general practitioner . Exclusion criteria were: (1) moderate to severe dementia (mini - mental state examination <18 points;20 (2) rheumatologic, orthopedic, or any other condition that could be aggravated by sport; (3) elevated systolic / diastolic blood pressure during exercise (> 230/110 mmhg); (4) cardiac arrhythmia; (5) epilepsy; or (6) legal care . The ethical review committee of the charit university medical center (berlin, germany) approved the study . Participants underwent a progressive resistance training program twice a week for 8 weeks using the following six gym machines: chest press, rowing machine, and butterfly reverse for the upper limb, leg press and leg extension for the lower limb, and a crunch trainer for the abdominals (gym80 international gmbh, gelsenkirchen, germany). Training was performed in three sets of eight repetitions of the individual lifted weight . Between the sets, participants had to pause for at least 1 minute ., all participants received an individual detailed introduction at each gym machine by qualified fitness trainers . The 15 participants were divided into two groups, which trained separately and were supervised by the trainers and the study investigator throughout the program . At each machine, height and angle of the seat participants who needed help to get on the seat were supported by the training staff . Sitting height and position as well as velocity and range of movements at each machine the lifted weight was set by the training staff, documented, and regularly adapted to the augmented muscle strength of participants . As soon as a participant could lift the weight more than eight times in a row in all of the three sets, the weight was increased to the next level . Primary outcome of the study was the change in mobility determined by the standardized and validated ems, a seven - item instrument with a good sensitivity and practicability, assessed by the study physician . The score ranges from zero (minimal mobility) to 20 (maximal mobility).2123 secondary outcomes were changes in muscle strength and quality of life . Muscle strength was measured indirectly by documenting the respective lifted weight . For the assessment of quality of life, the short form-36 health survey (sf-36) was used.24 all participants underwent a short medical exam including a resting electrocardiogram in the department of sports medicine at the charit university medical center . In the gym, the trainers determined the individual eight - repetition maximum, defined as the highest weight that a participant can lift a maximum of eight times in a row . It is comparable to 80% of the one - repetition maximum (the highest weight a person can lift only once). The latter was not used due to a higher risk of causing blood pressure peaks in comparison to the eight - repetition maximum . During this introductory exercise, blood pressure was measured after every weightlifting series at each machine to detect potential blood pressure peaks . All participants received a free 3-monthly membership for the gym including insurance, which covered possible accidents occurring during the stay at the gym . During transportation from the nursing home to the gym, a pre post comparison of assessed outcomes (mean standard deviation) was carried out by the wilcoxon signed - rank test for paired samples . The alpha - type error was set at 0.05, with a power of 80% . Data analysis was conducted with spss 12.0.0 (spss inc, chicago, il, usa). The outcome variables mobility and muscle strength were assessed as continuous variables from zero (minimum) to 20 (maximum ems score) and lifted weight in kilograms, respectively . Fifteen nursing - home residents aged 7797 years (mean age 84 years) were recruited . In the current analysis, ten participants who completed the 8-week program were included: four men (mean age 88 years, range 8095 years) and six women (mean age 81 years, range 7787 years). During the course of the 8 weeks, three men and two women dropped out of the study for the following reasons: back pain, which was not related to the intervention; depression; insufficient adjustment of insulin therapy to the exercise program; broken foot (at the nursing home); ripped catheter during changing of clothes before the training . The five participants who dropped out were comparable in age, sex, and body mass index to the ten participants who completed the 8 weeks of intervention, with an average of 15/16 completed sessions (table 1). After the 8-week intervention, mobility improved by 24% (p = 0.005; table 2). The effect was larger in men compared to women (27% versus 21%): baseline ems scores of 14 versus 18 and 15 versus 18 after 8 weeks for men and women, respectively . Muscle strength (eight - repetition maximum) increased at every machine (table 2). At the rowing machine and the leg extension machine, the number of sit - up repetitions increased fourfold (pre 10.5 3.1, post 41.7 12.1 repetitions; p = 0.027). Women showed a higher improvement in the eight - repetition maximum, particularly at the rowing machine and leg extension (figure 2). After the 8-week training program, quality of life did not change considerably regarding both the physical and emotional sum scales of the sf-36: pre 30/100, post 30/100 and pre 59/100, post 56/100 (p = 0.29), respectively . There were also no relevant improvements regarding the subscales of the sf-36 except for the subscale of physical functioning, which slightly improved (pre 27/100, post 32/100; p = 0.54). Fifteen nursing - home residents aged 7797 years (mean age 84 years) were recruited . In the current analysis, ten participants who completed the 8-week program were included: four men (mean age 88 years, range 8095 years) and six women (mean age 81 years, range 7787 years). During the course of the 8 weeks, three men and two women dropped out of the study for the following reasons: back pain, which was not related to the intervention; depression; insufficient adjustment of insulin therapy to the exercise program; broken foot (at the nursing home); ripped catheter during changing of clothes before the training . The five participants who dropped out were comparable in age, sex, and body mass index to the ten participants who completed the 8 weeks of intervention, with an average of 15/16 completed sessions (table 1). After the 8-week intervention, mobility improved by 24% (p = 0.005; table 2). The effect was larger in men compared to women (27% versus 21%): baseline ems scores of 14 versus 18 and 15 versus 18 after 8 weeks for men and women, respectively . Muscle strength (eight - repetition maximum) increased at every machine (table 2). At the rowing machine and the leg extension machine, the number of sit - up repetitions increased fourfold (pre 10.5 3.1, post 41.7 12.1 repetitions; p = 0.027). Women showed a higher improvement in the eight - repetition maximum, particularly at the rowing machine and leg extension (figure 2). After the 8-week training program, quality of life did not change considerably regarding both the physical and emotional sum scales of the sf-36: pre 30/100, post 30/100 and pre 59/100, post 56/100 (p = 0.29), respectively . There were also no relevant improvements regarding the subscales of the sf-36 except for the subscale of physical functioning, which slightly improved (pre 27/100, post 32/100; p = 0.54). This pilot study represents an approach to the development of an exercise program focused on the elderly . The progressive resistance program with two sessions per week over a period of 8 weeks improved mobility and muscle strength in nursing - home residents over 75 years of age with impaired mobility . This study showing that progressive resistance training increases muscle strength confirmed results from previous studies such as the randomized controlled trial by ferri et al who trained participants aged 6581 years at 80% of the one - repetition maximum with knee extension machines . The one - repetition maximum increased as well as the cross - sectional muscle area.25 rosendahl et al showed positive effects of 3 months resistance training (eight to twelve - repetition maximum) in participants with a mean age of 84 years, which were still present after 6 months.26 resistance training programs for nursing - home residents with a high - intensity program (eight - repetition maximum or 80% of the one - repetition maximum), as used in the current study, seem to be the most effective method to increase muscle strength.15,1719,26,27 however, the strong positive effect in muscle strength improvement shown in the current study was in accordance with only one of these studies,17 whereas the other studies showed smaller effects on muscle strength.18,19,26 regarding training frequency, sessions offered three times a week were used most often in studies focusing on the elderly.14,15 in contrast to this approach, the current study examined a training program with only two sessions per week and was able to show even stronger effects than in comparable studies.27,28 training duration did not seem to have a comparable influence as training intensity . Latham et al showed an effect size difference of 0.15 between short (12 weeks) and long training duration (> 12 weeks) in 41 trials with 1955 participants aged 60 years and over.14 as in the current study, most interventions had a duration of 812 weeks.18,19,27,29 progressive resistance training improves not only muscle strength and mobility but also physical abilities, including simple and more complex daily activities in the elderly . However, there is insufficient evidence on long - term effects as concluded by the authors of a cochrane review.30 overall quality of life, shown with the physical and emotional sum scale of the sf-36, did not improve in the current study . It is assumed that the 8-week duration of the intervention was too short to detect considerable changes in quality of life . Furthermore, the structure of the sf-36 may have been too complex for this age group (eg, heterogeneity of answering categories). Interviews had to be conducted with some participants who were not able to read the questionnaire by themselves . A shorter questionnaire with simpler answering categories such as the sf-12 may be an alternative to assess quality of life in future studies with the elderly.31 a particular feature of this study was the investigation of very elderly nursing - home residents with impaired mobility who have hardly been included in intervention studies as shown by the review of valenzuela.15 a strength of this study was the use of validated instruments like the ems and the sf-36.22,24 several potential limitations have to be considered when interpreting the results of this pilot study . Due to the lack of a control group, the possibility that other factors in addition to the resistance program contributed to the improved mobility and muscle strength cannot be ruled out . Furthermore, familiarization with the gym equipment may have contributed to the training effect, since most participants were not used to resistance training in a gym . The small study population of this investigation cannot be considered as representative for nursing - home residents in berlin . . For safety and legal considerations, several inclusion and exclusion criteria had to be defined in order not to put participants at risk and aggravate preexisting diseases . Persons under legal care (about 80% of nursing - home residents) were not eligible and were therefore not included in the study . Although several study participants dropped out, two - thirds completed the 8-week intervention, showing the feasibility of this resistance program in persons up to almost 100 years of age with impaired mobility . This pilot study represents an approach to the development of an exercise program focused on the elderly . The progressive resistance program with two sessions per week over a period of 8 weeks improved mobility and muscle strength in nursing - home residents over 75 years of age with impaired mobility . This study showing that progressive resistance training increases muscle strength confirmed results from previous studies such as the randomized controlled trial by ferri et al who trained participants aged 6581 years at 80% of the one - repetition maximum with knee extension machines . The one - repetition maximum increased as well as the cross - sectional muscle area.25 rosendahl et al showed positive effects of 3 months resistance training (eight to twelve - repetition maximum) in participants with a mean age of 84 years, which were still present after 6 months.26 resistance training programs for nursing - home residents with a high - intensity program (eight - repetition maximum or 80% of the one - repetition maximum), as used in the current study, seem to be the most effective method to increase muscle strength.15,1719,26,27 however, the strong positive effect in muscle strength improvement shown in the current study was in accordance with only one of these studies,17 whereas the other studies showed smaller effects on muscle strength.18,19,26 regarding training frequency, sessions offered three times a week were used most often in studies focusing on the elderly.14,15 in contrast to this approach, the current study examined a training program with only two sessions per week and was able to show even stronger effects than in comparable studies.27,28 training duration did not seem to have a comparable influence as training intensity . Latham et al showed an effect size difference of 0.15 between short (12 weeks) and long training duration (> 12 weeks) in 41 trials with 1955 participants aged 60 years and over.14 as in the current study, most interventions had a duration of 812 weeks.18,19,27,29 progressive resistance training improves not only muscle strength and mobility but also physical abilities, including simple and more complex daily activities in the elderly . However, there is insufficient evidence on long - term effects as concluded by the authors of a cochrane review.30 overall quality of life, shown with the physical and emotional sum scale of the sf-36, did not improve in the current study . It is assumed that the 8-week duration of the intervention was too short to detect considerable changes in quality of life . Furthermore, the structure of the sf-36 may have been too complex for this age group (eg, heterogeneity of answering categories). Interviews had to be conducted with some participants who were not able to read the questionnaire by themselves . A shorter questionnaire with simpler answering categories such as the sf-12 may be an alternative to assess quality of life in future studies with the elderly.31 a particular feature of this study was the investigation of very elderly nursing - home residents with impaired mobility who have hardly been included in intervention studies as shown by the review of valenzuela.15 a strength of this study was the use of validated instruments like the ems and the sf-36.22,24 several potential limitations have to be considered when interpreting the results of this pilot study . Due to the lack of a control group, the possibility that other factors in addition to the resistance program contributed to the improved mobility and muscle strength cannot be ruled out . Furthermore, familiarization with the gym equipment may have contributed to the training effect, since most participants were not used to resistance training in a gym . The small study population of this investigation cannot be considered as representative for nursing - home residents in berlin . . For safety and legal considerations, several inclusion and exclusion criteria had to be defined in order not to put participants at risk and aggravate preexisting diseases . Persons under legal care (about 80% of nursing - home residents) were not eligible and were therefore not included in the study . Although several study participants dropped out, two - thirds completed the 8-week intervention, showing the feasibility of this resistance program in persons up to almost 100 years of age with impaired mobility . A progressive resistance training program only twice a week over a period of 8 weeks seems to be a beneficial intervention to improve mobility and muscle strength in nursing - home residents with impaired mobility aged up to 97 years . The intervention did not seem to influence quality of life as assessed by the sf-36 over the 2-month study period; however, it cannot be excluded that a longer intervention may have beneficial effects on quality of life as well . Randomized controlled trials evaluating the benefits of resistance training in frail nursing - home residents are urgently needed . In these trials, the setting should be adapted and implementable in the daily routine of the elderly regarding intensity, duration, and frequency of the training; special attention should be paid to a presumably high dropout rate due to multimorbidity.
A common cause of brain injury during the perinatal period is hypoxic - ischemic injury, which frequently results in the chronic handicapping conditions such as cerebral palsy, mental retardation, learning disability, and epilepsy (1). Predicting the outcome of neonates with hypoxic - ischemic injury, however, is difficult, because some methods that predict outcome have not reliably or consistently predicted long term neurologic outcomes . Recently, h - magnetic resonance spectroscopy (mrs) has been used as a quantitative noninvasive tool to assess the biomechanical changes associated with central nervous system injury . H - mrs, which provides objective extent of hypoxic - ischemic insults, enhances predictability and provides a method to assess treatment effect (2 - 5). The exact mechanism by which hypoxic - ischemic brain injury occurs in neonates is not clear, although increasing evidences indicate that hypoxic - ischemic induced neuronal death includes both necrosis and apoptosis . Necrosis may predominate in acute damage, whereas apoptotic injury may take time to develop . Therefore, blocking the apoptotic cascade may prolong the therapeutic window after hypoxic - ischemic injury (6 - 8). There is evidence for involvement of multiple caspases in hypoxic - ischemic brain injury . And caspase inhibitors which were developed as antiapoptotic agents, are believed to play a key role in the delayed neuronal cell death observed after hypoxic - ischemic injury (7, 9). The effects of growth hormone (gh) on the central nervous system have become more apparent in the past decade . Not only it is involved in brain growth and development, but its qualities as a neuroprotective factor against injury are now appreciated . Recent studies have demonstrated that gh is involved in neuroprotection during hypoxic - ischemic brain injury (10, 11). These protective roles are supported by the ability of gh to accelerate glial cell division and myelinogenesis, and gh is also thought to have neuroprotective roles in neurogenesis (11). Although this neuroprotective mechanism is not completely known, is probably achieved by inhibiting of caspase activities (12, 13). The lipid peak in the h - mr spectrum has been reported to be a marker for apoptosis during hypoxic - ischemic injury (14). We have therefore used h - mrs to evaluate the effects of gh as a caspase inhibitor on hypoxic - ischemic injury in neonatal rat brains . The right common carotid arteries of 7-day old sprague - dawley rats (mean weight=13.3 g) were ligated under halothane anesthesia . After a recovery period of 3 hr gh (eutrophin, lgphd, korea) was administered just prior to hypoxic - ischemic insult . The rats were divided into four groups: control (10 l distilled water, n=29), intracerebroventricular (icv, 10 l gh in 10 l distilled water, n=23), intracerebroventricular / intraperitoneal (icv+ip, n=21), and intraperitoneal (ip, 10 mg / kg gh in distilled water, n=23). Localized in vivo h - mrs was performed on a bruker biospec 4.7 t mri / mrs system equipped with active shielded gradients and aspect3000 computer with tomikon hardware and software (bruker, fallanden, switzerland). Spectra were acquired in the right cerbral hemisphere of rats 24 hr after the onset of hypoxic - ischemic insult . Water suppressed h - mr spectra were acquired using a vosy sequence with detection of the double - refocused spin echo signal from the selected voxel (322 l, 12 l) using the following acquisition parameters: sw=5,000 hz, si=4,096 pts, ns=128, tr / te=3,000/30 and 135 msec . To identify the peak at 1.3 ppm, the spectra were acquired at echo times of 30 and 135 msec in order to differentiate the lactate peak from the lipid peak . Peak areas were measured and the lipid / n - acetyl aspartate (naa) and lipid / creatine (cr) ratios were used as apoptotic markers . After the h - mrs examinations on the 1st day, 6 brains from each group were perfused with 0.9% saline solution mixed with 2 units / ml of heparin, followed by perfusion with 4% paraformaldehyde in pbs solution . Each brain was isolated, and tunel staining was performed using an in situ cell death detection kit, pod (boehringer mannheim, germany), as described . Apoptotic cells were counted 3 times in the parietal area of the brain using a 200 lens, and the mean apoptotic cell numbers were calculated using image analyzer software . Gross morphologic changes were scored at 2 weeks using a 5 point grading system as method by palmer et al . (15), where 0 indicates no change and 4 indicates the most severe injury . Significance was assessed by unpaired t - test and anova followed by kruskall wallis test . Spearman correlation was used to investigate relationships between lipid / naa and lipid / cr ratio and morphologic score . The right common carotid arteries of 7-day old sprague - dawley rats (mean weight=13.3 g) were ligated under halothane anesthesia . After a recovery period of 3 hr gh (eutrophin, lgphd, korea) was administered just prior to hypoxic - ischemic insult . The rats were divided into four groups: control (10 l distilled water, n=29), intracerebroventricular (icv, 10 l gh in 10 l distilled water, n=23), intracerebroventricular / intraperitoneal (icv+ip, n=21), and intraperitoneal (ip, 10 mg / kg gh in distilled water, n=23). Localized in vivo h - mrs was performed on a bruker biospec 4.7 t mri / mrs system equipped with active shielded gradients and aspect3000 computer with tomikon hardware and software (bruker, fallanden, switzerland). Spectra were acquired in the right cerbral hemisphere of rats 24 hr after the onset of hypoxic - ischemic insult . Water suppressed h - mr spectra were acquired using a vosy sequence with detection of the double - refocused spin echo signal from the selected voxel (322 l, 12 l) using the following acquisition parameters: sw=5,000 hz, si=4,096 pts, ns=128, tr / te=3,000/30 and 135 msec . To identify the peak at 1.3 ppm, the spectra were acquired at echo times of 30 and 135 msec in order to differentiate the lactate peak from the lipid peak . Peak areas were measured and the lipid / n - acetyl aspartate (naa) and lipid / creatine (cr) ratios were used as apoptotic markers . After the h - mrs examinations on the 1st day, 6 brains from each group were perfused with 0.9% saline solution mixed with 2 units / ml of heparin, followed by perfusion with 4% paraformaldehyde in pbs solution . Each brain was isolated, and tunel staining was performed using an in situ cell death detection kit, pod (boehringer mannheim, germany), as described . Apoptotic cells were counted 3 times in the parietal area of the brain using a 200 lens, and the mean apoptotic cell numbers were calculated using image analyzer software . Gross morphologic changes were scored at 2 weeks using a 5 point grading system as method by palmer et al . (15), where 0 indicates no change and 4 indicates the most severe injury . Significance was assessed by unpaired t - test and anova followed by kruskall wallis test . Spearman correlation was used to investigate relationships between lipid / naa and lipid / cr ratio and morphologic score . The lipid / naa ratio was significantly lower in the icv (8.53.1) and icv / ip (9.22.5) groups than in the control group (12.24.6); although the lipid / naa ratio in the ip group (11.43.7) was lower than in the control group, this difference was not significant (fig . The lipid / cr ratio was also significantly lower in the icv group (8.13.5) than in the control group (10.94.4); the lipid / cr ratio was lower in the icv / ip (9.23.4) and ip (9.63.5) groups than in the control group, but these differences were not statistically significant (fig . Although the number of tunel positive cells did not differ between the control and ip groups, there were fewer in the icv and icv / ip groups (fig . We found that the morphologic scores were significantly lower in the icv group (1.41.3) and somewhat lower in the icv / ip group (1.81.4) than in the control group (2.21.4), but not in the ip group (2.21.2) (fig . Morphologic scores significantly correlated with the lipid / naa and lipid / cr ratios (fig . 5). Recently h - mrs has been used as a quantitative noninvasive assessment tool in monitoring of brain development and in the diagnosis of neurologically damaged infants (3 - 5). H - mrs can detect metabolites such as naa and other acetyl compounds, which serve as primarily neuronal markers; cr, including phosphocreatine and cr, which are bioenergetic markers; choline - containing compounds (cho), which are released during membrane disruption; and lactate (lac), which accumulates in response to anaerobic tissue metabolism (2, 16). Decreased naa / cho and naa / cr ratio and increased cho / cr ratio have were in asphyxiated neonates with poor neurologic outcomes after 1 yr (4, 17). H - mrs in asphyxiated neonates has also shown increased lactate and decreased naa in thalamus, as well as increased lac and decreased cr in basal ganglia (18, 19). During hypoxic - ischemic injury, there is a significant increase in the lipid peak, which correlates with apoptotic cell death, as well as in the intensity of the lipid peak, which is directly related to the apoptotic cell count (14). We therefore used the lipid / naa and lipid / cr ratios as apoptotic markers . Newborn infants subjected to transient hypoxic - ischemic injury during birth asphyxia are apparently relatively normal soon after resuscitation but show evidence of delayed cerebral injury hours later, the magnitude of which predicts the severity of later neurodevelopmental impairment (20, 21). The mechanism of delayed injury is unclear, but apoptotic cells are detected in brains of infants who died after birth asphyxia, suggesting that inappropriate activation of the apoptotic pathway accounts, at least in part, for the delayed cell death (6, 8, 21). Apoptosis was first described as a type of cell death distinct from necrosis, with no swelling or loss of membrane integrity, and no inflammatory response from the host tissue (22). Apoptotic cells undergo a ubiquitous physiologic process that is essential to the development and survival of multicellular organisms . This process takes place during embryologic development, turnover of gastrointestinal epithelium, and the regulation of the immune system . Many pathological events that cause necrosis, including hypoxic - ischemic injury, can also induce apoptosis (2, 4, 6, 23). Necrosis may predominate in more intense ischemic damage, whereas apoptosis may occur during milder ischemic damage and may take time to develop (24). Immature cortical neurons have been shown to be more susceptible to apoptosis than mature neurons, perhaps because cells of younger animals more readily undergo apoptosis than cells of more mature animals (21, 25). Thus, blocking the apoptotic cascade may prolong the therapeutic window after hypoxic - ischemic events, especially in the developing brain (6 - 8). The ability of specific therapeutic agents to reduce caspase inhibitors, which have antiapoptotic activity and are believed to play a key role in the delayed neuronal cell death after hypoxic - ischemic injury (7, 9). Caspase-3 is a terminal enzyme in the caspase family that activates an endonuclease (caspase - activated dnase), resulting in dna fragmentation (26). Caspase inhibitors, including inhibitors of caspase-3, may prolong the therapeutic window after hypoxic - ischemic injury (7, 27, 28). Recently, gh administration has been reported to inhibit neuronal death during hypoxic - ischemic injury, and to have a neuroprotective effect in the cerebral cortex, hippocampus, and thalamus (10, 11). Although the mechanism is not completely known, several reports suggest that hypoxic - ischemic injury induces neuronal death by downregulating bcl-2 protein levels, followed by sequential activation of the caspases, and that gh protects neuronal cells by inhibiting alterations in bcl-2 protein levels and caspase activities (12, 13). We found that the lipid / naa ratio was significantly lower in rats administered gh by the icv and icv / ip routes, that lipid / cr ratio was significantly lower in rats administered gh by the icv, and that the degree of morphologic changes in the brain was significantly correlated with the lipid / naa and lipid / cr ratios . In our results, lipid / naa ratio and lipid / cr ratio were not significantly changed by ip administration of gh . Because gh does not usually cross the blood - brain barrier, ip administration of gh taken together, these findings suggest that gh exerts neuroprotective effects in cerebral hypoxic - ischemic injury by inhibiting apoptosis, especially in the early stage after insult . Our results also suggest that gh, as a caspase inhibitor, can have therapeutic value in neuroprotective effect of hypoxic - ischemic brain injury.
Hepatitis e virus (hev) is the agent largely responsible for epidemic as well as sporadic hepatitis in the developing countries. (12) the virus is transmitted by the feco - oral route, often through contaminated water . Acute viral hepatitis is a major public health issue in the developing nations that have inadequate sanitary conditions, inadequate safe drinking water, and sewage disposal problems. (234) recognition of early warning signals, timely investigation and application of specific control measures can limit the spread of the outbreak and prevent deaths . In march 2013, we received the information from medical officer in - charge, lalkuan primary health center (phc), that several people were presenting to the hospital with acute hepatitis from ward no-5, of lalkuan . An investigation team visited the affected area immediately because an epidemiological investigation was required in this situation to determine the existence of epidemic of jaundice if any, to identify the source, and to recommend and adopt control measures . A criterion for diagnosis of acute hepatitis was defined as those cases that have / had jaundice with at least one of the following symptoms: dark urine, fever, pain in abdomen, vomiting, and loss of appetite between january and march 2013 . For all cases, information on personal details, time of onset, and source of drinking water were obtained . Randomly 13 sera from cases were collected for immunoglobulin m (igm) antibodies for hepatitis a virus (hav) and hev by enzyme - linked immunosorbent assay (elisa; dsi, italy provided by idsp, uttarakhand). Information regarding water quality, source of water supply, and drainage system were collected . Estimation of residual chlorine was performed at distribution level and from randomly collected consumer levels of the water distribution system . A total of 240 cases of jaundice (overall attack rate 8.62%) were reported in march 2013 . There was an initial cluster in the last week of february 2013 followed by a peak in the 3 week of march 2013 and then a gradual decline in the number of cases was observed . Epidemic curve of acute hepatitis cases in lalkuana, nainital district, 2013 the signs and symptoms of the affected persons included jaundice (100%), loss of appetite (60.2%), pyrexia (78.5%), malaise (36.6%), high colored urine (99.6%), vomiting / nausea (56%), and pain in right hypochondria (55.9%) [table 1]. Clinical profile in acute viral hepatitis cases (n = 240) in lalkuan, the attack rate in males was 10.21% and 7.18% for females and the difference was statistically significant (p <0.001) [table 2]. Age and sex wise distribution of cases of acute hepatitis in lalkuan, nainital the disease affected all the age groups, but the attack rate was much more among the age group of> 12 years (9.65%) and the difference was statistically significant (p <0.001) [table 2]. Out of the 13 sera collected, 10 were found positive for the hepatitis e igm antibody and rest three were positive for both hev and hav igm antibodies by elisa (dsi, italy). The higher attack rates of viral hepatitis was found in those, consuming water supplied from the leaking water pipelines passing adjacent to sewage pipeline, as compared to those who consumed water supplied from other pipelines indicating that the present outbreak was due to sewage contamination of drinking water supply . The difference in the attack rate was also found to be statistically significant (= 574.26, degrees of freedom (df) = 1, p <0.01) [table 3]. This study confirms the fact that there was an epidemic of infective hepatitis e in lalkuan, nainital district in march 2013 . Similarly other studies reported hev as the most important cause of all the clinical types of hepatitis commonly found in india. (56789101112) out of the 13 sera collected, 10 were found positive for the hepatitis e igm antibody and rest three were positive for both hev and hav igm antibodies by elisa (dsi, italy). In other studies also hepatitis e was the major cause of the outbreak. (71112) attack rate of acute viral hepatitis ranging from 1.9 to 17% have been reported from various studies from india. (891011121314) the overall attack rate in the present study was 8.61%, comparable with the other studies. (9) similar to other studies in india,(1516) we also found that all cases had jaundice, 78.5% had history of fever and 99.6% had dark colored urine . The attack rate in males was 10.21% and 7.18% for females and the difference was statistically significant (p <0.001), higher attack rates in males have also been reported in other studies from india. (1117) this study showed that population of> 12 years showed significantly more number of cases as compared to <12 years of age [table 2]. The age distribution of hev cases in our study was similar to previously described studies. (7891215) in developing countries hev is maintained as sporadic cases in the community and children acquire the infection in early life making them immune to another attack. (7) this outbreak started in last week of february 2013, reached peaked in 3 week of march 2013 and then started declining . No secondary peak was observed, similar finding were reported by varied studies. (61015) hepatitis e epidemics are frequently unimodal and short - lasting . Some have been multimodal, but even in such epidemics, new cases stopped appearing soon after water contamination was controlled. (18) hepatitis e outbreaks have been reported in urban areas whenever there is a break in the quality of water supplied including water chlorination. (67891011171920) in the present study out of 756 persons who gave the history of turbid water supply and leakage in water supply pipelines, 29.4% of them suffered from acute viral hepatitis . Whereas, rest 2,029 person who had not given the history of turbid water supply suffered less (0.84%) and this difference was statistically highly significant (= 574.26, p <0.01). Hence, clear association was observed between turbid water supply and occurrence of cases of hepatitis in the present study . Residual chlorine was also found less in most of the water sample tested in various affected areas during the time of outbreak investigation . The main source of water supply in lalkuan area is tap water supplied by the jal sansthan for 1 - 2 h in the morning and 1 h in evening there was also a history of leakages in drinking water pipelines and overflowing drains in the area . This result in entry of polluted water into the pipes when supply is closed. (21) we also confirmed the finding with the help of management staff at the jal sansthan in the affected areas . It was observed by the investigating team that many of the affected households had installed the electric motor system directly in the municipal water supply system as the pressure of the water was low during the supply hours . As soon as the epidemic was noted, safe water supply was made available to the inhabitants through mobile jal sansthan water tanks and the local people were advised to boil the drinking water . Chlorine tablets were distributed to all houses in the affected areas . Simultaneously, extensive health education was provided . A meeting was held with the district magistrate in which the local elected representative, officials from nagarpalika, jal sansthan, and health department participated . The appropriate authority (nagarpalika, jal sansthan) were requested to repair the leaking water pipe line . Nagarpalika, jal sansthan took initiative to repair the leaking pipe . Due to active intervention like health education, provision for safe water by tanks and chlorination, the outbreak subsided . Based on the above observations, it was concluded that, the present outbreak was due to fecal contamination of drinking water supplied to the affected areas, which occurred due to old and corroded leaking pipelines passing close to old leaking sewage lines . The recognition of early warning signals, timely investigation, and application of specific control measures can contain the outbreak and decrease morbidity and mortality . Water samples were not tested for the coliform count which could have further supported our findings in this study . Water samples were not tested for the coliform count which could have further supported our findings in this study.
Almost 32% of us children aged 2 to 19 years have a body mass index (bmi) at the 85th percentile or greater . Childhood obesity is a risk factor for concurrent diabetes mellitus, nonalcoholic fatty liver disease, hyperlipidemia, and cardiovascular disease, and increases risk for adult obesity . Expert committee guidelines from the american academy of pediatrics (aap) published in 2007 outline recommendations for the prevention, assessment, and treatment of child and adolescent overweight and obesity (table 1). Aap recommendations for the prevention, assessment, and treatment of child and adolescent overweight and obesity . 2007 aap obesity and overweight definitions under 2 years: overweight = weight - for - length> 95th percentile for age / sex over 2 years: overweight = bmi 85 - 94th percentile for age / sexobese = bmi> 95th percentile for age / sex overweight = bmi 85 - 94th percentile for age / sex obese = bmi> 95th percentile for age / sex 2007 aap serum screening recommendations for obese / overweight patients any overweight or obese patient should have fasting lipids if 10 years old: any obese patient or any overweight patient with risk factors for type 2 diabetes should have fasting lipids, aspartate aminotransferase / alanine aminotransferase (ast / alt), and fasting glucose . Type 2 diabetes risk factors include the following: family history of diabeteshigh - risk racial / ethnic background (black, hispanic, or native american)polycystic ovarian syndromeacanthosis nigricanscardiovascular disease risk factors family history of diabetes high - risk racial / ethnic background (black, hispanic, or native american) polycystic ovarian syndrome cardiovascular disease risk factors if serum screening laboratory studies are normal, may repeat every 2 years after age 10 abbreviations: aap, american academy of pediatrics; bmi, body mass index . Appropriate counseling and treatment for overweight and obesity begins with health care provider recognition of elevated bmi and screening for obesity - related comorbidities . Pediatric providers underrecognize overweight and obesity and do not perform evaluations and interventions consistent with expert committee recommendations . A review of nationally representative data from 1997 to 2000 found that providers diagnosed obesity in only 0.93% of well - child visits for children aged 2 to 18 years . More recent data from 2005 to 2007 report that physicians documented a diagnosis of obesity in 18% of youth aged 2 to 18 years who had bmi above the 95th percentile for age and sex, which is an improvement in documentation but remains far below the actual prevalence of obesity and overweight . Identification of overweight and obesity is particularly problematic among young children and those with milder degrees of obesity . An evaluation of health supervision visits at an academic pediatric hospital showed providers identify obesity as a problem for only one - half of obese children aged 3 months to 15 years, with the lowest rates of identification among children <5 years of age and those with milder degrees of obesity . In a retrospective chart review of outpatient visits at 2 academic hospitals, children under age 5 years and with bmi percentile of 85% to 94% were least likely to receive diagnosis and intervention for overweight . Surveys of pediatric providers have found variable adherence to guidelines for screening for obesity - related comorbidities . A study of children seen for well - child care in a diverse group of pediatric practices in chicago from 2002 to 2003 showed rates of laboratory screening for obesity - related comorbidities among children with bmi 85th percentile to be low (7% to 13%), but screening rates improved when providers documented overweight in the medical record . Since the release of the aap 2007 recommendations for pediatric obesity management, a survey of pediatricians behaviors and beliefs reported variability in the use of laboratory screening and referrals for children with overweight and obesity . The use of an electronic medical record (emr) may facilitate weight - related evaluations in pediatrics . The kaiser permanente southern california pediatric weight management initiative evaluated more than 700 000 patients using computer - assisted decision tools that standardized pediatric weight management . In this setting, diagnosis of overweight or obesity increased significantly from 12% to 61%, and documented counseling rates for exercise and nutrition increased significantly from 1% to 50% . Furthermore, a systematic review in pediatrics evaluated 13 studies that used information technology (including emr use, telemedicine counseling, telephone support, and text - messaging) to deliver obesity screening or treatment to children aged 2 to 18 . The use of emrs was associated with improvements in bmi documentation and counseling about nutrition and physical activity; however, these studies did not demonstrate a significant improvement in laboratory screening . We conducted a study to determine how often pediatric residents correctly diagnose overweight / obesity and order laboratory screening tests based on the aap 2007 expert committee recommendations at pediatric health supervision visits . We hypothesized that residents are more likely to correctly order serum - based screening tests when they correctly diagnose overweight or obesity in the emr . We conducted a retrospective chart review of patient visits at 1 of the 2 pediatric resident continuity clinic sites affiliated with our large, tertiary care, academic pediatric hospital, the ann & robert h. lurie children s hospital of chicago (formerly children s memorial hospital), the pediatric teaching hospital affiliated with northwestern university feinberg school of medicine . Each clinic is staffed by up to 9 residents per session (varies due to resident schedules and duty hours). Each resident is assigned to a half - day session each week that does not change during residency training . Residents are supervised by 2 to 3 attending physicians in each session; some are full - time academic generalist pediatric faculty and some are volunteer community - based pediatricians . The clinic serves a predominately urban, minority population (49% hispanic, 33% african american); 90% are insured by illinois medicaid . Using our emr we identified patients aged 2 to 18 years who were seen for a well - child visit from march 1, 2010, through august 31, 2010, and had both a height and weight documented at the visit . All subjects had date of birth, date of visit, reason for their visit, and bmi extracted from the hospital emr . Bmi was calculated and plotted automatically on centers for disease control bmi growth charts by the emr (weight in kilograms divided by height in centimeters squared). Overweight was defined as bmi 85th and <95th percentile; obesity was bmi 95th percentile; and normal was bmi from 5th to <85th percentile . If the patient had a bmi 85th percentile for age and sex, the visit record was evaluated for (a) the appropriate diagnosis of either overweight or obesity in the emr and (b) proper screening tests ordered in the previous 2 years . Subjects were grouped by age into 2 to 9 years and 10 years to correspond to the aap s laboratory screening recommendation age groups . We defined correct diagnosis as the notation of terms such as obese, overweight, or elevated bmi in the visit icd-9 code, in the provider s assessment at the time of the visit, or in the emr problem list for the patient . If any concern about excess weight was documented, regardless of whether the patient s associated bmi was correctly categorized as overweight or obese . The emr was then examined for screening laboratory tests for obesity - related comorbidities ordered in accordance with the 2007 aap panel recommendations within the previous 2 years . We determined that patients had appropriate screening laboratory tests if an overweight patient had fasting lipids, if an obese patient aged 2 to 9 had fasting lipids, or if an obese patient aged 10 years had fasting lipids, aspartate aminotransferase / alanine aminotransferase (ast / alt), and fasting glucose . Patients who had laboratory studies in addition to the aap s recommended labs were considered to have had appropriate screening . Physician compliance with the guidelines was assessed on the basis of laboratory test ordering and not on whether patients actually went to the laboratory to have the screening tests performed . We used tests to examine associations between diagnosis of overweight or obesity, recommendations for laboratory screening testing consistent with aap panel recommendations, and patient age . We conducted a retrospective chart review of patient visits at 1 of the 2 pediatric resident continuity clinic sites affiliated with our large, tertiary care, academic pediatric hospital, the ann & robert h. lurie children s hospital of chicago (formerly children s memorial hospital), the pediatric teaching hospital affiliated with northwestern university feinberg school of medicine . Each clinic is staffed by up to 9 residents per session (varies due to resident schedules and duty hours). Each resident is assigned to a half - day session each week that does not change during residency training . Residents are supervised by 2 to 3 attending physicians in each session; some are full - time academic generalist pediatric faculty and some are volunteer community - based pediatricians . The clinic serves a predominately urban, minority population (49% hispanic, 33% african american); 90% are insured by illinois medicaid . Using our emr we identified patients aged 2 to 18 years who were seen for a well - child visit from march 1, 2010, through august 31, 2010, and had both a height and weight documented at the visit . All subjects had date of birth, date of visit, reason for their visit, and bmi extracted from the hospital emr . Bmi was calculated and plotted automatically on centers for disease control bmi growth charts by the emr (weight in kilograms divided by height in centimeters squared). Overweight was defined as bmi 85th and <95th percentile; obesity was bmi 95th percentile; and normal was bmi from 5th to <85th percentile . If the patient had a bmi 85th percentile for age and sex, the visit record was evaluated for (a) the appropriate diagnosis of either overweight or obesity in the emr and (b) proper screening tests ordered in the previous 2 years . Subjects were grouped by age into 2 to 9 years and 10 years to correspond to the aap s laboratory screening recommendation age groups . We defined correct diagnosis as the notation of terms such as obese, overweight, or elevated bmi in the visit icd-9 code, in the provider s assessment at the time of the visit, or in the emr problem list for the patient . If any concern about excess weight was documented, regardless of whether the patient s associated bmi was correctly categorized as overweight or obese . The emr was then examined for screening laboratory tests for obesity - related comorbidities ordered in accordance with the 2007 aap panel recommendations within the previous 2 years . We determined that patients had appropriate screening laboratory tests if an overweight patient had fasting lipids, if an obese patient aged 2 to 9 had fasting lipids, or if an obese patient aged 10 years had fasting lipids, aspartate aminotransferase / alanine aminotransferase (ast / alt), and fasting glucose . Patients who had laboratory studies in addition to the aap s recommended labs were considered to have had appropriate screening . Physician compliance with the guidelines was assessed on the basis of laboratory test ordering and not on whether patients actually went to the laboratory to have the screening tests performed . We used tests to examine associations between diagnosis of overweight or obesity, recommendations for laboratory screening testing consistent with aap panel recommendations, and patient age . A total of 1075 charts of patients seen for well - child care visits were reviewed . Of these, included subjects had a mean age of 8.06 years (standard deviation 4.47 years); 66% of subjects were aged 2 to 9 years . Two hundred and fifteen of the 522 subjects (41%) met criteria for either overweight or obesity; 19% were overweight and 22% were obese . Children aged 10 years were more likely to be obese or overweight than younger children (table 2). Ninety - nine subjects (19.0%) were overweight; 17/99 (17.2%) carried a correct diagnosis of overweight in the emr . One hundred sixteen subjects (22.2%) were obese; 74/116 (63.7%) carried a correct diagnosis of obesity in the emr . Two hundred fifteen subjects were overweight or obese; 91 (42.3%) were correctly diagnosed . Older children were more likely to have a diagnosis of overweight or obesity . Among the 91 children 10 years, 52 (57.1%) had a correct diagnosis in the emr, while among the 124 children aged 2 to 9 years, only 39 (31.5%) had a correct diagnosis in the emr (p <.001; table 3). Overall, screening tests consistent with 2007 aap guidelines were ordered for 21.9% of patients with obesity and overweight . Test ordering was significantly more common for those with an overweight or obesity diagnosis in the emr: aap guideline - recommended tests were ordered for 39 of 91 (42.3%) patients with a correct diagnosis versus 8 of 124 (6.5%) missing a correct diagnosis (p <.001; table 4). In addition, test ordering was more common when bmi was at or above the 95th percentile; recommended tests were ordered for 31.9% of obese children and 10.0% of overweight children (p <.001). Laboratory screening and emr diagnosis for overweight and obese subjects . Abbreviation: emr, electronic medical record . Overweight and obesity were common in our urban, predominantly medicaid - insured clinic population, with a combined prevalence of 41% . Similar to findings in other studies, rates of documentation of overweight and obesity by practitioners were low and worse for younger children and for those with milder degrees of excess weight . In addition, physicians in our clinic frequently failed to order recommended laboratory tests: labs consistent with the 2007 aap guidelines were ordered for 21.9% of patients with bmi 85th percentile . This rate of testing is actually higher than the 13% reported by obrien et al in an academic continuity clinic population and the 7% to 13% reported by dilley et al in a chicago - area community - based sample . Our higher screening rates could be related to greater pediatric provider awareness of the 2007 aap guidelines or a function of increasing public and professional awareness of obesity . It is also possible that the opportunity to discuss a patient with another physician, as occurs when a resident discusses a patient with a clinic attending, increases the likelihood of recognition and evaluation of medical concerns . Office - based tools to support documentation of bmi and nutrition / activity counseling can help improve adherence to obesity recognition and treatment recommendations . Emrs that include automatic bmi plotting, as ours does, are associated with increased documentation of overweight . While prior studies have not demonstrated that, in itself, the use of an emr improves rates of serum laboratory screening for overweight and obese patients, these studies did not investigate an interaction between provider recognition of excess weight, the emr - based prompt, and subsequent ordering of screening tests . Our results, despite the fairly low overall rates of laboratory screening, show that recording a correct diagnosis of excess weight in the emr was highly associated with appropriate ordering of screening tests . It seems logical that the first step in identifying the need for screening for obesity - related comorbidities is recognizing and documenting the presence of overweight / obesity . However, our study does not identify the way in which the presence of an emr diagnosis facilitates ordering appropriate laboratory studies . It is unclear whether providers record the diagnosis in the emr when they order laboratory tests, having already identified a patient as obese or overweight, or whether they use a preexisting documented diagnosis as a prompt to order appropriate screening . Additional investigation of clinician recognition of overweight / obesity and adherence to guidelines for screening for obesity - related complications is needed . It is a single - site study and represents the practices of residents in training, which may limit its generalizability . Prior studies have found that resident physicians are more likely than attending physicians to document and plot bmi, which may suggest that the trainees in our study were more likely than the average physician to focus on weight - related issues . However, the residents were directly supervised by board - certified attending pediatricians who practice in a variety of settings and who were responsible for the quality of care provided in the clinic . It is possible we misclassified some children with bmis in the overweight range who should have had screening labs in addition to fasting lipids on the basis of diabetes risk factors from their personal or family history these limitations should be considered in interpreting our results but they would be unlikely to significantly alter the implications of our findings . We anticipate that introduction of an emr - based automatic prompt to alert providers to a patient s weight status based on patient height and weight will improve serum - based screening and increase the diagnosis and treatment of overweight / obesity - related comorbidities . However, in our study, rates of laboratory screening were low even among patients with an emr diagnosis of overweight or obesity . Cabana et al cite 3 barriers for physician adherence to clinical practice guidelines: lack of familiarity, lack of awareness, and lack of agreement with guidelines . Their research shows that lack of adherence can be due to differences in the interpretation of the evidence, believing the benefits are not worth the risk, discomfort, or cost, or believing that guidelines decrease clinician autonomy . Physicians may not agree with the screening recommendations, may not believe adhering to the guidelines will change patient care or improve health outcomes, or may not know how to react appropriately to abnormal laboratory values . Ideally, a streamlined emr with automated prompts would help physicians recognize obesity, evaluate abnormal values, reinforce healthy eating behaviors with their patients, and increase awareness of obesity - related dangers . In addition to the potential for direct benefit to individual patients, the affordable care act (aca) includes provisions for increased reimbursement for meaningful use of emrs, including documenting overweight and obesity and providing counseling to patients . The effectiveness of such provisions in the aca, emr - based prompt strategies, and their subsequent impact on patient or family adherence to recommended changes in diet and exercise will require further study.
The herd comprised approximately 550 dairy cows of the israeli - holstein breed raised in a zero - grazing management system . Heifers were transferred at the age of 2 months to another farm and returned pregnant 14 months later . All the cows and heifers were observed at least twice daily by the herd s personnel and at least twice weekly by the attending veterinarian . Cases of bnvv were observed during january through march 2002 . Clinical signs of bnvv developed in 32 of the 39 heifers . Other age groups and heifers before calving were not affected . Treatment consisted of vaginal rinses with potassium permanganate or h2o2 daily from calving for as long as clinical symptoms persisted (9). Local antimicrobial therapy was not attempted since it was ineffective during the outbreaks on the other farms . A penicillin / streptomycin combination was given to 12 cows with temperatures> 39.5c, to provide broad - spectrum protection and prevent sepsis and limit economic losses (metronidazole is the drug of choice to treat infections caused by anaerobic bacteria, but its use is prohibited in israel). Five of these cows developed metritis and peritonitis and had to be slaughtered, whereas the temperature of the remaining seven cows returned to normal . No difference between convalescence periods of cows treated with potassium permanganate or h2o2 was observed . Eighteen cows recovered within 4 weeks after onset of infection; in 9, a more chronic infection, characterized by a mucopurulent vaginal discharge lasting up to 10 weeks, developed . The swabs were kept in amies transport medium (copan, italy) and processed within 5 hours from sampling . Swabs for mycoplasma and ureaplasma were taken in mycoplasmal transport medium and hayflick s medium, respectively (10). Swabs for virologic examination were placed in eagle s medium supplemented with antimicrobials and fetal calf serum and chilled . After vortexing, the medium was used for polymerase chain reaction (pcr) assays and virus isolation . Vaginal biopsies were suspended directly in 10% formalin and stained with hematoxylin - eosin in the laboratory . Bacteriologic examination included aerobic, microaerophilic, and anaerobic cultures, and resulting microorganisms were identified by standard methods (10). Samples were examined for chlamydia by direct immunofluorescence (cellab, australia) and for coxiella burnetti by the stamp (11) staining method . The only microorganisms cultured consistently from affected cows but not from healthy ones were pigmented, gram - negative, non spore - forming, anaerobic rods . The number of pigmented colonies was directly related to the severity of the vulvovaginal lesions, and they were not cultured from cows after their recovery . The pigmented bacteria were weakly saccharolytic; negative for indole, catalase, esculin hydrolysis, -fucosidase, and -galactosidase; and positive for -galactosidase and n - acetyl--glucosaminidase positive (as determined by the api rapid i d 32a kit, [biomrieux, france]). Identification of pigmented isolates as p. levii was done according to the manual of clinical microbiology (12). Since susceptibility to vancomycin distinguishes porphyromonas spp . From other gram - negative anaerobic rods (12) the mic of the isolates to this antibiotic was determined by the etest method (ab biodisk, sweden) and was found to be 3 g / ml, indicating susceptibility . Other potentially pathogenic bacteria isolated occasionally included arcanobacterium pyogenes, -hemolytic streptococci, and several enterobacteriaceae, but they could not be correlated with either the clinical signs or the presence of p. levii or bovine herpesvirus 4 (bohv-4). For the virologic examination, samples were added to confluent monolayers of madin - darby bovine kidney cells, observed daily for cytopathic effect, and passaged every 7 days . Bohv-4 was detected in samples from 27 cows with bnvv and from two cows in which the syndrome did not develop . Association of the bacteriologic or virologic findings and the clinical status of the cows was assessed statistically by the mantel - haenszel test (statistix, analytical sotware, tallahassee, fl). The test indicated a significant association between cows with bnvv and the presence of p. levii, adjusted for bohv-4 (p = 0.0006). No statistically significant association between bohv-4, adjusted for p. levii, and bnvv was found (p = 0.2359). Previous publications (13) also reported difficulties establishing a clear relationship between the presence of bohv-4 and, among other syndromes, bovine metritis, vaginitis, and abortions . Uterine biopsies were not taken, as injuring the mucosa in the presence of vaginal lesions could increase the risk for metritis . Bacterial colonies were seen in the section of vaginal biopsies, but typical changes of bohv-4 infection (13) were absent . The single necropsied cow showed peritonitis, resulting from rectal rupture, the cause of which could not be determined . The lesions affected the vulva and caudal part of the vagina but not the uterus (figure 2). Histopathologic examination of the vaginal lesions showed extensive necrosis of the epithelium and severe infiltration with a large number of mostly degenerative neutrophils and foamy macrophages . In the submucosa, since p. levii was isolated from all bnvv cases and very few healthy cows, it likely caused the lesions . All the cases observed during this outbreak occurred in primiparous cows in a restricted geographic area (within a radius of 20 km) during a limited period of time, indicating that one or more risk factors, alone or in conjunction, predisposed cows to infection . Outbreaks were observed after a large number of cattle were introduced, affecting primarily the animals introduced into the host farm . Transportation and social conflict may have acted as stressors (with stress immunosuppressive effects) (14) and predisposed the introduced cows to infection, with only social conflict affecting the local cattle . Calving is a well - known stressor (15), especially in primiparous cows (16). Moreover, lesions of the genital tract caused by calving tend to be more severe in primiparous cows, thus making them more prone to infection . After bnvv was described and its putative etiologic agent was identified, several sporadic cases were diagnosed on other farms, indicating that it might be underdiagnosed, and further studies of the syndrome may be warranted.
Studies have shown that only about 50.00%80.00% of the patients seeking care in emergency departments are real emergency cases . Triage is the process of prioritizing patients seeking emergency care based on their condition severity and initial need for care . This process is intended to allow the truly urgent cases to be handled first, when there are not sufficient resources for all patients to be treated immediately . The canadian triage and acuity scale (ctas) was developed in the mid-1990s by a group of physicians in new brunswick based on the australian national triage . It separates patients into the following five levels based on clinical presentation and severity: level 1, resuscitation required; level 2, urgent; level 3, semi - urgent; level 4, emergency; and level 5, nonemergency . Under this system, all patients needs are assessed rapidly upon arrival and the level 1 and level 2 patients are treated immediately . Pediatric patients can have particularly rapid changes in their condition, and parents expect them to receive appropriate treatments immediately . Canada developed a pediatric ctas, based on the original adult ctas, in 2001 . The 2008 revision of the pediatric ctas is a standard procedure in children's hospitals and pediatric emergency departments across canada . In china, emergency triage lacks operational efficiency and there is a shortage of well - trained triage nurses . Consequently, triage becomes backed up, resulting in some unstable children not being treated on time and avoidable tragic outcomes . In september of 2011, the chinese national health and family planning commission published the emergency patients triage guiding principle guide for classifying patients into the following four levels based on severity and resource demands: level 1, imminently endangered; level 2, critical; level 3, emergency; and level 4: nonemergency . The guide provides reference ranges for heart rate, breathing, systolic blood pressure, and oxygen saturation in adult patients . However, the guide is not appropriate for pediatric care due to the developmental, physiological differences between adults and children, particularly with respect to airway function, circulatory system, and nervous system . China lacks a standard pediatric triage system and its emergency departments often function primarily like 24-h outpatient clinics . As a result, medical professionals, researchers, and chinese families alike are keen to see more efficient emergency care for sick and injured children . We implemented the chinese pediatric emergency triage system (cpets) in our hospital's pediatric emergency room (per) in october 2013 and examined its effects on patient care parameters and patient satisfaction . The aim of the present study was to examine the utility of the cpets in real - time clinical use in our per . Toward this aim, we developed a new cpets modeled on the pediatric ctas and informed by domestic conditions, as summarized in table 1 . To reduce data artifacts related to seasonality all patients who visited our per in a 6-month period before (january 2013june 2013) and a 6-month period after (january 2014june 2014) implementation of the newly developed cpets served as the control group and experimental cpets group, respectively . The control group patients experienced our prior two - level triage system which segregated patients into only two groups, namely, triage into the per for care versus shunting to the outpatient care clinic . Summary of chinese pediatric emergency triage system levels rd: respiratory distress; e / m: ear / mouth; rc: rectal; svr: severe; plr: pupillary light reflex; spo2: peripheral capillary oxygen saturation; fb: foreign body; w/: with; cp: chest pain; bp: blood pressure; bldg: active bleeding; gi: gastrointestinal . The control group included 114,040 per patients (57,523 boys and 56,517 girls) and the cpets group included 102,969 per patients (51,936 boys and 51,033 girls). The study design was approved by the review board of first affiliated hospital of xiamen university . Briefly, triage level 1, 2, and 3 patients are directed to waiting area a, near the triage nurse station, and triage level 4 and 5 patients are directed to waiting area b. triage nurses completed a 2-month cpets training course (120 instructional hours) before using the cpets and associated software in the per . Second, if a patient does not meet the criteria for level 1 or 2, then the nurse evaluates whether the patient is level 5 . If so, then the patient's case is deemed a nonemergency and shunted to outpatient care . Subsequently, if the patient did not meet level 1 or 2 criteria and also did not meet level 5 criteria, then the patient is subjected to further evaluation to distinguish whether a level 3 or level 4 classification is more appropriate . T: body temperature; bp: blood pressure; spo2: peripheral capillary oxygen saturation; p: pulse . The dependent variables compared between the control and cpets groups included number of per visits, triage rate, triage accuracy, overall patient wait time, level 1/2 patient waiting time, and care satisfaction of the patients families . Triage rate was the percentage of check - in patients for whom a complete triage evaluation, terminating in shunting or per admission, was made . Triage accuracy was determined by per physicians confirming and correcting the nurse - assigned triage level . Wait times were calculated for randomly extracted samples from the populations assigned triage levels of 3, 4, or 5 (n = 100 per level for each group) and all patients assigned triage levels of 1 or 2 . The patients parents satisfaction was determined by telephone survey carried out by an independent third party surveyor . Data for the evaluated parameters were entered into a microsoft excel database by two researchers independently . The data entries were cross - referenced, and any gap and inter - user differences were corrected by referring back to the original case records . Quantitative data were compared between the two groups with student's t - tests . For those nonparametric data mann - whitney u - test and chi - square test the aim of the present study was to examine the utility of the cpets in real - time clinical use in our per . Toward this aim, we developed a new cpets modeled on the pediatric ctas and informed by domestic conditions, as summarized in table 1 . To reduce data artifacts related to seasonality all patients who visited our per in a 6-month period before (january 2013june 2013) and a 6-month period after (january 2014june 2014) implementation of the newly developed cpets served as the control group and experimental cpets group, respectively . The control group patients experienced our prior two - level triage system which segregated patients into only two groups, namely, triage into the per for care versus shunting to the outpatient care clinic . Summary of chinese pediatric emergency triage system levels rd: respiratory distress; e / m: ear / mouth; rc: rectal; svr: severe; plr: pupillary light reflex; spo2: peripheral capillary oxygen saturation; fb: foreign body; w/: with; cp: chest pain; bp: blood pressure; bldg: active bleeding; gi: gastrointestinal . The control group included 114,040 per patients (57,523 boys and 56,517 girls) and the cpets group included 102,969 per patients (51,936 boys and 51,033 girls). The study design was approved by the review board of first affiliated hospital of xiamen university . Briefly, triage level 1, 2, and 3 patients are directed to waiting area a, near the triage nurse station, and triage level 4 and 5 patients are directed to waiting area b. triage nurses completed a 2-month cpets training course (120 instructional hours) before using the cpets and associated software in the per . Second, if a patient does not meet the criteria for level 1 or 2, then the nurse evaluates whether the patient is level 5 . If so, then the patient's case is deemed a nonemergency and shunted to outpatient care . Subsequently, if the patient did not meet level 1 or 2 criteria and also did not meet level 5 criteria, then the patient is subjected to further evaluation to distinguish whether a level 3 or level 4 classification is more appropriate . T: body temperature; bp: blood pressure; spo2: peripheral capillary oxygen saturation; p: pulse . The dependent variables compared between the control and cpets groups included number of per visits, triage rate, triage accuracy, overall patient wait time, level 1/2 patient waiting time, and care satisfaction of the patients families . Triage rate was the percentage of check - in patients for whom a complete triage evaluation, terminating in shunting or per admission, was made . Triage accuracy was determined by per physicians confirming and correcting the nurse - assigned triage level . Wait times were calculated for randomly extracted samples from the populations assigned triage levels of 3, 4, or 5 (n = 100 per level for each group) and all patients assigned triage levels of 1 or 2 . The patients parents satisfaction was determined by telephone survey carried out by an independent third party surveyor . Data for the evaluated parameters were entered into a microsoft excel database by two researchers independently . The data entries were cross - referenced, and any gap and inter - user differences were corrected by referring back to the original case records . Chicago, il, usa). Mean and standard deviation (sd) were determined for quantitative data . Quantitative data were compared between the two groups with student's t - tests . For those nonparametric data mann - whitney u - test and chi - square test the patients visited to per in control group and cpets group were similar in terms of gender ratio (= 0.004, p> 0.05) and age (t = 0.590, p> 0.05). The percentages of children admitted to and treated in the per (emergency rate%), rather than being shunted to outpatient care, decreased after implantation of the cpets (cox - stuart trend test, t = 0, p = 0.0156). The monthly numbers of children subjected to triage rating (total), admitted and treated in the per (portion of total deemed emergency cases), and referred for outpatient treatment (portion of total deemed not emergency cases) during and between the study periods are reported in table 2 . As shown in table 3, patients wait times for care in the per were significantly shorter in the cpets group than in the control group, both overall and for severe (level 1 or 2) patients . Numbers of pediatric emergency room visitors by month note that emergency rate encompasses all patients not shunted to the outpatient clinic (i.e., cpets levels 14 in the post - cpets period). Effects of chinese pediatric emergency triage system on pediatric emergency room waiting times (min) data are presented as mean standard deviation or median (interquartile range). * highly significant (t = 11.27, p <0.001); significant comparison outcomes demonstrated reduced waiting times for care in the per in the cpets group relative to the control group, both overall and for severe (level 1 or 2) patients (z = 2.057, p = 0.040). The numbers of patients triaged, triage accuracy, and patient satisfaction for each group are reported in table 4 . Briefly, the triage rate for the cpets group (93.40%) was significantly increased compared to that of the control group (90.75%). In addition, triage accuracy calculations indicated that triage accuracy rate was also significantly improved in the cpets group (96.32% of a sample of 9679 cases) relative to that in the control group (85.09% of a sample of 8978 cases). Finally, patient satisfaction in the cpets group (94.23%) was significantly greater than that observed in the control group (92.21%). Group comparisons of number of patients triaged, triage accuracy, and satisfaction of pediatric emergency room patients parents * the cpets experienced by the experimental group was associated with a higher triage rate; better triage accuracy; higher family satisfaction rates . In the present study, we examined the effects of implementing a five - level cpets on patient care variables and patient satisfaction . We observed positive effects of the cpets on every variable assessed, including triage rate, triage accuracy, overall waiting time, level 1/2 waiting times, and parent satisfaction . Interestingly, although the number of patients who were treated in the per in the cpets group was less than that in the control group, there were actually more pediatric patients treated in our hospital during the cpets study period (275,378 patients, january 2014july 2014), when the cpets was in place, than during the control period (243,536 patients, january 2013july 2013). The reduction in per visits can be attributed to an addition of 142.40% number of people rather than treating in the outpatient clinic in the post - cpets study period (172,409 out patients) versus during the control period (71,127 out patients). The downward trend in per - admitted patients seen per month became apparent in october 2013 [table 2], the 1 month that the cpets was active . These shifts in patient numbers indicated that the cpets shunted nonemergency (level 4/5) patients to the outpatient clinic effectively . The resultant reduction in per cases should alleviate overcrowding and help ensure that appropriate resources are available for truly urgent cases . Given that parents often lack the medical knowledge to determine when their children should be brought to the per, triage rate is a key nursing quality evaluation index of emergency department care . A higher triage rate, such as we found with the cpets versus in the control period, represents more patients being treated in accordance with their needs . To help improve our triage rate, the cpets includes a reminder function to recall (through a loud speaker) checked - in patients in the per for reevaluation . Triage accuracy is critical to improve the quality of care delivered in the per, particularly with respect to ensuring that truly urgent cases are prioritized immediately . Indeed, one of the leading causes of death among pediatric patients is poor outcome when patient is in critical condition . If critical cases are recognized promptly, urgently needed treatments can be delivered without delay, preventing serious complications and deaths . Currently, in china, triage quality is variable ranging from more attentive administrators to less attentive administrators . Commonly, administrators staff triage desks with their most inexperienced nurses, preferring to assign the most experienced nurses to the resuscitation room . Indeed, a prior survey conducted among triage nurses in a swedish emergency department indicated that 20% of triage - station nurses were lacking triage knowledge, leaving their patients vulnerable to triage misjudgments . In the present study, the improved triage accuracy combined with the lower emergency rate indicate that our per had likely been over - triaging patients and that the cpets reduced over - triage incidents . As implemented in our per, the cpets provides clear computer - administered guidance to triage nurses, leading them to check patient variables item by item, resulting in a more comprehensive assessment, regardless of the triage nurse's experience . Notably, the present results indicate that utilization of the cpets resulted in a triage accuracy rate that was 11.23% higher than that obtained in the control period, demonstrating the system's value in a live clinical setting . In the chinese healthcare system, there are not a sufficient number of family doctor offices for family doctors to serve as the primary administrators of healthcare for children nationwide . Moreover, although the chinese government has highlighted the importance of general practices to encourage people to visit physicians in community hospitals, many parents prefer to bring their children to a per in a tertiary hospital because they are skeptical of the competence of community hospital physicians . Consequently, a larger proportion of children in china receive medical care primarily in the per than that in the western world . An important component to improving this situation is the implementation of a high - quality triage system that shunts nonemergency cases from the per to outpatient clinics while facilitating the delivery of emergency care in true emergency cases parents in china have even resorted to violent behavior when they felt that their child was not attended to properly . The intense reactions of parents might be exacerbated by the fact that most chinese families have only a single child . Previously, partovi et al . Found that implementation of a professional triage system in their hospital emergency department reduce length of stay from an average of 445 s (n = 814 cases) to an average of 363 s (n = 920 cases), an 18.40% reduction in average wait time . A computerized triage system enables general information collected by the triage nurse (e.g., demographics, vital signs, and history) to be viewable by the physician on his or her computer, reducing redundancy of communication and data, thereby saving time . The significant reductions in waiting times observed in this study support the idea that computerized triage systems can help reduce wait times . Moreover, we believe that the reduced waiting times observed in the present study were likely due to a combination of the outpatient shunting effect (discussed above) and reduced redundancy . Given the critical status of patients seeking treatment in pers, treatment delays can have life or death consequences: the shorter the wait time, the higher the success rate of lifesaving treatments . In the interest of minimizing delay, if a patient meets the criteria for level 1 or 2, he / she is sent to the resuscitation room immediately, without further triage assessment . Level 1 and 2 cases are relatively rare, and such patients can receive treatment immediately upon being identified . That being said, because patients triaged as level 3 can progress rapidly into a level 2 or 1 condition, they must be monitored closely . Indeed, partovi sn et al . Found that conditions changed for 25% of level 3 patients while they were waiting to be seen . Therefore, it is important that level 3 patients be seated in the immediate vicinity of the triage station, as is done in our cpets procedure, so that if their conditions worsen, the triage nurse is immediately accessible to update their level designation and expedite delivery of lifesaving treatments . There are several aspects of our cpets procedures that we believe have contributed to an overall improvement in per care and patient satisfaction . First, the cpets - associated reduction in per patient flow helps reduce wait times as discussed above . Second, under the new system, the triage station is now located at the per entrance, such that families arriving from multiple directions encounter the triage nurse immediately and receive triage and guidance promptly upon their arrival, which should help alleviate families anxieties . Third, the nurse now patrols patients regularly in accordance with reminders from the cpets software . Accordingly, patients vital signs are rechecked, enabling nurses to recognize condition changes that would otherwise be missed; this increased patient in addition, a rubric of the triage levels is posted in the waiting room for parents to see, which should help alleviate their worries about the care their child is receiving . Finally, having a standardized software - led triage form makes the triage process less arbitrary and less at risk of errors related to triage nurse inexperience . Regarding study limitations, it is important to note that this study was conducted at a single center in a tertiary hospital in xiamen . Consequently, it might not be representative of the whole city's per characteristics, not to mention the whole province's or nation's per characteristics . However, the study site is the first place where the ctas has been introduced in the mainland of china, after being modified to fit the chinese per setting with an integration plan . Thus, given its significant benefits, this first implementation provides an important example case for other cities in china to study in implementing their own per triage systems . In conclusion, the present study demonstrated that implementation of the cpets, which was developed in accordance with international standard five - level per triage practices, results in a shunting of nonemergency patients to outpatient care (thereby reducing per patient flow), improves triage accuracy, reduces wait times, especially for critical patients for whom reduced delays amount to a better rescue success rate, and improves the satisfaction of the patients parents . The present comparison of 6-month periods indicates that this system is clinically useful and suggests that its broader implementation should be promoted . This work was supported by the grant from the state of foreign experts bureau (no . This work was supported by the grant from the state of foreign experts bureau (no.
The most common combination of oral antihyperglycaemic agents used for patients with type 2 diabetes is metformin and sulphonylurea [1, 2]. Despite good initial efficacy, for most patients, this dual therapy is associated with progressive worsening of blood glucose control over time, requiring additional medication . Clinicians and researchers currently debate the question of which additional agent is best in this situation [4, 5]. In clinical studies, dpp-4 inhibitors have been shown to improve hba1c significantly when administered as monotherapy or as dual therapy in conjunction with metformin or a sulphonylurea [611]. However, information on its use as a third agent in triple therapy is relatively scant . Two studies have demonstrated the efficacy of dpp-4 inhibitors when added to the metformin and sulphonylurea combination [12, 13]. Also of interest is a meta - analysis of 55 studies that has suggested that dpp-4 inhibitors may be more effective in asian patients . As type 2 diabetes is becoming more common in asia with the progressive increase in affluence of this region, it is important to determine if the better response to dpp-4 inhibitors in asian patients can be confirmed . The purpose of the present study is, therefore, to examine the efficacy of dpp-4 inhibitors when given as add - on therapy to the combination of metformin and sulphonylurea in patients with type 2 diabetes and suboptimal glycaemic control . Whether asian patients respond better to triple therapy was also determined . Patients were selected from those who attended the diabetes centre of royal prince alfred hospital, sydney, australia . Their clinical and demographic data were extracted from a purpose built diabetes database which included approximately 25,000 patients collected over two decades . A total of 688 patients with type 2 diabetes were found to be treated with dpp-4 inhibitor (sitagliptin, linagliptin, saxagliptin, or vildagliptin) over the previous 5 years (from 2008 to 2013). Amongst these, 169 patients were on triple oral therapy using sulphonylurea, metformin, and dpp-4 inhibitors . From this group, patients were selected for this analysis if they met the following exclusion and inclusion criteria: added dpp-4 inhibitor at the maximum recommended dosage to the combination of metformin and sulphonylurea treatment (metformin> 1.5 gm / day or> 1.0 gm / day if on xr preparation, or maximum tolerated dose; diamicron> 160 mg / day, diamicron mr 60 mg / day, amaryl 4 mg / day, or minidiab 10 mg / day);had no change in the dosage of sulphonylurea or metformin after dpp-4 inhibitor was added;remained on triple therapy for 12 weeks;had hba1c results available (i) within the 12 months prior to and (ii) between 3 to 12 months after addition of dpp-4 inhibitor therapy;were not on insulin or glp agonist or other diabetes medications.eighty-nine patients were excluded from the analysis . Of these, 49 were on dpp-4 inhibitors as the second agent of the triple therapy and 40 had hba1c measured at a time outside the specified window . Ethnicity of the individuals was determined by self - report of the patients (asian group n = 41: 36 chinese and 5 from the indian subcontinent; non - asian group n = 39: 21 anglo - celtics, 3 middle eastern, 1 indigenous australians, and 14 europeans). Permission to record and analyze the computerized data was given by the ethics committee of the hospital . Added dpp-4 inhibitor at the maximum recommended dosage to the combination of metformin and sulphonylurea treatment (metformin> 1.5 gm / day or> 1.0 gm / day if on xr preparation, or maximum tolerated dose; diamicron> 160 mg / day, diamicron mr 60 mg / day, amaryl 4 mg / day, or minidiab 10 mg / day); had no change in the dosage of sulphonylurea or metformin after dpp-4 inhibitor was added; remained on triple therapy for 12 weeks; had hba1c results available (i) within the 12 months prior to and (ii) between 3 to 12 months after addition of dpp-4 inhibitor therapy; were not on insulin or glp agonist or other diabetes medications . Numerical data are presented as percentage or mean standard deviation or median and interquartile range . The change in hba1c before and after dpp-4 inhibitor add - on treatment was the primary endpoint and tested by student's t - test, wilcoxon signed rank test, or analysis of variance (anova). The change in hba1c was examined as the dependent variable and tested against duration of diabetes, gender, body mass index (bmi), age, ethnicity, and baseline hba1c as independent variables by multiple regression analysis . The change in hba1c was also studied by analysis of covariance adjusted for baseline hba1c, duration of diabetes, bmi, and ethnicity . Statistical significance was based on 2-sided tests and accepted at the p <0.05 level . Amongst the 80 patients studied, 64 were taking sitagliptin, 12 were on saxagliptin, and 4 were on vildagliptin . After triple oral therapy duration of 4.6 (3.66.6) months, their mean hba1c decreased from 8.3% (7.78.9) [67 mmol / mol, 6174] to 7.2% (6.87.6%) [55 mmol / mol, 5160 mmol / mol] with 26 patients (32.5%) achieving an hba1c of <7% (53 mmol / mol). Weight before (79.6 19.5 kg) and after (78.8 19.3 kg) triple therapy did not change significantly in the 66 patients with readings available before and after this treatment . The mean changes in hba1c from pre- to posttriple therapy, according to clinical characteristics, are shown in table 2 . Patients with higher baseline hba1c, absence of obesity, and younger age showed a greater response . By multiple regression analysis gender, the duration of diabetes, and bmi analyzed as a continuous variable were not significant factors . The duration of triple therapy analyzed categorically (or <6 months) or by regression did not affect response (r = 0.17). The asian and non - asian groups have similar age, duration of diabetes, and baseline glycaemic control . The hba1c reduction after the implementation of triple therapy was not different between the asian (1.00, 0.61.3%) and non - asian group (0.90, 0.41.6%). Metformin and sulphonylurea are commonly prescribed together in patients with type 2 diabetes [1, 2]. When such combination therapy can no longer maintain acceptable glycaemic control, many other antihyperglycaemic agents are available to be added as the third agent of triple therapy . Mcintosh et al . Conducted a systematic review and meta - analysis of 33 controlled trials to evaluate the comparative safety and efficacy of various classes of antihyperglycaemic therapies in this scenario . Insulins, dpp-4 inhibitor, glucagon - like peptide-1 (glp-1) analogues, and thiazolidinediones (tzds) all produced statistically significant reductions in hba1c ranging from 0.89% to 1.17%, whereas meglitinides and alpha - glucosidase inhibitors did not . In their analysis, insulins and tzds were associated with weight gain of 1.855.00 kg, glp-1 analogues were associated with modest weight loss, and dpp-4 inhibitor was weight - neutral and did not show any increase in hypoglycaemia which was found when insulin was used . In the analysis of mcintosh, only one dpp-4 inhibitor (sitagliptin) was included and when used in triple therapy, a fall in hba1c of 0.89% was demonstrated . In another study not included in the analysis, linagliptin administrated for a 24-week period as triple therapy significantly improved glycaemic control (hba1c, 0.62%) and was well tolerated . Study showed that sitagliptin as the 4th agent also reduced hba1c by about 1% when added to the combination of metformin, sulphonylurea, and glucosidase - inhibitor . Based on these studies, dpp-4 inhibitorsas a third - line oral antihyperglycaemic agent appear to be effective and safe, without weight gain . Overall, despite its increasing usage, there have been few reports on the efficacy of dpp-4 inhibitors as the third oral agent in a clinical setting . Also of considerable interest, kim et al . In their review of dpp-4 inhibitors provided evidence that this class of agents may be more effective in asian patients . However, the great majority of the studies included in the analysis pertained to mono- or dual therapies . The categorization of ethnicity was not based on an individual patient basis . Instead, it assigned the total cohort of a trial as asian or non - asian ethnicity according to whether the study included more or less than 50% asian participants or was conducted in an asian dominant country . The superiority of any pharmacological agent in an asian population could have important medicoeconomic implications due to the high and progressively increasing prevalence of diabetes in asia . Based on these considerations we have audited the efficacy of dpp-4 inhibitors as the third oral agent in our clinic and also made comparison of the response in asian and non - asian patients . Our results confirmed the usefulness of dpp-4 inhibitors in improving glycaemic control in this context . One - third of patients reached an optimal level of glycaemic control, as defined by an hba1c of less than 7% (53 mmol / mol). The efficacy of dpp-4 inhibitors in this clinical setting seems quite effective although we could not dissect out the impact of a concerted effort at lifestyle changes by the patients when faced with the possibility of insulin requirement . Baseline glycaemic control, degree of obesity, and age of patients were identified by both univariate and multivariate analyses to be important determinants of response . Not unexpectedly, the drop in hba1c was greater in those with worse initial glycaemic control . There was also a better response in those with lesser obesity, perhaps a reflection that these subjects are relatively more insulin deficient than insulin resistant in the pathogenesis of their diabetes . They would be expected to respond better to an agent that can increase insulin availability . Our observation that the younger patients appeared to respond better is a potential advantage of the ddp-4 inhibitors as the need for better glycaemic control is obviously more in this age group . It is interesting that duration of diabetes does not seem to impair response, suggesting that the ability of dpp-4 inhibitors to augment meal associated insulin secretion does not decline with time of having diabetes . Moreover, there was no evidence of a significant decline in its efficacy up to 12 months of such triple therapy . However, the benefits of such treatment regimen for the longer term would need to be examined . The findings of seino et al . Suggest a better response of asian patients to dpp-4 inhibitors, raising the possibility that patients of asian ethnicity have relatively more defects in meal associated insulin secretion [17, 18]. A better response to glucagon - like peptide analogues in asians has also been shown in a meta - analysis . Our study did not provide evidence to support this notion of difference in ethnic response . In comparison with the reviews and meta - analyses mentioned above, our study has the advantage of categorizing ethnicity specifically at an individual level but had examined only 80 subjects . Therefore, we cannot exclude the possibility that a small difference may be missed . Our study also only examined patients on triple oral agent therapy which was not comprehensively evaluated in the kim analysis . So the possibility remains that asian patients would respond better to dpp-4 inhibitors in mono- or dual therapies . Being a retrospective study, we cannot be certain that there was no selection bias for using dpp-4 inhibitor as the third agent but, during the period examined, the safety of the thiazolidinediones had come into question and the sglt-2 inhibitors were not yet available . It would have been informative to know the relative efficacy of the dpp-4 inhibitor in comparison with insulin or glp - agonist in this clinical scenario . However, this would require a randomized clinical trial and is beyond the scope of this analysis . However, our study confirmed the effectiveness of dpp-4 inhibitors as the third oral agent in improving glycaemic control in type 2 diabetes.
The history of total knee arthroplasty (tka) designs demonstrates attempts at implants that more closely match patient anatomy for improved treatment results . Traditional total knee implant systems were designed to provide components of a wide range of sizes to achieve a proper fit in almost all patients . However, selection of a femoral component of correct size for a particular patient can still be challenging not only because it is common to have a distal femoral anteroposterior (ap) dimension going in the middle of usual 3 - 4 mm gap between each sizes of traditional femoral components but also because medial lateral (ml) overhang is sometimes encountered even with a traditional femoral component with a precisely matching ap dimension . In sizing of a femoral component, the ap dimension is important to maintain the flexion - extension gap and optimal tension in the extensor mechanism, whereas the ml dimension determines adequate coverage of the resected bone surface, allowing more even stress distribution and smooth tracking of the patellofemoral joint . Although there is no evidence on the clinical effect of underhang and overhang of implants, theoretical assumption is possible . If a component is too small (underhang), there will be higher contact stresses on the reduced bone - implant interface, increasing the risk of subsidence and loosening . Conversely, if a component is too large (overhang), it may impinge on the surrounding capsular tissues and ligaments, causing pain and limiting the range of motion (rom) of the joint . Anatomic variations between male and female are well - documented in several anatomic and radiographic studies1,2,3,4,5). In general, females tend to have smaller distal femora and different shapes with a narrower ml dimension for a given ap dimension when compared with males1). In addition, females are thought to have an increased quardriceps angle and a less prominent anterior femoral condyle than males even though controversy still remains6,7). The recent introduction of a gender - specific implant (gender solutions nexgen high - flex; zimmer inc ., warsaw, in) designed specifically for females started the debate that has focused on the effect of gender on the results of tka using traditional implant systems . The gender solutions implant was designed to better accommodate the anatomic differences noted in females with a narrower ml dimension for any given ap dimension . Besides, the angle of trochlear groove was increased and the anterior flange thickness and width were reduced to more closely reproduce the native female anatomy . With this background, we designed this present study in order to find out if gender - specific implants reduce the incidence of overhang during operation and there is any clinical or radiological advantage of gender specific implants over traditional implants . We hypothesized: 1) the incidence of femoral component overhang would be reduced with the use of gender - specific femoral components and 2) clinical and radiological results would show no difference between two types of implants in a short - term follow - up because no clinical disadvantage of overhang has been reported so far . Between may 2007 and january 2008, 136 primary tkas were undertaken in a consecutive series of 92 patients . There were 7 males (8 knees) and 85 females (128 knees). The criteria for inclusion into the study consisted of female gender, a primary diagnosis of osteoarthritis and a minimum 3-year follow - up . Patients with severe bone loss requiring structural bone grafting or metal augmentation were excluded from the present study . Their mean age at the time of the operation was 67.9 years (range, 52 to 80 years) and their mean body mass index (bmi) was 26.8 kg / m (range, 20.2 to 33.3 kg / m). The mean follow - up duration was 41.3 months (range, 36 to 50 months). There were 28 unilateral procedures and 38 bilateral procedures that were performed two weeks apart . We compared the clinical and radiological outcomes of tka using a gender - specific implant (gender solutions nexgen high - flex) and a traditional implant (nexgen legacy posterior stabilized flex [lps - flex], zimmer inc .) At 3 years after surgery . We also analyzed intraoperative measurements, including the overhang of femoral component and patella tracking, after tka using two different trial components . All the operations were performed by the senior author (bin) using the same technique . After eversion of the patella, resection of the femoral condyles was performed to remove a thickness of bone equal to that of the femoral component to be inserted . The femoral cutting block was placed in 3 to 5 of external rotation to the posterior condyles, and this was ascertained by the ap trochlear sulcus . The valgus angle of femoral resection was made using an intramedullary guide, and ap cutting was performed using an anterior referencing system . Femoral component sizing was based on the ap dimension of the femur, which was measured with a femoral ap sizer during operation . If a measurement of the ap dimension fell between two sizes, the smaller size femoral component was chosen . Using an intramedullary tibial guide, a 10 mm - thick tibial bone resection was performed to obtain a surface that was perpendicular to the shaft of the tibia in the coronal plane, with a 7 posterior slope in the sagittal plane . Two different trial femoral components (gender solutions nexgen high - flex prosthesis and nexgen lps - flex prosthesis) of the same size were inserted sequentially after femoral and tibial bone cuts . At this moment, overhang of each trial femoral component was examined medially and laterally at three cut surfaces (anterior, anterior chamfer and distal cut surfaces). Patellar tracking was also checked with each trial component using the' no thumb' test . The rom in which the patella showed congruent tracking was recorded with each trial femoral component and designated as congruent patella tracking rom . Based on the coverage of cut surface and patellar tracking, appropriate type of permanent femoral implant was chosen by the surgeon . The patients who received a gender - specific implant (gender solutions) were assigned to group i and those with a traditional implant (nexgen lps - flex) were to group ii . Two days after surgery, drain was removed and continuous passive motion and tolerable weight bearing were started . The entire data were collected prospectively from the beginning of the present study and were compared and analyzed retrospectively . This study was approved by the institutional review board at our hospital and informed consent was waived . Presence of overhang at the three femoral cut surfaces and the congruent patella tracking rom of each prosthesis were assessed intraoperatively and compared between groups . Postoperatively, the patients were reviewed at six weeks, three months, six months, and annually after surgery . Clinical parameters including hospital for special surgery (hss) knee score8), flexion contracture, maximal flexion, and complications were evaluated and compared preoperatively and at 3 years after surgery . Flexion contracture and maximal flexion were measured using a manual goniometer with the arms aligned along the long axes of the femur and tibia on the lateral side of the knee joint . Patients were told to straighten and bend their knee until they felt a slight degree of pain in supine position . All the data at follow - up examinations were recorded by an orthopedic surgeon and compiled by a research assistant who was not a part of the surgical team and had no information about the surgical and radiological findings . Radiographic evaluation included standing ap, lateral, and merchant views of both knees at three months, six months, and annually after surgery, which were analyzed using the radiological evaluation system of the knee society9) to delineate radiolucency around the components . Postoperative patellar tracking was assessed by measuring patella tilt angle and patella displacement as described by gomes et al.10). All the radiographs were analyzed by two of the co - authors (kim jm and kim sb) who were blinded to patient information . Between may 2007 and january 2008, 136 primary tkas were undertaken in a consecutive series of 92 patients . There were 7 males (8 knees) and 85 females (128 knees). The criteria for inclusion into the study consisted of female gender, a primary diagnosis of osteoarthritis and a minimum 3-year follow - up . Patients with severe bone loss requiring structural bone grafting or metal augmentation were excluded from the present study . Their mean age at the time of the operation was 67.9 years (range, 52 to 80 years) and their mean body mass index (bmi) was 26.8 kg / m (range, 20.2 to 33.3 kg / m). The mean follow - up duration was 41.3 months (range, 36 to 50 months). There were 28 unilateral procedures and 38 bilateral procedures that were performed two weeks apart . We compared the clinical and radiological outcomes of tka using a gender - specific implant (gender solutions nexgen high - flex) and a traditional implant (nexgen legacy posterior stabilized flex [lps - flex], zimmer inc .) At 3 years after surgery . We also analyzed intraoperative measurements, including the overhang of femoral component and patella tracking, after tka using two different trial components . All the operations were performed by the senior author (bin) using the same technique . After eversion of the patella, resection of the femoral condyles was performed to remove a thickness of bone equal to that of the femoral component to be inserted . The femoral cutting block was placed in 3 to 5 of external rotation to the posterior condyles, and this was ascertained by the ap trochlear sulcus . The valgus angle of femoral resection was made using an intramedullary guide, and ap cutting was performed using an anterior referencing system . Femoral component sizing was based on the ap dimension of the femur, which was measured with a femoral ap sizer during operation . If a measurement of the ap dimension fell between two sizes, the smaller size femoral component was chosen . Using an intramedullary tibial guide, a 10 mm - thick tibial bone resection was performed to obtain a surface that was perpendicular to the shaft of the tibia in the coronal plane, with a 7 posterior slope in the sagittal plane . Two different trial femoral components (gender solutions nexgen high - flex prosthesis and nexgen lps - flex prosthesis) of the same size were inserted sequentially after femoral and tibial bone cuts . At this moment, overhang of each trial femoral component was examined medially and laterally at three cut surfaces (anterior, anterior chamfer and distal cut surfaces). Patellar tracking was also checked with each trial component using the' no thumb' test . The rom in which the patella showed congruent tracking was recorded with each trial femoral component and designated as congruent patella tracking rom . Based on the coverage of cut surface and patellar tracking, appropriate type of permanent femoral implant was chosen by the surgeon . The patients who received a gender - specific implant (gender solutions) were assigned to group i and those with a traditional implant (nexgen lps - flex) were to group ii . Two days after surgery, drain was removed and continuous passive motion and tolerable weight bearing were started . The entire data were collected prospectively from the beginning of the present study and were compared and analyzed retrospectively . This study was approved by the institutional review board at our hospital and informed consent was waived . Presence of overhang at the three femoral cut surfaces and the congruent patella tracking rom of each prosthesis were assessed intraoperatively and compared between groups . Postoperatively, the patients were reviewed at six weeks, three months, six months, and annually after surgery . Clinical parameters including hospital for special surgery (hss) knee score8), flexion contracture, maximal flexion, and complications were evaluated and compared preoperatively and at 3 years after surgery . Flexion contracture and maximal flexion were measured using a manual goniometer with the arms aligned along the long axes of the femur and tibia on the lateral side of the knee joint . Patients were told to straighten and bend their knee until they felt a slight degree of pain in supine position . All the data at follow - up examinations were recorded by an orthopedic surgeon and compiled by a research assistant who was not a part of the surgical team and had no information about the surgical and radiological findings . Radiographic evaluation included standing ap, lateral, and merchant views of both knees at three months, six months, and annually after surgery, which were analyzed using the radiological evaluation system of the knee society9) to delineate radiolucency around the components . Postoperative patellar tracking was assessed by measuring patella tilt angle and patella displacement as described by gomes et al.10). All the radiographs were analyzed by two of the co - authors (kim jm and kim sb) who were blinded to patient information . The incidence of overhang of the trial femoral component was significantly higher in the knees with the traditional component than those with the gender - specific trial component (table 1). Of the 104 knees, overhang was observed at least in one area in 62 knees with the traditional trial component (59.6%) compared to 26 knees (25.0%) with the gender - specific trial component (p<0.001). Among the six areas where overhang was examined, the lateral anterior cut surface was the area where overhang of the femoral component was most frequent regardless of the type of the trial component . Distal medial area was the only exception that did not show statistically significant difference (p=0.250). Sixty - two knees (59.6%) that demonstrated overhang with traditional trial components were replaced with a gender - specific implant (group i) and the remaining 42 knees (40.4%) that showed no overhang with traditional trial components were replaced with a traditional implant (group ii). Therefore, the incidence of overhang after final implantation was 26/62 knees (41.9%) in group i and no overhang (0/42 knees) was observed in group ii . As a result, 36 knees in group i were able to avoid overhang with use of the gender - specific implants instead of the traditional ones, whereas the remaining 26 knees in group i still showed overhang even with gender - specific implants . Ten patients received a gender solutions prosthesis on one side and a lps - flex prosthesis on the other side . The difference of the congruent patella tracking rom between traditional and gender - specific trial components was not significant (p>0.05) (table 3). Hence, there was no case where a gender - specific implant was chosen for better patella tracking . The distribution of the femoral component size ranged from c to e and showed a tendency of selecting larger sizes in the knees with the gender - specific implant (p=0.045) (table 4). Clinical evaluation was performed in all patients including 10 patients who received different implants in bilateral tka . The mean flexion contracture, mean maximal flexion and mean rom improved significantly after operation in both groups . The mean hss score also improved after operation in both groups and the difference between two groups was not significant (table 5). Lateral release was not performed in both groups . At the final follow - up, the mean patella tilt angle and mean patella displacement showed no significant difference between two groups (table 6). We observed no clinical complications in both groups, including delayed wound healing, deep infection or instability . No indications of component loosening and progressive osteolysis were noted in any zone on the postoperative radiographs . In the present study, the incidence of overhang was reduced by 34.6% in the gender - specific implant group in which overhang was inevitable during trial insertion of the traditional implant . However, clinical disadvantage of overhang was not demonstrated in the short - term follow - up . In 1996, poilvache et al.4) indicated some female femora were narrower than average and the available femoral component with adequate ap dimension might be too broad in these patients . Several years later, chin et al.1) investigated 200 consecutive knees (100 males and 100 females) during primary tka and reported female femora showed a tendency to have a narrower ml dimension for any given ap dimension than male femora (ap / ml ratio, 0.82 vs. 0.79). Shortly after this study, hitt et al.2) performed a well - designed multicenter study enrolling 337 knees (128 males and 209 females) to compare six different contemporary prosthetic systems of traditional design . They reported that all the prostheses tended to present more or less ml overhang in female femora and the tendency was more evident in larger sizes . Moreover, they introduced the concept of aspect ratio (ml / ap ratio) and demonstrated that the aspect ratio of female femora tended to decrease in larger knees while male femora showed relatively constant aspect ratio throughout the sizes . After this study, the aspect ratio became a standard parameter to describe distal femoral morphology . They also stated manufacturers should consider gender - specific implants or decrease the ml dimension to prevent overhang in females . Stimulated by these reports, gender - specific implants were launched and marketed, which lead to the debate as to the relevance of this system6,7,11). Proponents of gender - specific implants presented accumulated data on sexual dimorphism of distal femur during decades and possible negative effects of component overhang6,11). On the contrary, opponents pointed out that most recent studies had failed to eliminate or correct for other biases between both sexes such as average height or size of the studied femora and concluded that the unproven use of gender - specific implants may pose ethical or medico - legal dilemmas7). In spite of this, there seems to be no disagreement at least on one point that the reported studies had neither directly demonstrated negative effects of overhang or overstuffing nor investigated actual merits of gender - specific knee arthroplasty with real implants . The paucity of data on negative effects of overstuffing has been taken for granted due to the difficulty of measuring the amount of overhang and controlling multiple possible prognostic factors other than overhang . However, no theoretical advantage was expected with regard to the overhang of femoral implant . Thus, we decided to investigate the reduced incidence of overhang in tka using gender - specific implants in asian female patients even though the practical advantage of reduced overhang remained unclear . In this study, the incidence of ml overhang after tka using traditional and gender - specific trial components showed a significant difference (59.6% vs. 25.0%). In other words, 34.6% of the patients were able to avoid femoral component overhang because gender - specific implants were available . Regarding the incidence of 25.0% of overhang even with the gender - specific trial component, these patients would have had greater overhang if it had not been for a gender - specific implant . With respect to the incidence of overhang in six separate areas, only distal medial cut surface did not show significant differences between two groups . This may be attributable to our efforts to lateralize femoral components during operation in order to improve patella tracking regardless of the type of implant . The distribution of selected sizes in each group showed a tendency of higher selection rate of a gender - specific implant in larger sizes (p=0.045). Our data is consistent with those of hitt et al.2) that showed a higher aspect ratio in smaller knees and a proportionally lower ratio in larger knees . Song et al.12) compared clinical and radiologic results of tka using gender - specific implants and conventional unisex design with a minimum follow - up of two years . They found no difference in clinical and radiologic outcomes between two groups and concluded gender - specific implants demonstrated no advantage over standard unisex design . Guy et al.13) compared 50 males and 50 females to investigate the difference in the morphology of distal femur and the incidence of femoral component overhang . The mean aspect ratio was larger in females (1.02) than males (0.98) (p=0.005). Standard implants in tka resulted in a significant increase of overhang in females in terms of both incidence and magnitude than did gender - specific implants . Tanavalee et al.14) performed a prospective study that compared standard and gender - specific implants with an average follow - up of two years . The selection rate of gender - specific implants was significantly higher in females (60.8%) than males (8.2%) and significantly increased with increasing femoral size . The latter finding was exactly concordant with that in our present study and the selection rate of gender - specific implants in females (60.8%) was very similar to that of our study (59.6%). They evaluated 122 knees in three subdivided groups (42 females with unisex implants, 41 males with unisex implants and 39 females with gender - specific implants) and concluded that traditional unisex femoral components did not accurately match female anatomy . However, they were not able to demonstrate better radiographic results with gender - specific implants . Therefore, the overall conclusion of the study was similar to ours in that traditional femoral components did not seem pertinent to reproduce female anatomy and gender - specific components did not provide a perfect solution, either . Asymmetrical component sizes are known to be frequently used in bilateral tka, and the prevalence of which ranges from 6.7% by brown et al.16) to 31% by hitt et al.2). In our study, 10 patients had a gender solutions prosthesis in one knee and a lps - flex prosthesis in the contralateral knee . This accounted for 10/38 (26.3%) of all bilateral surgeries in the present study . Not only the size but also the shape of both distal femora of a patient may be different from each other according to our data, which alerted us to the risk of the use of the same implant in bilateral tka . We believe the phenomenon of asymmetric component implantation probably originated from the awareness of the fact that anatomy is different from one side to the other . The clinical outcomes of two groups did not present significant differences in terms of motion, performance of patella and hss score in this study . Because these parameters are not considered to deteriorate rapidly in short term, long - term observation seems to be necessary to elucidate differences, if any, in clinical outcomes between two groups . One of the limitations of this study is that it is not a true comparative study: implant selection relied on the extent of overhang of each trial femoral component and therefore the true independent clinical outcomes of each implant system were not thoroughly investigated . Besides, the minimum 3-year follow - up period may be considered insufficient to determine the longevity of clinical outcomes . However, this study is based on solid evidence from prospective data, which can be regarded as a strength of the study . In the present study, the gender - specific femoral component explicitly reduced the incidence of overhang compared to the traditional femoral component . However, we could not find any correlation between this apparent advantage and more favorable clinical and radiologic outcomes . Potential advantages of gender specific implants, such as avoidance of overstuffing in the patellofemoral joint, soft tissue irritation and anterior knee pain, were not identified in this study . There was significant difference in the incidence of overhang of femoral component between the gender - specific implant and the traditional implant . It was observed that 34.6% of the patients were able to avoid femoral component overhang with gender - specific implants . After three years of follow - up, there was no significant difference in clinical and radiological outcomes between two groups . Potential advantages of gender specific implants, such as avoidance of overstuffing in the patellofemoral joint, soft tissue irritation and anterior knee pain, should be evaluated in a long - term follow - up study.
Shivering associated with neuraxial anesthesia is a frequent complication; occurring in up to 55% of patients . Shivering is uncomfortable for the patient and may interfere with monitoring of electrocardiogram, blood pressure (bp), and oxygen saturation . The metabolic and hemodynamic consequences of shivering include increased disbursement of cardiac and systemic energy, increased oxygen consumption and carbon dioxide production, and increased cardiac work . The mechanisms chiefly responsible for shivering in patients undergoing surgery are intraoperative temperature loss, increased sympathetic tone, pain and systemic release of pyrogens . Spinal anesthesia for cesarean section still continues to be a popular technique because it provides many advantages such as rapid onset, high success rate, minimal maternal and fetal drug exposure and minimal maternal aspiration . In spite of several investigations used to treat shivering after spinal anesthesia little is known about the exact etiology of shivering and the best way of prevention . Meperidine has been used for a long time to treat and prevent shivering, with somecontroversy of its optimal dose without producing undesired side - effects . Recommended use of intrathecal meperidine to decrease the incidence of shivering without considerable side - effects in the obstetrical population but in another study khan et al . Found that intrathecal meperidine cannot be recommended in caesarean section to prevent shivering as its use however, these controversy and lack of meta - analysis study to clear the risk and benefit of injecting meperidine into intratechal spaceforced us to do this study . The purpose of this study was to compare the effect of adding different doses of intrathecal meperdine on the incidence, intensity and other side - effects during delivery under spinal anesthesia . This randomized double - blinded clinical trial was conducted between august 2009 and august 2010 in the educational hospitals of busheher university of medical sciences, bushehr, iran . This study was approved by the ethics committee of busheher university of medical sciences and is registered at the clinicaltrials.gov database (reference no . This study was performed according to the requirements of the declaration of helsinki . After attaining informed consent 156 parturient women (asa physical status i or ii) scheduled for elective cesarean delivery under spinal anesthesia parturient women with contraindications to regional anesthesia, hypersensitivity to amide local anesthetics or meperidine, history of headache, smoking, previous postoperative nausea and vomiting, opioid use or severe preeclampsia were excluded . Patients were randomly assigned to four equal groups for spinal anesthesia according to numbers inserted in sealed envelopes . The temperature of the operating room was kept instantly using a calibrated temperature in cases and control groups . Before the spinal anesthesia was administered, patients were placed under standard monitoring (blood pressure, electrocardiogram, heart rate and oxygen saturation) and received iv lactated ringer's solution 10ml / kg . During anesthesia, oxygen was given, and patients were covered with drapes but not activelywarmed . Standard group received only 2 cc heavy bupivacaine 0.5% (10 mg) (group i) and we added free preservative meperidine in three experimental groups . Heavy bupivacaine 0.5% (10 mg) plus 0/2 mg / kg free preservative meperidine (group ii), heavy bupivacaine 0.5% (10 mg) plus 0/3 mg / kg free preservative meperidine (group iii), heavy bupivacaine 0.5% (10 mg) plus 0/4 mg/ kg free preservative meperidine (group iv). Spinal anesthesia was administered in sitting position at the l4 - 5 interspace with a midline approach by using a 25-gauge quincke needle . Solutions were prepared by another anesthesiologist so that the anesthesiologist performing the spinal block was blind to which drug was injected . Active support of ventilation was provided if there was any sign of respiratory depression (spo2 less than 95%) in the case and control groups . Sensory anesthesia was evaluated by pinprick at one - minute intervals for 10 min, five - minute intervals for 35 min, and then at ten - minute intervals until regression to l4 . Once patients were in the post - anesthesia care unit, motor blockade was assessed with the bromage scale:1, unable to move feet; 2, able to move feet only; 3, just able to move knees; and 4, full flexion of knees and feet . Hypotension was defined as a decrease in systolic blood pressure to <90 mm hg or 30% less than baseline value . It was treated with 5 - 10 mg of ephedrine iv and bradycardia (heart rate<50) was treated with intravenous atropine 0.5 mg . Vomiting was scored yes or no; nausea was scored none, mild, moderate or severe on a verbal patient / examiner / scale and metoclopramide 10 mg iv was administered for moderate to severe nausea and vomiting . Supplemental intraoperative analgesia was limited to iv fentanyl (1 - 2 g / kg), which was standardized and used as a rescue dose if necessary . The tympanic temperature was monitored every 20 min from one side (right ear). The operating room temperature was maintained at 21c23c for all patients using a calibrated temperature . Shivering was graded with a scale described by crossley and mahajan: 0, no shivering; 1, piloerection or peripheral vasoconstriction but no visible shivering; 2, muscular activity in only one muscle group; 3, muscular activity in more than one muscle group but not generalized shivering; and 4, shivering involving the whole body . All patients were asked on the first and second postoperative day about occurrence of headache, backache, paraesthesia, pain in thighs, buttocks or leg etc . Demographic data was collected by an observer unaware of the study groups; and demographic data except vomiting and maximal intensity of shivering were compared using kruskal wallis h. the maximal intensity of shivering and vomiting were compared using chi - square . To obtain at least 50% reduction in expected incidence, with error of 0.05 and a error of 0.2, a sample size of 39 patients per group was needed . There was no significant difference between groups in terms of demographic and surgical data [table 1]. Time to highest sensory level and maximum number of segments blocked also regression of sensory and motor blocks showed no difference between the groups . Demographic and surgical data vomiting was increased with higher dose of meperidine and was most common in group iv . Finally, the usage of methoclopramide as antiemetic drug was more frequently in experimental groups . The systolic bp was similar between groups for each time interval, as was patient temperature . We lost one patient in the control group and two patients in the experimental groups because of failure of spinal block . There was no need for rescue dose of fentanyl in the case and experimental groups . First need forpostoperative analgesia in groups ii, iii, iv compared to standard group was (median time to need analgesic drug):130,140,140 versus 80 min,(p<0.05). Need for analgesic drugs was more in the standard group (17.6%)(p = 0.000) but was not significantly different between experimental groups (4.6%, 4.8% and 4.3%, respectively (p = 0.641). The incidence and intensity of shivering between the groups of study decreased as the dose of meperidine increased (incidence: 47.5%, 37.5%, 27.5% and 15.0%, respectively) (p=0.002) [figure 1]. The incidence of pruritis also increased to 25.64, 28.21, 38.46, and 48.72 respectively) (p=0.000). According to the obtained results, adding meperidine, in increasing small dosages, to the intrathecal mixture anesthesia for cesarean delivery reduces the incidence and intensity of shivering but increases undesirable side - effects that bother the patients . Although a lot of iv drugs were used for the treatment of postoperative shivering iv meperidine is the gold standard . The main consequences of postoperative shivering are an increase of oxygen consumption and of co2 production . Otherwise, it also produces an increase in intracranial pressure, interferes with electrocardiographic monitoring, and causes general discomfort, including a sensation of feeling cold . Postoperative shivering produces physiological changes and constitutes a significant risk during the early postoperative period in high - risk patients . The prevention of shivering is more important than its treatment and has not been well - investigated . When we compare treatment of shivering with intrathecal route and iv route we might find two advantages in the former: first, it is a preventive treatment, therefore, patients are spared the negative experience associated with shivering . Second, the administration of iv drug is associated with unwanted side - effects which can be harmful, especially in obstetrics women . Although the etiology of post - spinal shivering is inadequately understood, various risk factors have been evaluated . Among these, hypothermia, stress, uncontrolled pain, uninhibited spinal reflexes, and decreased sympathetic activity are frequently mentioned . Meperidine, which binds to both mu and k - opioid receptors, is frequently recommended for the treatment of postoperative shivering and the anti - shivering action of meperidine has previously been attributed to its action on -opioid receptors. [1115] patel et al . Compared intrathecal meperidine and lidocaine in 42 asa physical status ii or iii patients who were candidates for endoscopic urological procedures and found that mean arterial blood pressure decreased significantly in the lidocaine group but not in the meperidine group . Motor block was absent in ten patients in the meperidine group but was present in all the patients in the lidocaine group . Complications such as nausea, vomiting, itching, drowsiness and respiratory depression were similar in the two groups . It is concluded that low - dose meperidine, 0.5 mg kg1 is effective as a spinal anesthetic agent and has few complications . Some of the findings of this study are against those of our study . In ours there is no bp change between groups and the incidence of side - effects is related to the dose of meperidine and is least in the control group . Studied the effect of a low dose of intrathecal meperidine on shivering in 40 parturient women scheduled for no emergent cesarean delivery . Spinal anesthesia consisted of hyperbaric bupivacaine (0.75%; 10.5 mg), morphine 0.15 mg, and, in the experimental group, meperidine (0.2 mg/ kg) or, in the control group, normal saline . Time to highest sensory level, maximum number of blocked segments, sensory and motor blockade regression, and systolic blood pressure showed no difference between groups . The incidence of shivering was less in the meperidine group, as was its intensity . They found no difference between doses of diphenhydramine, and metoclopramide administered to the patient . Finally, they recommended use of intrathecal meperidine to decrease the incidence of shivering without considerable side - effects in the obstetrical population . Intrathecal meperidine (0.2 mg / kg) was effective in reducing the incidence and intensity of shivering associated with spinal anesthesia for cesarean delivery . Some findings of this study confirm our findings but we found a higher incidence of post operative nausea and vomiting and pruritis in the meperdine group that was dose - dependent . Although, meperidine readily cross the placenta and block performed just a few minutes before delivery of fetus but none of newborns was depressed at the time of delivery because a highly lipid - soluble drug like meperidine is readily absorbed by lipid tissues and reabsorption into capillaries of the spinal cord is very slow . In one study the effect of intrathecal meperidine on the incidence of shivering after spinal anesthesia was studied . Sixty patients with asa class i - ii were divided into intrathecal meperidine group (case group) and control group (group ii). Case group received spinal tetracaine with meperidine 0.2 mg / kg and control group received spinal tetracaine without meperidine . There was a significant reduction in the incidence of shivering in the case group (16.7%) when compared with the control group (56.7%). In one prospective randomized double - blinded study 72 parturient women, scheduled for elective caesarean section under spinal anesthesia, were enrolled in three different groups . Spinal anesthesia consisted of bupivacaine 0.5% (10 mg) for the control group, and the same dose of bupivacaine with meperidine 12.5 or 25 mg for the experimental groups . They found the highest incidence of shivering in the control group (16.7%) in comparison with the experimental groups (0/24). They concluded that intrathecal meperidine cannot be recommended for the prevention of shivering during spinal anesthesia for caesarean section as its use is associated with increased incidence of nausea and vomiting .. some result of this study confirm our findings . In our study and we found a higher incidence of pruritis that was also dose - dependent in another study the effect of adding meperidine 0.5% hyperbaric bupivacaine was investigated in 50 patient candidates for elective transurethral resection operations under spinal anesthesia . Expect vomiting the single serious adverse effect of intrathecal opioid is respiratory - depressant but reported with much larger doses (50 mg) of intrathecal meperidine . There has been no report of delayed respiratory depression with intrathecal meperidine, also some investigations confirm early respiratory depression in higher dosages than we used . We found no respiratory depression and need for mechanical ventilation in any of the groups . First: in our hospital it was impossible to determine meperidine level of cerebrospinal fluid . Second: it was impossible to monitor the temperature of csf and compare it with the tympanic temperature obtained from the right ear and intensity of shivering . It could help us to know more about the relation between the temperature of the spinal cord, core temperature, intensity, incidence of shivering and add to our knowledge about the etiology of shivering . According to this study, shivering continues to be a common problem after spinal anesthesia for cesarean delivery, with unknown etiology and no definite treatment . Although increasing the dose of intrathecal meperidine can decrease the incidence and intensity of shivering it can also increase the incidence of nausea and vomiting that can be a major problem in these patients . Finally, the use of intrathecal meperidine cannot be recommended for the prevention of shivering during spinal anesthesia for caesarean section as its use is associated with increased incidence of nausea and vomiting and we need to treat shivering with safer drugs.
Fear of hypoglycaemia and gain in body weight act as barriers for initiation of insulin therapy . Modern insulin analogues are a convenient new approach or tool to glycaemic control, associated with low number of hypos and favourable weight change . A1chieve, a multinational, 24-week, non - interventional study, assessed the safety and effectiveness of insulin analogues in people with t2 dm (n = 66,726) in routine clinical care . Please refer to editorial titled: the a1chieve study: mapping the ibn battuta trail . The patient characteristics for the entire cohort divided as insulin - nave and insulin users is shown in the table 1 . The majority of patients (71.28%) started on or switched to biphasic insulin aspart . Other groups were insulin detemir (n = 28), insulin aspart (n = 24), basal insulin plus insulin aspart (n = 13) and other insulin combinations (n = 3). Overall demographic data after 24 weeks of treatment, overall hypoglycaemic events reduced from 2.1 events / patient - year to 0.0 events / patient - year in insulin nave group and from 6.8 events / patient - year to 0.0 events / patient - year in insulin users group . The hypoglycaemia incidence in insulin naive group at 24 weeks was lower than that observed in insulin users at baseline . Major hypoglycaemic events decreased from 0.5 events / patient - year to 0.0 events / patient - year in insulin nave group while no change from baseline (1.3 events / patients - year) was observed for insulin user group . Mean hba1c and fpg values improved from baseline to study end in the insulin nave group [table 4]. Overall efficacy data of the total cohort, 340 patients started on biphasic insulin aspart ogld, of which 153 (45%) were insulin nave and 187 (55%) were insulin users . After 24 weeks of starting or switching to biphasic insulin aspart, hypoglycaemic events reduced from 2.3 events / patient - year to 0.0 events / patient - year in insulin nave group and from 6.3 events / patient - year to 0.0 events / patient - year in insulin users . Quality of life improved at the end of the study [table 5 and 6]. Biphasic insulin aspartoral glucose - lowering drug safety data mean hba1c and fpg values improved from baseline to study end in those who started on or were switched to biphasic insulin aspart for insulin nave group [table 7]. Biphasic insulin aspartoral glucose - lowering drug efficacy data of the total cohort, 7 patients started on basal + insulin aspart ogld, of which 2 (28.6%) were insulin nave and 5 (71.4%) were insulin users . After 24 weeks of treatment, hypoglycaemic events reduced from 10.4 events / patient - year to 0.0 events / patient - year in insulin users whereas hypoglycaemia remained nil in insulin naive group, similar to baseline . Quality of life improved after 24 weeks of treatment [table 8 and 9]. Basal+insulin aspartoral glucose - lowering drug safety data mean hba1c and fpg values improved from baseline to study end in those who started on or were switched to basal + insulin aspart oglds for insulin nave group . Of the total cohort, 90 patients started on insulin detemir ogld, of which 57 (63.3%) were insulin nave and 33 (36.7%) were insulin users . After 24 weeks of starting or switching to insulin detemir, hypoglycaemic events reduced from 2.5 events / patient - year to 0.0 events / patient - year in insulin nave and from 6.3 events / patient - year to 0.0 events / patient - year in insulin user groups [table 10 and 11]. Insulin detemiroral glucose - lowering drug safety data mean hba1c and fpg values improved from baseline to study end in those who started on or were switched to insulin detemir oglds for insulin - nave group [table 12]. Insulin detemiroral glucose - lowering drug efficacy data of the total cohort, 37 patients started on insulin aspart ogld, of which 26 (70.3%) were insulin nave and 11 (29.7%) were insulin users . After 24 weeks of treatment, hypoglycaemic events reduced from 14.2 events / patient - year to 0.0 events / patient - year in insulin users whereas, hypoglycaemic events remained nil in insulin users, similar to baseline . Quality of life improved at the end of the study [table 13 and 14]. Insulin aspartoral glucose - lowering drug safety data mean hba1c and fpg values improved from baseline to study end in those who started on or were switched to insulin aspart oglds for insulin nave group [table 15]. Of the total cohort, 340 patients started on biphasic insulin aspart ogld, of which 153 (45%) were insulin nave and 187 (55%) were insulin users . After 24 weeks of starting or switching to biphasic insulin aspart, hypoglycaemic events reduced from 2.3 events / patient - year to 0.0 events / patient - year in insulin nave group and from 6.3 events / patient - year to 0.0 events / patient - year in insulin users . Quality of life improved at the end of the study [table 5 and 6]. Biphasic insulin aspartoral glucose - lowering drug safety data mean hba1c and fpg values improved from baseline to study end in those who started on or were switched to biphasic insulin aspart for insulin nave group [table 7]. Of the total cohort, 7 patients started on basal + insulin aspart ogld, of which 2 (28.6%) were insulin nave and 5 (71.4%) were insulin users . After 24 weeks of treatment, hypoglycaemic events reduced from 10.4 events / patient - year to 0.0 events / patient - year in insulin users whereas hypoglycaemia remained nil in insulin naive group, similar to baseline . Quality of life improved after 24 weeks of treatment [table 8 and 9]. Basal+insulin aspartoral glucose - lowering drug safety data mean hba1c and fpg values improved from baseline to study end in those who started on or were switched to basal + insulin aspart oglds for insulin nave group . Of the total cohort, 90 patients started on insulin detemir ogld, of which 57 (63.3%) were insulin nave and 33 (36.7%) were insulin users . After 24 weeks of starting or switching to insulin detemir, hypoglycaemic events reduced from 2.5 events / patient - year to 0.0 events / patient - year in insulin nave and from 6.3 events / patient - year to 0.0 events / patient - year in insulin user groups [table 10 and 11]. Insulin detemiroral glucose - lowering drug safety data mean hba1c and fpg values improved from baseline to study end in those who started on or were switched to insulin detemir oglds for insulin - nave group [table 12]. Of the total cohort, 37 patients started on insulin aspart ogld, of which 26 (70.3%) were insulin nave and 11 (29.7%) were insulin users . After 24 weeks of treatment, hypoglycaemic events reduced from 14.2 events / patient - year to 0.0 events / patient - year in insulin users whereas, hypoglycaemic events remained nil in insulin users, similar to baseline . Quality of life improved at the end of the study [table 13 and 14]. Insulin aspartoral glucose - lowering drug safety data mean hba1c and fpg values improved from baseline to study end in those who started on or were switched to insulin aspart oglds for insulin nave group [table 15]. Our study reports improved glycaemic control (hba1c, fpg) following 24 weeks of treatment with any of the insulin analogues (biphasic insulin aspart; basal + insulin aspart; insulin detemir; insulin aspart) with or without ogld . Major hypoglycaemic events decreased from 0.5 events / patient - year to 0.0 events / patient - year in insulin nave group while no change from baseline (1.3 events / patients - year) was observed for insulin user group . Overall, body weight reduced in insulin nave group whereas no change in body weight was observed for insulin users . Though the findings are limited by number of patients, still the trend indicates that insulin analogues can be considered effective and possess a safe profile for treating type 2 diabetes in rajasthan, india.
A 64-year - old woman with no previous medical history sustained a motor vehicle accident (mva) on january 23, 2012 (she crashed into a truck from behind). When examined in the field, the patient noted numbness and weakness in her arms and numbness in her lower limbs . She was put in a philadelphia collar and transferred to the nearest hospital, where head and spine images were obtained . The cervical spine x - rays and magnetic resonance imaging (mri) showed a spared posterior longitudinal ligament with ruptured posterior elements (interspinous and supraspinous ligaments; fig . Based on her imaging findings, she was advised to undergo surgery within 48 hours . The patient refused surgical treatment and was discharged home 4 days after the accident on the philadelphia collar . During the weeks following the mva, she consulted other neurosurgeons, who also recommended surgery . She was referred to our department 38 days after the accident . At that time, she was neurologically intact (frankel grade e); however, she experienced neck pain, which was moderately controlled with nonsteroidal anti - inflammatory drugs . After carefully explaining her condition to her, she finally decided to proceed with surgery a few weeks later and was operated on march 22 (59 days after the mva). Considering the facet remodeling found in these kind of cases1 and the fact that failure of closed reduction is more common with facet dislocation injuries,2 we decided not to attempt traction before the procedure . (a) initial plain x - ray showing a c56 subluxation with bilateral locked facets . (b) preoperative sagittal computed tomography (ct) scan with bone windows; degeneration at the c45 level is clearly seen . (c) parasagittal ct scan with bone windows; although the c56 facet joint appears to be separated, they looked fused intraoperatively . (d) preoperative sagittal t2-weighted magnetic resonance imaging showing an intact posterior longitudinal ligament without disk extrusion . Because the posterior longitudinal ligament was intact and the posterior ligaments were disrupted (fig . 1d), we performed a posterior approach under neurophysiologic monitoring of motor and somatosensory evoked potentials (meps and sseps). The patient was placed in a mayfield head holder with her neck slightly extended . After opening and exposing the posterior elements, the locked c5c6 facets appeared ossified, and no joint space could be identified (fig . 3a). Using a high - speed drill and with the aid of the c - arm, we performed a wide bilateral foraminotomy to refracture the partially ossified facets and release both c6 roots . After this, we placed four lateral mass screws (two on c5 and two on c6; vertex system; medtronic sofamor - danek, memphis, tennessee, united states). Initially, we thought the c5 body could be easily brought back with a towel clamp, as described by lim et al.3 however, the c5 vertebral body stayed in that position and could not be reduced by this maneuver . Because the c45 space was already degenerated (figs . 1b and d), we decided to extend the instrumentation to c4 (fig . 3b), and we secured a rod from c4 to c6, spanning the c5 lateral mass screw . Resembling the technique utilized in the reduction of high - grade lumbar spondylolisthesis with reduction screws,4 we used a rod reducer to bring the c5 screw head back toward the rod (fig . 3c), thus realigning the lateral mass screw heads and reducing the subluxation (fig . After both rods were tightened with six lock head screws, bone was harvested from the posterosuperior iliac crest to achieve posterolateral fusion . (c, d) postoperative t2-weighted magnetic resonance imaging taken 3 months after the operation, showing complete reduction . (a) artist's drawing depicting the c56 bilateral facet subluxation . Note the almost fused facet joint, which is usually found in old cases . The rod reducer (vertex system; medtronic sofamor - danek, memphis, tennessee, united states) allowed a gentle and controlled reduction, one side at a time . (d) final construct showing complete c56 reduction . Following an uneventful 3-hour procedure, the patient was transferred to the postanesthetic care unit and discharged 3 days later . On her immediate postoperative exam, a mild (4/5) right c6 paresis was noted, which resolved completely within 48 hours . On postoperative follow - up, she remained neurologically intact and pain free (figs . A 64-year - old woman with no previous medical history sustained a motor vehicle accident (mva) on january 23, 2012 (she crashed into a truck from behind). When examined in the field, the patient noted numbness and weakness in her arms and numbness in her lower limbs . She was put in a philadelphia collar and transferred to the nearest hospital, where head and spine images were obtained . The cervical spine x - rays and magnetic resonance imaging (mri) showed a spared posterior longitudinal ligament with ruptured posterior elements (interspinous and supraspinous ligaments; fig . Based on her imaging findings, she was advised to undergo surgery within 48 hours . The patient refused surgical treatment and was discharged home 4 days after the accident on the philadelphia collar . During the weeks following the mva, she consulted other neurosurgeons, who also recommended surgery . She was referred to our department 38 days after the accident . At that time, she was neurologically intact (frankel grade e); however, she experienced neck pain, which was moderately controlled with nonsteroidal anti - inflammatory drugs . After carefully explaining her condition to her, she finally decided to proceed with surgery a few weeks later and was operated on march 22 (59 days after the mva). Considering the facet remodeling found in these kind of cases1 and the fact that failure of closed reduction is more common with facet dislocation injuries,2 we decided not to attempt traction before the procedure . (a) initial plain x - ray showing a c56 subluxation with bilateral locked facets . (b) preoperative sagittal computed tomography (ct) scan with bone windows; degeneration at the c45 level is clearly seen . (c) parasagittal ct scan with bone windows; although the c56 facet joint appears to be separated, they looked fused intraoperatively . (d) preoperative sagittal t2-weighted magnetic resonance imaging showing an intact posterior longitudinal ligament without disk extrusion . Because the posterior longitudinal ligament was intact and the posterior ligaments were disrupted (fig . 1d), we performed a posterior approach under neurophysiologic monitoring of motor and somatosensory evoked potentials (meps and sseps). The patient was placed in a mayfield head holder with her neck slightly extended . After opening and exposing the posterior elements, the locked c5c6 facets appeared ossified, and no joint space could be identified (fig . 3a). Using a high - speed drill and with the aid of the c - arm, we performed a wide bilateral foraminotomy to refracture the partially ossified facets and release both c6 roots . After this, we placed four lateral mass screws (two on c5 and two on c6; vertex system; medtronic sofamor - danek, memphis, tennessee, united states). Initially, we thought the c5 body could be easily brought back with a towel clamp, as described by lim et al.3 however, the c5 vertebral body stayed in that position and could not be reduced by this maneuver . Because the c45 space was already degenerated (figs . 1b and d), we decided to extend the instrumentation to c4 (fig . 3b), and we secured a rod from c4 to c6, spanning the c5 lateral mass screw . Resembling the technique utilized in the reduction of high - grade lumbar spondylolisthesis with reduction screws,4 we used a rod reducer to bring the c5 screw head back toward the rod (fig . 3c), thus realigning the lateral mass screw heads and reducing the subluxation (fig . After both rods were tightened with six lock head screws, bone was harvested from the posterosuperior iliac crest to achieve posterolateral fusion . (c, d) postoperative t2-weighted magnetic resonance imaging taken 3 months after the operation, showing complete reduction . The rod reducer (vertex system; medtronic sofamor - danek, memphis, tennessee, united states) allowed a gentle and controlled reduction, one side at a time . Following an uneventful 3-hour procedure, the patient was transferred to the postanesthetic care unit and discharged 3 days later . On her immediate postoperative exam, a mild (4/5) right c6 paresis was noted, which resolved completely within 48 hours . On postoperative follow - up, she remained neurologically intact and pain free (figs . About 200,000 people currently live with spinal cord injuries in the united states, with 15 to 40 new cases per million people estimated to occur annually.5 6 more than 50% of these involve the cervical spine, and half of these present with unilateral or bilateral subluxations . The vast majority of these cases are treated within a week, either by traction, open reduction, or both . When the interval between the accident and diagnosis is longer than 3 weeks, the injury is considered old.7 8 some authors consider injuries old when the interval is longer than 8 weeks.1 there are few reports on the management of old dislocations of the subaxial cervical spine in the literature,1 7 9 10 11 but these injuries still occur . Cervical traumatic subluxations and usually occur in older populations.1 10 this could explain the relatively mild neurologic status of these patients (frankel c or more) because, in this age group, the facet dislocation may be produced by less severe forces, which are less likely to damage the cord.12 in one of the biggest series published to date, hassan reported the surgical treatment of 12 patients with an old dislocation of the lower cervical spine.10 the time from injury to presentation averaged 3.5 (range 1.5 to 12) months, and the majority of the dislocations were between c4 and c5 . The author's treatment protocol started with 1 week of skull traction followed by anterior fusion with plate fixation . If reduction was not achieved, the traction was continued for another week and then followed by anterior diskectomy and fusion with plate fixation . In cases in which reduction by traction did not succeed, a posterior partial facetectomy was performed, followed by a posterior fusion with plate fixation . The average follow - up was 34 (range 12 to 54) months, and all patients developed bone fusion and showed neurologic improvement . Jain et al described the open reduction of four patients in whom the mean delay between injury and presentation was 4 months (range 1 to 5).7 the mean age of the patients was 48.2 years old (range 27 to 60), and three of them had a myelopathy . They performed a two - stage procedure under the same anesthetic . On the first stage, they performed a posterior soft tissue release and partial facetectomy to allow partial reduction of the dislocation, supplemented by interspinous wiring and corticocancellous graft . During the second, anterior stage, they did a diskectomy, tricortical bone grafting, and anterior cervical plating . All the patients achieved a nearly anatomical reduction with a mean follow - up of 2.6 years (range 1 to 4). The myelopathy settled completely in three patients, and there was no graft dislodgement or graft - related problems . In the authors' experience, the posteroanterior procedure for neglected traumatic bilateral dislocation of the subaxial cervical spine is a good method of achieving sagittal alignment . Bartels and donk reported the surgical treatment of three cases.1 they treated the first two cases through a three - stage approach (anterior - posterior - anterior). Because these cases failed and a fourth stage had to be added to correct misalignment, the third case was done through a posterior - anterior - posterior approach . As in our case, the authors stated that facet joints were covered with fibrous tissue and the joint space was not seen . Based on their experience, they suggest that in cases of nonacute bilateral cervical facet dislocations, the operating sequence should be posterior - anterior - posterior . It should be noted that the cohort's mean age was 71, suggesting again that patients who present subacutely tend to be older . For this reason, an open single - stage posterior approach under neurophysiologic monitoring dramatically reduces operating time, which is important to reduce morbidity in these cases . Note that this technique can be added to the decision - making armamentarium only for cases without disk herniation . When signs of frank disk herniation are found on the preoperative mri, anterior diskectomy is mandatory to avoid neurologic deterioration during reduction, as recommended by eismont et al.13 although this technique could be easily criticized due to the fact that a rostral level needs to be included in the fusion, we believe this does not add morbidity and has been done for years in lumbar spondylolisthesis.4 moreover, because this entity is more frequent in older patients, the chances of finding an already degenerated level rostral to the subluxation are high . Whereas a 2-month subluxation is regarded as old by most authors, it is uncertain whether this technique would work on most chronic dislocations . Perhaps dislocations older than 3 months could not be easily reduced through this technique, as a result of ossification of the uncovertebral joints . Finally, it is critical to note that this technique is somewhat different from its lumbar counterpart . Given the fact that lumbar pedicle screws achieve a much better purchase than cervical lateral mass screws, there is a potential risk for screw pullout during the reduction technique.
Patients with heart failure (hf) older than 65 years have a two - fold increased risk of cognitive impairment than elders without hf (1). The prevalence of cognitive impairment in adults with chronic heart failure is recognized as a factor contributing to the complexity of care for these patients (2). Cognitive impairment may impact the ability to perform heart failure self - care procedures and is associated with an increased risk of re - hospitalization and mortality (3). Despite the prevalence of these two conditions, cognitive impairment in hf patients is usually underestimated by physicians (4) and currently, there is insufficient evidence to develop recommendations for strategies to improve cognitive impairment for hf patients (5). This study was designed to determine factors related to cognitive impairment among elder with hf . In this descriptive, correlational, cross - sectional study, 184 patients with chronic heart failure were selected from four mazandaran university of medical sciences teaching hospitals using convenience sampling: imam khomeini hospital in behshahr, fatemeh zahra heart center in sari, imam khomeini hospital in fereydunkenar and imam khomeini hospital in noor . Patients hospitalized for symptomatic heart failure between october 2013 and january 2014 were included in this study and confirmed by the cardiologists . Inclusion criteria were a history of at least six months involvement with heart failure, age 60, and remaining stable 1 - 2 days after admission . Exclusion criteria were communication problems such as severe hearing impairment (without hearing aids), speech problems, severe cognitive impairment with abbreviated mental test (amt) scores <4 (6) and uncooperativeness . After providing written informed consent, each patient was interviewed by an independent data collector who was not involved in the patient s care . Socio - demographic variables consisted of age, gender, location, living status, education level, and income . Clinical variables consisted of left ventricular ejection fraction (ef), poly - pharmacy (5 different drugs), comorbidities (charlson comorbidity index), blood pressure, depressive symptoms, body mass index (bmi), number of hospitalizations during the previous six months, and some biochemical characteristics of the blood . These variables were collected from patients medical records and by interviews . Cognitive status was measured using the iranian version of the abbreviated mental test . The ideal cut - off point reported 6, while sensitivity and specificity identified at 88 and 99%, respectively (6), using a 10-item scale . Each correct answer received a score of 1 and incorrect answers were scored as 0 . A total score of 6 indicates the presence of cognitive impairment (a score of 0 - 3 indicates severe cognitive impairment and 4 - 6 indicates moderate cognitive impairment). The severity of comorbid conditions was assessed using the charlson comorbidity index (7), which classifies comorbidities based on the number and seriousness of one - year survival, with higher scores indicating greater risk of death . Most diseases are assigned a score of 1 on the index, but more severe conditions are given a weight score of 2, 3 or 6 . Fisher s exact test and logistic regression were used to compare cognitive status scores with demographic characteristics and disease related variables . The independent samples t - test was used to compare biochemical characteristics of the blood from the subjects both with and without cognitive impairment . Socio - demographic variables consisted of age, gender, location, living status, education level, and income . Clinical variables consisted of left ventricular ejection fraction (ef), poly - pharmacy (5 different drugs), comorbidities (charlson comorbidity index), blood pressure, depressive symptoms, body mass index (bmi), number of hospitalizations during the previous six months, and some biochemical characteristics of the blood . These variables were collected from patients medical records and by interviews . Cognitive status was measured using the iranian version of the abbreviated mental test . The ideal cut - off point reported 6, while sensitivity and specificity identified at 88 and 99%, respectively (6), using a 10-item scale . Each correct answer received a score of 1 and incorrect answers were scored as 0 . A total score of 6 indicates the presence of cognitive impairment (a score of 0 - 3 indicates severe cognitive impairment and 4 - 6 indicates moderate cognitive impairment). The severity of comorbid conditions was assessed using the charlson comorbidity index (7), which classifies comorbidities based on the number and seriousness of one - year survival, with higher scores indicating greater risk of death . Most diseases are assigned a score of 1 on the index, but more severe conditions are given a weight score of 2, 3 or 6 . Test and logistic regression were used to compare cognitive status scores with demographic characteristics and disease related variables . The independent samples t - test was used to compare biochemical characteristics of the blood from the subjects both with and without cognitive impairment . The amt mean was 6.18 2.002 (minimum 4 and maximum 10), with 95% ci (5.89 to 6.48). The majority of the elders in the study (110 persons, 59.8%) had scores of 4 - 6 and the others (74 persons, 40.2%) had scores of 7 - 10 . Females made up 61.4% of participants (113 persons) and 38.6% (71 persons) were male . The 60 - 75 age group comprised 70% of the subjects (128 persons), 29% (54 persons) were in the 75 - 90 age group and 1% (2 persons) were in the 90 - 94 age group . The mean ages in women and men were 70.7 8.35 and 70.01 8.99, respectively (p = 0.595). The ci odds ratio was higher in older patients than in the younger ones (p = 0.002). In addition, the ci odds ratio was higher in female subjects than male (p <0.001) (table 1). There were significant relationships between cognition status scores and living status (p <0.001) and also between cognition status scores and education level (p <0.001) (table 2). Values are expressed as mean sd . The ci odds ratios were higher in subjects with an ejection fraction of <40% compared to subjects with an ejection fraction of 40% (p = 0.002) (table 1). Fisher s exact test results showed a significant relationship between cognitive status scores and hypertension (p = 0.039) and also between cognitive status scores and anemia (p = 0.046). Although the ci odds ratio was higher among subjects with hypertension and anemia, there was not a significant relationship (table 1). There was no significant correlation between biochemical characteristics of the blood and cognitive status scores . However, there was a near - significant level for hemoglobin, blood urea nitrogen (bun) and systolic blood pressure (table 3). Values are expressed as mean sd . The mean of charlson comorbidity index and depression symptoms were higher among subjects with cognition impairment (p <0.001) (table 4). The amt mean was 6.18 2.002 (minimum 4 and maximum 10), with 95% ci (5.89 to 6.48). The majority of the elders in the study (110 persons, 59.8%) had scores of 4 - 6 and the others (74 persons, 40.2%) had scores of 7 - 10 . Females made up 61.4% of participants (113 persons) and 38.6% (71 persons) were male . The 60 - 75 age group comprised 70% of the subjects (128 persons), 29% (54 persons) were in the 75 - 90 age group and 1% (2 persons) were in the 90 - 94 age group . The mean ages in women and men were 70.7 8.35 and 70.01 8.99, respectively (p = 0.595). The ci odds ratio was higher in older patients than in the younger ones (p = 0.002). In addition, the ci odds ratio was higher in female subjects than male (p <0.001) (table 1). There were significant relationships between cognition status scores and living status (p <0.001) and also between cognition status scores and education level (p <0.001) (table 2). The ci odds ratios were higher in subjects with an ejection fraction of <40% compared to subjects with an ejection fraction of 40% (p = 0.002) (table 1). Fisher s exact test results showed a significant relationship between cognitive status scores and hypertension (p = 0.039) and also between cognitive status scores and anemia (p = 0.046). Although the ci odds ratio was higher among subjects with hypertension and anemia, there was not a significant relationship (table 1). There was no significant correlation between biochemical characteristics of the blood and cognitive status scores . However, there was a near - significant level for hemoglobin, blood urea nitrogen (bun) and systolic blood pressure (table 3). Values are expressed as mean sd . The mean of charlson comorbidity index and depression symptoms were higher among subjects with cognition impairment (p <0.001) (table 4). In this study, the prevalence of cognitive impairment was higher among elderly subjects with a lower level of education, patients living alone or with their children, and females . It seems that fewer years of education contribute to processing speed (2). Providing social support another important factor that may decrease patient stress and improve cognitive status . The results of the present study show that comorbidities contribute to cognitive impairment in hf patients . These findings are consistent with other studies (2, 9, 10), but differ from that of pressler et al . Who found no association between comorbidities and cognitive status (11). In this study, there were significant relationships between cognitive impairment with depression, hypertension, and anemia . These findings are consistent with some studies (9, 10). In a clinical setting, it is hard to differentiate mild stages of dementia and late life depression, because both situations may have similar presentations (12). Few studies have directly examined the association between cognitive impairment and depression in persons with heart failure, despite the likelihood of common mechanisms (13). Persons with heart failure exhibit numerous pathological changes on neuro - imaging, including greater atrophy and the presence of white matter hyper - intensities, frequently in frontal brain regions (14). Hypertension has a significant impact on cardiovascular function, cerebral structural integrity and associated cognitive deterioration (15, 16). The most common explanation for the deleterious effect of hypertension on cognition is that hypertension increases cerebrovascular disease (17). Some longitudinal studies have shown a positive association between hypertension and cognitive impairment (15, 16). These effects were independent of clinical strokes and the association was stronger in individuals who did not use antihypertensive drugs (15). The results of some studies indicate that hypertension may be a risk factor dementia, especially alzheimer s disease (ad) (18, 19). However, the factors that predispose individuals with hypertension to developed ad are unknown (20). There have been very few studies on the association of anemia with cognitive status (22). The literature on the association between anemia and dementia is also limited and the results are inconsistent (23, 24). Anemia in heart failure may have multiple origins, which are thought to involve reduced erythrocyte production, decreased bmi, and hemodilution (25). Further contributors to the risk of anemia in hf are comorbid renal disease and increased inflammation (26). Elevated levels of pro - inflammatory cytokines may also inhibit hematopoietic proliferation (27). In addition, some medications, such as angiotensin - converting enzyme (ace) inhibitors and angiotensin receptor blockers, reduce erythropoietin production (25). First, this is a cross - sectional study and the correlations cannot imply causation relationships between parameters . Moreover, the data represent only the subjects who agreed to participate in the study . Identifying factors affecting cognitive impairment in hf may help clinicians in directing educational interventions on disease management to families to possibly prevent readmission of their loved ones . Further research to determine the feasibility and acceptability of cognitive assessment in routine clinical care is suggested . First, this is a cross - sectional study and the correlations cannot imply causation relationships between parameters . Moreover, the data represent only the subjects who agreed to participate in the study . Identifying factors affecting cognitive impairment in hf may help clinicians in directing educational interventions on disease management to families to possibly prevent readmission of their loved ones . Further research to determine the feasibility and acceptability of cognitive assessment in routine clinical care is suggested.
Autoimmune hepatitis (aih) is a chronic progressive hepatitis characterized by positive antinuclear antibody (ana) and hypergammaglobulinemia, portal inflammatory cell infiltration with plasmacyte dominance, and piecemeal necrosis 1 . Acuteonset cases of aih account for up to 25% of all cases, some of which present as fulminant liver failure 2 . The diagnosis of aih is established by the criteria defined by the international autoimmune hepatitis group and by the exclusion of other causes 3 . However, acuteonset aih is not typical, and this diagnosis is difficult 4, 5, 6 . There are few reports of complications after recovery from fulminant liver failure secondary to aih . On the other hand, risk factors of actinomycosis are poor oral hygiene resulting in tooth decay, chronic lung disease, alcoholism, and other disorders resulting in reduced immunocompetence 7 . The present report describes an interesting case of an osteolytic lesion (actinomycosis) after recovery from fulminant liver failure induced by aih . The patient was a 77yearold woman whose chief complaints were jaundice, general fatigue, and brown urine . The patient was seeing a local doctor for chronic gastritis and was given lansoprazole 15 mg once a day, mosapride citrate hydrate 5 mg three times a day, etizolam 0.5 mg once a day, and an unknown herbal medicine, without signs of hepatic injury . On 15 may 2009, she had general fatigue, but no laboratory dysfunction was shown . On 23 june 23 2009, she had jaundice and brown urine, and laboratory data showed liver dysfunction . While she had eaten raw foods within the previous month, she had not eaten raw oysters . On admission, her consciousness was clear, and although yellowing of the bulbar conjunctiva was noted, no other abnormalities were identified . Laboratory findings on admission (table 1) included aspartate aminotransferase (ast), 1735 iu / l; alanine aminotransferase (alt), 1707 iu / l; total bilirubin (tb), 7.13 mg / dl; prothrombin time rate (pt%, international normalized ratio [inr]), 45%, 1.5; hepatitis b surface antigen (hbsag) negative; hepatitis c virus (hcv) rna negative; antiliver / kidney microsome type 1 antibody(lkm1) negative, and other virus antibodies negative . Furthermore, as levels of ana (320) and immunoglobulin g (igg) (1793 mg / dl) were elevated, aih was suspected . Although ast and alt levels began to improve after the injection of glycyrrhizin, tb and pt% deteriorated to 17 mg / dl and 35%, respectively . While we wanted to perform liver biopsy for the diagnosis of aih, it was deemed too dangerous due to severe liver failure, especially with regard to the abnormal pt% . However, the patient developed fulminant liver failure with stage 2 hepatic encephalopathy on the eighth clinical day, and plasma exchange was performed for 3 days . Levels of ast, alt, tb, and the symptomatology of hepatic encephalopathy improved, but freshfrozen plasma (ffp) was transfused every day from 12th clinical day due to a lack of response in pt% . On the 19th clinical day, general fatigue and stage 2 hepatic encephalopathy developed, and plasma exchange was again performed for 3 days from the 24th clinical day . The ast and alt levels subsequently normalized, and the prednisolone dose was decreased to 20 mg / day on the 28th clinical day . The dose was decreased to 15 mg / day 1 week later, as ast and alt remained normal . On the 60th clinical day, she was discharged, and the prednisolone dose was decreased to 7.5 mg / day . At the outpatient examination on the 78th clinical day, abdominal ultrasound and helical dynamic ct scan were performed, showing a 4cm osteolytic tumor of the 10th right rib (fig . The suspicion of metastasis was raised, but gastrointestinal endoscopy and colon endoscopy did not reveal evidence of any malignant tumor . (b, c) ultrasound finding of tumor in the 10th right rib is heterogeneous . (d) doppler ultrasound finding of tumor in the 10th right rib is hypovascular . (e h) abdominal unenhanced and enhanced computed tomography (ct) scan findings at 78 days after first admission . (e) plane, (f) arterial phase, (g) equilibrium phase, and (h) venous phase . An increase in serum cancer antigen 125 (ca125) level was noted, but gynecologic evaluation failed to detect any malignant tumor . On the 125th clinical day, the patient presented with severe pain of the right hip joint and was readmitted with suspicion of the osteonecrosis of the femoral head . However, she was subsequently diagnosed with suppurative inflammation of the hip joint due to methicillinsensitive staphylococcus aureus (mssa) and was treated by an orthopedist . Her hip joint pain improved by the 146th clinical day, and a biopsy of the liver and the 10th right rib tumor was performed . Examination of the liver specimen showed that the portal tract was infiltrated with inflammatory cells (including plasma cells), the parenchymal architecture had been partially disrupted, and surviving hepatocytes had formed glandlike rosettes . Examination of the 10th right rib specimen showed infiltration of lymphocytes and plasma cells, fibrosis, and the druse (arrow) with abscess formation (fig . Liver biopsy finding: the portal tract is infiltrated with inflammatory cells, including plasma cells . The parenchymal architecture is partially disrupted . Surviving hepatocytes form glandlike rosettes (arrow). Right rib tumor biopsy finding: infiltration of lymphocytes and plasma cells and fibrosis are seen . Druse (arrow) with abscess formation is seen . In conclusion, during recovery from fulminant liver failure induced by acute onset aih, the patient was diagnosed with actinomycosis of the 10th right rib . This patient had been treated with amoxicillin . However, due to the development of amoxicillin allergy, the treatment was discontinued immediately, and treatment with levofloxacin hydrate was initiated . On admission, the present patient was suspected of having acuteonset aih, because ana was 320, eight times the normal value, and igg was 1793 mg / dl, 1.1 times the normal value . However, since she had a history of taking various medicines, the possibility of druginduced hepatic injury could not be excluded . As the patient scored <2 points on the diagnostic criteria scale for acute druginduced hepatic injury established by digestive disease week japan (ddwj) 8, we believed that druginduced hepatic injury seemed unlikely . According to her clinical course and the international diagnostic criteria for aih 1, she was finally diagnosed with recovery from fulminant liver failure induced by acuteonset aih, likely type 1 aih . A nationwide survey of patients with fulminant hepatitis and lateonset hepatic failure in japan revealed that outcomes were especially poor in aih patients (the survival rate was 17.1% without liver transplantation) 9 . As this patient was older than 75 years and repeatedly developed stage 2 hepatic encephalopathy, we believed that she had poor prognosis . Although this patient might have benefitted from corticosteroids and plasma exchange at the early stage of fulminant aih, there is a little evidence for the efficacy of these treatments 6, 10 . Although this patient recovered from fulminant liver failure due to aih, she developed suppurative inflammation of the hip joint due to mssa and developed a right rib tumor due to actinomyces . Actinomycosis occurs in the cervicofacial (6070%), abdominal (2040%), and thoracic (920%) areas . Risk factors of actinomycosis are poor oral hygiene resulting in tooth decay, chronic lung disease, alcoholism, and other disorders resulting in reduced immunocompetence 7 . In the present case, immunosuppression due to corticosteroids might have contributed to the development of the right rib actinomycosis . However, the frequency of actinomycosis development secondary to the use of corticosteroid is unclear . Actinomyces are part of the normal oral flora and colonize the human digestive tract 7, 13, 14 . Although the aspiration of saliva containing actinomyces is considered to be responsible for cases of pulmonary actinomycosis, the right rib actinomycosis in this patient might have occurred via hematogenous spread, as there was no invasion from the right lung . This immunocompromised patient might have also developed hip infection due to hematogenous spread of mssa . The diagnosis of thoracic actinomycosis is difficult, and the differential diagnosis includes pulmonary malignancy 7, 13, 14 . Thus, the diagnosis of actinomycosis depends on growth of the bacteria in culture and the discovery of sulfur granules in the pathologic specimen . In this patient, when treating patients with fulminant liver failure due to aih, the possibility of such rare occurrences should be kept in mind . In conclusion, this report described an interesting case of an osteolytic lesion secondary to actinomycosis after fulminant liver failure induced by aih.
Stress cardiomyopathy (scm), a disease with many names like the broken heart syndrome, takotsubo cardiomyopathy, and apical ballooning syndrome, is characterized by regional myocardial dysfunction, typically occurring in the wake of a significant physical or emotional stressor . The pathophysiology of the condition remains incompletely understood, yet the effects of catecholamines on select portions of myocardium are thought to play an integral role . The degree of cardiac dysfunction in this condition is variable, and in the largest contemporary registry of patients with scm, only 9.9% developed cardiogenic shock . Although uncommon, this severe presentation of the disease is critical to appreciate, and we present a case of the disease at its extreme, with a patient in cardiogenic shock . A 79-year - old woman with a history of an infratentorial meningioma was admitted to the coronary care unit in cardiogenic shock . The morning of admission her past medical history was significant only for modest essential hypertension and no known coronary ischemia or dysrhythmias . Following routine induction of anesthesia with securement of the airway and institution of mechanical ventilation, sinus bradycardia associated with profound hypotension (70/40 mm hg) ensued, and this hemodynamic perturbation subsequently progressed to cardiac standstill with absence of peripheral pulses . Cardiopulmonary resuscitation (cpr) was initiated and return of spontaneous circulation (rosc) was achieved after 90 s with one round of cpr and 1 mg of intravenous epinephrine . After rosc, her vital signs were notable for sinus bradycardia with a systolic blood pressure initially between 140 and 170 mm hg, which quickly declined to less than 90 mm hg despite continuous infusions of high - dose norepinephrine (3 g / kg / min) and dopamine (20 g / kg / min). The surface electrocardiogram post - rosc revealed new t - wave inversions in the precordial leads . Intra - operative transthoracic echocardiography shortly after cardiac arrest demonstrated a left ventricular ejection fraction (lvef) of 15 - 20% with severely hypokinetic mid and distal segments and a hyperkinetic left ventricular base consistent with scm (fig . 1 and supplementary video 1, www.cardiologyres.org). In the setting of her continued hemodynamic instability, left heart catheterization was performed and an intra - aortic balloon pump was placed . The coronary anatomy was notable for only mild, non - obstructive disease (fig . The right coronary artery has minimal disease, and supplies the posterior descending artery (c). In the subsequent 6 h serum troponin i levels peaked at 1.67 ng / ml and a newly prolonged qt interval developed (fig . 3). Inotropic and mechanical supports were rapidly weaned off over the following 6 h and she was successfully extubated . Repeat echocardiography 12 h after the initial study revealed a lvef of 75 - 80%, near cavity obliteration during systole, and no regional wall motion abnormalities (supplementary video 2, www.cardiologyres.org). At 6-month follow - up, the patient denied any symptoms of heart failure and her ef normalized to 55% . Note the low voltages, inferior and lateral precordial lead st segment depressions, and the prolonged qt interval . This case highlights many of the classic elements of scm: a post - menopausal female with a primary neurologic disease; a temporal correlation with significant stress and exposure to catecholamines; prolongation of the qt interval; low - level cardiac enzyme elevations; and reversible, often transient left ventricular dysfunction, which at the extreme, results in cardiogenic shock . Although reversal of left ventricular dysfunction is the norm in scm, the time course of this patient s improvement is remarkable . Most studies of scm cite echocardiographic and symptom improvement occurring within days to weeks of diagnosis, and to our knowledge, the earliest reported echocardiographic resolution occurred 5 days following diagnosis in these case series [2, 3]. Scm has been associated with administration of catecholamines (i.e. Dobutamine during stress testing or epinephrine for treatment of anaphylaxis), and has occurred in the wake of cpr, which we believe occurred in this case . From a pathophysiological standpoint, a catecholamine surge, iatrogenic or otherwise, can impair myocyte contractility, particularly at the apex where the highest concentrations of adrenergic receptors are localized . In summary, we describe a case of transient, albeit severe scm that reversed rapidly . To our knowledge, this is the fastest recovery ever reported . This case highlights the role of catecholaminergic excess, one of the proposed mechanisms underlying the disease s pathophysiology, and also reinforces the need of aggressive supportive therapy early in the disease . The authors declare that there are no conflicts of interest regarding the publication of this paper.
Three types of calibrated microspheres have recently been approved in japan as embolic agents for hypervascular tumors and arteriovenous malformations . To date, two types of embolic microspheres capable of being loaded with drug have been introduced: dc bead (biocompatibles, farnham, uk) and hepasphere (biosphere medical, roissy cdg cedex, france). In a phase ii randomized controlled study of transarterial chemoembolization (tace) using a drug - eluting bead (deb - tace) for hepatocellular carcinoma (hcc), the precision - v study, the response rate after 6 months was 52% in the deb - tace group and 44% in the tace group using a lipiodol (laboratoire guerbet, aulnay - sous - bois, france)/anticancer drug mixture solution (lip - tace), showing no significant difference or superiority of deb - tace . However, complication by postembolization syndrome, such as abdominal pain, fever, nausea, vomiting and liver enzyme elevation, developed in 2080% after lip - tace [3, 4], whereas symptoms developed in only 1837% after deb - tace [5, 6], showing that the complications were milder than those after lip - tace . On the other hand, deb - tace induced liver abscesses at a relatively high frequency, which could be fatal [5, 7, 8, 9]. Adverse events after deb - tace have been reported occasionally [5, 7, 8, 9, 10, 11], but as it has just been introduced in japan, many points regarding adverse events are unclear with respect to the developmental mechanism and risk factors . We encountered a hcc patient who underwent deb - tace and experienced delayed intratumoral hemorrhage . An 81-year - old male regularly visited his physician for treatment of hepatitis c virus - related cirrhosis . On routine abdominal ultrasonography (us), on esophagogastroduodenoscopy, a type 0-iia early gastric carcinoma with a diameter of 25 mm was detected in the lesser curvature of the middle body . The patient was referred to our hospital for further examination and treatment . During the first examination, the liver function was classified as child - pugh class a, and the tumor markers were normal (afp level: 3.4 ng / ml, afp - l3 fraction level <0.5%), but the pivka ii had a level of 118 mau / ml, showing a mild elevation . It was capsulated and enhanced in the arterial phase on dynamic computed tomography (ct) (fig . 1), and showed a mosaic pattern accompanied by a marginal hypoechoic zone on gray - scale us . The mass was enhanced in the vascular phase (040 s) on contrast - enhanced us (ceus) using sonazoid (daiichi sankyo, tokyo, japan), and showed a hypoechoic area in the postvascular phase (10 min after an intravenous injection of sonazoid). Based on the tumor marker and imaging findings, the patient was diagnosed with hcc . Deb - tace was performed because the patient declined to undergo surgical resection of hcc . Access for tace was performed under sterile conditions and under local anesthesia, via the right femoral artery using a 3-fr sheath (medikit, tokyo, japan) and in a retrograde fashion . When a microcatheter was advanced to the anterior segmental artery of the right hepatic artery and contrast imaging was applied, an intensely stained tumor was observed, with a5 as the feeding artery . A solution impregnated with 100300 m dc bead and 50 mg epirubicin hydrochloride, with a dc bead volume of 0.35 ml, was administered via the feeding artery to perform deb - tace . Disappearance of the intensely stained tumor image was confirmed by right hepatic arterial angiography, and the treatment was completed . The course after deb - tace was favorable, and no adverse events above grade 3 according to the common terminology criteria for adverse events (ctcae) version 4.0 were observed . Endoscopic submucosal dissection (esd) of the early gastric carcinoma was performed about 1 month after deb - tace . Right hypochondriac pain suddenly developed 3 days after esd, but no adverse events were assumed to be caused by esd (i.e., free air, noted on dynamic ct). However, the tumor diameter had increased from that before deb - tace, and the tumor showed a high - intensity area on unenhanced ct (fig . 2a), which was not enhanced in the arterial phase on dynamic ct, suggesting intratumoral hemorrhage . The hemodynamics of the tumor were followed using ceus over the time before and after deb - tace . On ceus carried out on the day after deb - tace, the whole intratumoral enhancement decreased, and nonenhanced patchy regions, assumed to be necrosis, were noted in the tumor in the vascular phase . On ceus performed 4 weeks after deb - tace, i.e., immediately before esd, the tumor size was 33 31 mm, and the enhancement area was increased in the vascular phase (fig . However, the tumor had enlarged to 41 36 mm on ceus when the right hypochondriac pain developed (5 weeks after deb - tace), and changes in the echogenicity on gray - scale us and nonenhancement of almost the entire tumor in the vascular phase on ceus were noted (fig . Based on the above findings, the cause of right hypochondriac pain may have been deb - tace - associated intratumoral hemorrhage . Since the hemorrhage was limited to inside the tumor, it stopped spontaneously with rest . The advantages of deb - tace are as follows: the infusion dose of the chemotherapeutic agent can be modified by adjusting the chemotherapeutic agent carrier, while altering the depth of embolization with beads, the duration of tumor exposure to chemotherapeutic agent can be fine - tuned through sustained release of the chemotherapeutic agent, and systemic adverse reactions of chemotherapeutic agent can be attenuated by continuous release of high - dose chemotherapeutic agents into the tumors . Deb has proven to be extremely potent and safe by allowing large amounts of the chemotherapeutic agent to concentrate within the tumor over time, thereby maximizing the cytotoxic effect, which also makes it attractive as an effective treatment for large lesions . Favorable therapeutic effects have been reported occasionally [8, 9] and have increased the expectations of hcc treatment with deb - tace; however, the beads exhibit characteristic physical properties and behavior that are different from those of gelatin particles . It is important to fully investigate these characteristics to avoid inadequacy of the therapeutic effect and to prevent the onset of adverse events . Previous studies have reported that the incidence of mild adverse events, such as postembolization syndrome, is lower in the deb - tace group compared with the lip - tace group [3, 4, 5, 6]. Meanwhile, liver abscesses were induced at a relatively high frequency (about 1.67.4%) [5, 7, 8, 9]. Malagari et al . Reported that the 30-day mortality was 1.26% among 237 hcc patients treated with deb - tace with dc bead loaded with doxorubicin, and that the cause was liver abscess in all cases . Regarding other adverse events, acute pancreatitis and cholecystitis developed with incidences of 15 and 16.3%, respectively . Previous studies have shown that rare adverse events included liver failure and infection, bile duct injury, upper gastrointestinal bleeding, pulmonary embolism, splenic infarction, or spinal embolization [1, 7, 10, 11]. Unlike lip - tace, many adverse events occurred due to ischemic changes induced by the inflow of beads into organs other than the hcc lesion, which is characteristic of deb - tace . Tumor hemorrhage has also been reported after deb - tace [1, 7, 10, 11, 13] with large tumors, with a diameter of 516 cm, and the tumor locations were subcapsular in all previous reports . To our knowledge, there has been no case of a tumor with a diameter <5 cm distinct from the subcapsular, as was observed in our patient . There has been no report of a mechanism underlying the deb - tace - specific tumor hemorrhage . In general, the mechanism of tumor rupture after tace might be related to increased intratumoral pressure as a result of rapid edematic expansion due to tumor necrosis or vascular injury, secondary to embolization . The risk is particularly high in subcapsular lesions, and we must be careful because the spread of hemorrhage in the abdominal cavity cannot only lead to hemorrhagic shock, but also peritoneal metastasis . Unlike the reported cases of tumor hemorrhage after deb - tace, in the present case, the tumor diameter was not large and the tumor was distinct from the subcapsular region . However, intratumoral hemorrhage occurred about 1 month after deb - tace . Local pooling of the contrast agent within the target tumor could occur during deb - tace, and seki et al . Reported that it occurred in 25.2% of 135 hcc patients treated with deb - tace (epirubicin - loaded hepasphere). While the cause of this local pooling remains unclear, it is similar to extravasation or blood sinus, suggesting that it was a local hemorrhage due to intratumor vascular collapse as a consequence of the treatment . When local pooling of the contrast agent was noted, seki et al . Added a small amount of gelatin sponge particles, with a diameter ranging from 1 to 2 mm, to the feeding artery until the pooling disappeared . Based on the above findings, the observation of local pooling of the contrast agent within the target tumor during treatment suggests that intratumoral pressure has already increased, which was unlikely in our patient . Treatment - induced intratumoral hemorrhage continued immediately after deb - tace because no pooling was noted during treatment or on confirmation angiography following treatment . However, it is possible that the amount of dc bead was insufficient and embolization was incomplete . It was assumed that incomplete embolization subsequently caused intratumoral vascular collapse, i.e., a phenomenon similar to pooling, and that the hemorrhage expanded to the necrotic intratumoral area by sustained release of the chemotherapeutic agent and embolization, leading to complication of intratumoral hemorrhage despite the low risk of tumor hemorrhage . Many points remain unclear, i.e., the appropriate bead sizes for each case and the level of embolization, but it may be important to apply sufficient embolization because insufficient embolization is likely to lead to tumor hemorrhage . Deb - tace is considered to rarely cause adverse events, but fatal complications have been reported [5, 7, 8, 9], and reports of adverse events may increase with augmentation of the numbers of deb - tace - treated patients . If reports of tumor hemorrhage after deb - tace increase, the mechanism of intratumoral hemorrhage in cases similar to our patient may be elucidated . Intratumoral hemorrhage was suspected due to the presence of a high - intensity area in the tumor on unenhanced ct and a rapid increase in the tumor diameter and accompanying enlargement of the unenhanced area on ceus performed before and after deb - tace . Based on these findings it was difficult to assess the therapeutic effect of deb - tace by ct, based on lipiodol accumulation, unlike that after conventional lip - tace, and evaluation of the intratumoral hemodynamics could be important . Ceus is superior in evaluating intratumoral hemodynamics in real time, and the utility of ceus to evaluate the therapeutic effect after deb - tace was suggested . We encountered a patient who experienced intratumoral hemorrhage about 1 month after deb - tace . Deb - tace for hcc is a new treatment method in japan, and reports of adverse events may increase in the future . To obtain the therapeutic effect of deb - tace while preventing the adverse events, it may be important to understand the characteristics of the beads themselves and to apply the appropriate embolization to each individual case . It was also suggested that ceus is useful to evaluate the therapeutic effect after deb - tace.
We report a case of iga pemphigus with iga antibodies to desmoglein 1 (dsg1) and desmoglein 3 (dsg3). We report the case of an 60-year - old man with intraepidermal neutrophilic iga pemphigus with iga antibodies to dsg1 and dsg3 . The disease was not effectively controlled by conventional therapeutic regimens (colchicine, dapsone). Systemic treatment with isotretinoin 25 mg / d and prednisone 20 mg / d achieved only a slight effect after six months . Our case confirmed the recalcitrant nature of iga pemphigus in response to distinct therapies, indicating that further research focusing on therapeutic approaches for this type of pemphigus is needed . An otherwise healthy 60-year - old man was referred to our department with a one - year history of recurrent pruritic vesiculo - pustular lesions on both axillae and groin . The lesions improved after topical application of steroids but reappeared and gradually spread to trunk and extremities . Examination revealed a symmetric eruption on the proximal extremities and trunk with prominent involvement of the axillae and groin . It was composed of grouped vesiculo - pustular lesions mostly on well - circumscribed erythematous patches (figure 1). Direct immunofluorescence microscopy (dif) of perilesional skin detected intercellular deposits of iga throughout the epidermis (figure 3). Igg - elisa for desmogleins (dsg) showed no igg antibodies to either dsg1 or dsg3 . Iga - elisa for dsgs was then performed and the results indicated that the patient s serum was positive for iga anti - dsg1 and anti - dsg3 antibodies . Colchicine, 0.5 mg 3 times daily was begun, but this had no effect after one month so it was substituted by dapsone 100 mg / d, which also failed to produce any improvement and his disease remained active . Subsequently, isotretinoin 25 mg / d and prednisone 20 mg / d were initiated achieving only a slight effect after six months . Based on pathology and dif findings, iga pemphigus can be further divided into two subtypes, namely, subcorneal pustular dermatosis (spd) and intraepidermal neutrophilic dermatosis (ien) types . Clinically, as seen in our patient, both subtypes of iga pemphigus present with small blisters and pustules overlaying well - circumscribed erythemas . A herpetiform appearance has also been reported . The whole body can be involved, with a predilection for flexures, such as axilla, groin and submammary area . While spd - type iga pemphigus shows subcorneal pustules, the ien type is characterized by pustule formation throughout the entire epidermis . In dif, spd - type iga pemphigus involves cell surface iga binding only in the upper epidermis, where as ien - type iga pemphigus shows binding throughout the epidermis . Although the histological features of our case are consistent with those of the spd type of iga pemphigus, ien - type iga pemphigus was diagnosed in the present patient because of iga deposits throughout the epidermis . In contrast, in the ien type no reactivity of auto antibodies with desmocollin 1, 2, and 3 has been found, whereas desmoglein 1 and 3 were suggested as putative target antigens of ien type in single case reports . The result of immunoelectron microscopic study revealed that the antigen of ien type may not be a desmosomal component . In our patient, iga - elisa for dsgs showed reactivity with dsg1 and dsg3, suggesting that his pemphigus most likely belongs to the ien type . Iep should be performed because iga pemphigus has been reported to be associated with monoclonal iga gammapathy . Treatments of iga pemphigus are performed based on the disease pathomechanism and on anecdotal reports . Dapsone is commonly the drug of choice due to its effect in suppressing neutrophilic infiltration . Recently, adalimumab and mycophenolate mofetil, have also been reported to be useful in treating iga pemphigus . Our case confirmed the recalcitrant nature of iga pemphigus in response to distinct therapies, indicating that further research focusing on therapeutic approaches for this type of pemphigus is needed.
Topical bimatoprost is a popular prostaglandin analog used primarily as a powerful ocular hypotensive agent for treatment of glaucoma (approved in 2001). It is also used cosmetically to induce hypertrichosis, approved by the us food and drug administration in 2008 as latisse (allergan, inc, irvine, ca, usa). However, it may cause complications such as periocular skin hyperpigmentation, intraocular inflammation, and conjunctival hyperemia.1 these mainly affect superficial tissues and, to date, there has been no report on involvement of deeper periocular tissues as far as we are aware . Herein, we report an unusual case of periocular muscle atrophy and weakness secondary to unilateral topical bimatoprost use . A 58-year - old chinese female was referred to the oculoplastics services of national university hospital, singapore, for right upper eyelid ptosis of 1.5 years duration and difficulty in closing her right eye . She had a history of bilateral primary angle closure diagnosed in 2003, for which bilateral laser peripheral iridotomies were performed . She was started on topical latanoprost 0.005% once every night to the right eye, as intraocular pressure (iop) was suboptimal . Subsequently, she underwent an uneventful right cataract extraction with intraocular lens implantation that same year . Her condition remained stable until 2008 when there were progressive defects in her right visual field with increased iop . Thus, she was started on topical bimatoprost 0.03% (lumigan, allergan, inc) once every night to her right eye . However, she reported an onset of right upper eyelid drooping 2 months after starting treatment . The ptosis did not show any diurnal variability or fatigability, and there was no diplopia . A detailed evaluation by the neuro - ophthalmology team revealed no underlying neurological cause . Four months after starting treatment, the treated eye was found to have long eyelashes and ocular pigmentation . Subsequently, the patient reported worsening of her right ptosis after 12 months on treatment and was keen for referral to the oculoplastics team . By the time the patient was reviewed by the oculoplastics team in 2010 measurements of the palpebral aperture (pa) of her right and left eye were 6 mm and 8 mm, respectively . Measurements of marginal reflex distance 1 (mrd1) for her right and left eyes were 0.5 mm and 2 mm, respectively . Levator palpebrae function was 8 mm for the right eye and 14 mm for the left eye . Lid creases measured 7 mm and 5 mm for the right and left eye, respectively . Eventually, the patient underwent a surgical correction of her ptosis with levator muscle advancement in august 2011 . Seven months after the operation, her pa measured 9 mm and 8 mm while mrd1 measured 4 mm and 3 mm for the right and left eye, respectively . The pre- and postoperative clinical photos of her eyelids are shown in figures 1 and 2, respectively . Preoperatively, she had clinically significant right ptosis obscuring her visual axis, and right lagophthalmos with orbicularis oculi muscle weakness . Topical bimatoprost is highly efficacious and superior to many other anti - glaucoma eye drops.2,3 however, it commonly causes side effects such as ocular hyperemia, periocular and iris pigmentary changes, although these are mostly reversible with cessation of use.4 periorbital lipodystrophy resulting in periorbital hollowing, eyelid retraction, and enophthalmos from chronic use of bimatoprost has also been well - documented.58 upper eyelid sulcus deepening has been shown to occur significantly more frequently (objectively and subjectively) with bimatoprost use than with other prostaglandin analogs, namely latanoprost, tafluprost, and unoprostone.9 aihara et al reported an increased incidence of upper eyelid sulcus deepening in patients who were switched from latanoprost to bimatoprost, suggesting that the latter has a more potent effect on periorbital fat tissue.10 the term prostaglandin - associated periorbitopathy (pap) has been coined to describe the constellation of periorbital soft - tissue changes resulting from the use of topical prostaglandin analogs . The clinical features of pap include upper eyelid ptosis, deepening of the upper eyelid sulcus, involution of dermatochalasis, orbital fat atrophy, mild enophthalmos, flattening of lower eyelid bags, inferior scleral show, and tight orbits.11 these effects can appear after a period of several weeks to several years, and have been reported to be reversible upon cessation of use.11 most of the anatomical changes in pap are thought to be caused by orbital fat atrophy, while dehiscence of levator aponeurosis or mller muscle may account for the upper lid ptosis.11 it has been postulated that upregulation of prostaglandin f2- results in significant anti - adipogenic effect.12 prostaglandin f2- blocks adipogenesis through the activation of mitogen - activated protein kinase, resulting in the inhibitory phosphorylation of peroxisome proliferator - activated receptor - gamma, a nuclear hormone receptor central to adipocyte differentiation.13,14 topical prostaglandin analogs have been shown to downregulate the expression of peroxisome proliferator - activated receptor - gamma and inhibit the accumulation of intracytoplasmic lipid droplets.15 the reversal of fat atrophy upon cessation of use may be due to removal of this inhibition, allowing adipogenesis to recommence . Imaging of orbits (computed tomography and magnetic resonance imaging) has ruled out orbital pathology in the fellow eye (resulting in pseudo - enophthalmos) and other disease processes such as cicatrizing changes.5,6 periorbital changes may be underestimated, as they may be subtle or attributed to age - related volume depletion . Bilateral topical use of prostaglandin analog eye drops in glaucoma may lead to equivocal changes in both eyes, which may be less obvious and thus under - reported . However, in asians, who tend to have greater upper eyelid fullness, unilateral periorbital fat atrophy may result in more obvious disfigurement.16 to date, as far as we are aware, there has been no report of topical bimatoprost use resulting in periorbital muscle atrophy . In figure 1, it can also be seen that the patient had deepening of her upper eyelid sulcus and dermatochalasis involution . Clinical eyelid measurements clearly showed reduced function of the right upper eyelid, suggesting that the effects of topical bimatoprost may not be limited to periorbital fat . Reduced levator function and ptosis may be masked by the more common eyelid retraction and deepening of the upper eyelid sulcus . Peplinski and albiani smith postulated that mller muscle (smooth muscle) may be directly affected by prostaglandin - mediated pathways, but whether there is a similar effect on levator muscle (striated muscle) remains unknown.17 other reasons for the induced right ptosis might have been stretching of the eyelid due to initiation of glaucoma - drop usage, disruption of the whitnall s suspensory ligament from the relative enophthalmos, or the patient s previous cataract surgery, even though there was no fixing of patient s superior rectus during the surgery . Further studies are warranted . In this case, the patient was advised to stop topical bimatoprost and right ptosis correction surgery was performed successfully . In figure 2, it can be seen that the upper eyelid sulcus deepening was much reduced, with a marked improvement in the dermatochalasis involution after the surgery . It may be worthwhile to note that topical bimatoprost used cosmetically can also cause unwanted side effects such as conjunctival hyperemia and iop reduction.18 therefore, while considered generally safe when applied superficially to the base of eyelashes, ocular side effects can still occur with risk of instillation onto the eye surface . Herein, we report an unusual and previously unreported case of upper eyelid ptosis secondary to levator muscle atrophy as a result of the chronic unilateral use of topical bimatoprost . Clinicians and patients should be aware of this, as it may compromise visual field and be potentially disfiguring, especially when used monocularly . Patients using latisse should similarly be warned, since the cosmetic and ophthalmic preparations are identical . Although this side effect may not be completely reversible with discontinuation of medication, this case shows that it may be successfully corrected with surgical intervention
Esophageal cancer is a relatively common form of cancer in the eastern world (3). The disease is highly lethal, with overall five - year survival rates less than 10% . Histologically, there are two main types; one type grows in the inside layer of the lining of the esophagus squamous cell carcinoma (scc) a second cancer that starts in gland cells are called adenocarcinoma (ac) (5). A worldwide increasing incidence for ac but not for scc has been reported in north america, europe and japan (6). In kerman province in iran, an increased incidence of adenocarcinoma of the esophagus 1991 to 2002 has been reported while the incidence of esophageal scc hardly increased . (7). In ardabil province in 2007, the one- and five - years survival rates in patients with upper gastrointestinal cancer were estimated to be 40.5% and 0.8% respectively (8). Geographic variations in iran show that the incidence and mortality of gastric cancer is higher in west and north west regions and in the kurdistan province in particular . Previous survival studies in iran have focused on esophageal cancer, indicating the importance of gender, age, residence (rural and urban) and risk factors whereas inconsistent results have been observed for education, and income (810). Factors such as histological type and grade, location and stage of tumor, surgical treatments have not been studied in relation to survival . Therefore, the present study aimed to investigate the effects of histological factors on the survival of patients with esophageal cancer . Data were sourced mainly from the patient reports of pathology laboratories and hospital database record . Through a retrospective cohort study using censes method; all eligible patients with esophageal cancer (134 gastric cancers) who had been hospitalized at the towhid hospital, sanandaj city, kurdistan province western iran were recruited . Inclusion criteria were patients with definite diagnosis of esophageal cancer during a five - year period from 2006 onward . Samples were coded under the direct supervision of clinical pathologists according to the international classification of diseases for oncology . Clinical data such as practice treatment were obtained through a structured questionnaire and the patients clinical records . Vital status and date of death were determined through the by official death certificates, with a maximum follow - up of 90 months . Survival time (in months) was calculated from the date of diagnosis through the date of death or last follow - up . A failure was defined as death by any cause during the follow - up period and patients alive at the end of the follow - up period were censored . 7 patients were excluded from the analyses according to exclusion criteria (4 patients lost follow - up, 2 illegible data, and 1 patient due to migration). Clinical and pathologic variables, which were sub - layered into age, gender, setting, histological type of tumor and practice treatment were entered into parametric regression models (by considering and not considering heterogeneity) for multivariate analysis in order to assess the relationships between the characteristics and prognostic factors for survivors . Ilam university of medical sciences, ethics committee on considering of publication data result in general approved the study (code no: 91002, date: 22.08.2012). The kaplan meier and log rank statistic methods were used to compare survival rates in different subgroups . Using life table, the breslow (generalized wilcoxon) statistics was used to compare median survival time in three age groups . The cox hazards regression analysis was also used to investigate the effect of the variables and adjusting the effect of age . The graphical (diagram log (s) t vs. time) and analytical (time - varying covariate) methods was applied to test the appropriateness of cox's proportional hazard (11). The multivariate cox regression analysis was used to identify independent predictors for patient survival using a backward stepwise approach with an entry limit of p <0.1 and a removal limit of p <0.05 . The survival time of patients stratified by this grouping method were analyzed by the kaplan - meier analysis and cox regression as described earlier . The kaplan meier and log rank statistic methods were used to compare survival rates in different subgroups . Using life table, survival rates and survival density function was assessed at year intervals . The breslow (generalized wilcoxon) statistics was used to compare median survival time in three age groups . The cox hazards regression analysis was also used to investigate the effect of the variables and adjusting the effect of age . The graphical (diagram log (s) t vs. time) and analytical (time - varying covariate) methods was applied to test the appropriateness of cox's proportional hazard (11). The multivariate cox regression analysis was used to identify independent predictors for patient survival using a backward stepwise approach with an entry limit of p <0.1 and a removal limit of p <0.05 . The survival time of patients stratified by this grouping method were analyzed by the kaplan - meier analysis and cox regression as described earlier . A total number of 127 patients (55.1% males) with esophageal cancer were recruited . Mean age standard deviation was 65.38 11.62 years with a range of between 35 to 86 years . Based on histological type of tumor, 23 (18.1%) had ac type, 94 patients (74.0%) had scc and other histological type 10 (7.9%). For 44 patients (36.6%), the tumors were located in the lower third of the esophagus . Meanwhile, the tumors were located in the middle third and upper third of the esophagus for 28 (22.0%) and 48 patients (37.8%) respectively . The tumors were well differentiated for 42 patients (48.8%), moderately differentiated for 35 patients (27.6%), poorly differentiated in 16 patients (12.6%) and unknown differentiated for 14 patients (11.0%). Gender and setting of patients had no significant effects on survival rate variation in univariate analysis . Age, location of tumor, histological type, tumor grade, stage of diagnosis and practice treatment showed a significant association on the survival rates variation (table 1). The kaplan - meier analysis revealed significant differences in five - year survival rate and age of diagnosis (log - rank= <0.001) (figure 1a), histological type (log - rank = 0.004) (figure 1b), tumor grade (log - rank=0.017) (figure 1c), and tumor locate (log - rank=0.008) (figure 1d). The association between demographic, histological factors and survival in esophageal cancer patients . (a) by age of diagnosis (b) by histology of tumor (c) by histology grade (d) by location of tumor . Clinic - pathological characteristic of the patients with esophageal cancer cox - regression analysis using histological factors 45> year as reference revealed that patients whose 46 - 65 year at diagnosis (hr= 3.43, 95% ci = 1.03 - 11.41, p= 0.044), 66 <year (hr = 9.78, 95% ci = 2.93 - 32.64, p = <cox - regression analysis for poor grade tumors as reference revealed that patients whose tumors with moderately differentiate (hr= 0.52, 95% ci = 0.25 - 1.07, p= 0.078) and well differentiate (hr = 0.98, 95% ci = 0.51 - 1.85, p= 0.951) had a decreased risk for death from esophageal cancer . Cox regression coefficient () analysis shows patient with tumor located in middle of esophaus (= -0.91) and upper esophagus (= -0.13) have lower death rates compression to tumors that are located in the lower esophagus . Multivariate cox regression analyses; f: cox regression analysis for age, gender, setting, stage at diagnosis and practice treatment; e: cox - regression analysis for age, gender, setting, histology grade and location of tumor stage of tumor and practice treatment in step 2 of cox - regression analysis flowing demographic characteristic in step 1 using treatment surgery as reference revealed that patients who only had chemotherapy in process of treatment (hr = 2.79, 95% ci = 1.35 - 5.73, p = 0.005), and patients who only had radiotherapy (hr = 7.27, 95% ci = 1.53 - 34.39, p = 0.012) had an increased risk of deaths from esophageal cancer . Coefficient value analysis shows patient who had only chemotherapy (= 1.98) have increase death rates in comparison to patients who had surgical operation only . Similar results were obtained for patient who only had chemotherapy (table 2f). The five - year survival rates (by year) were 49%, 27%, 24%, 22% and 19% respectively . Overall five - year survival in the cases of esophageal cancer in the present study was 49.0%, with survival rates being higher in the group of women compared to the men (19.7% versus 13.8%) (p = 0.48), higher in clinical localized stages (41.2%) compared to regional stages (14.3%) (p = <0.001), one survival rate higher in cases submitted to curative surgery (53.2%) compared to those submitted to chemotherapy care (13.5), (p = <0.001), as demonstrated in table 3 and figure 2 . Cumulative survival rate in patients with esophageal cancer the 1 - 5 year survival rates and med time survival in patients with esophageal cancer in a recent review of survival analyses, it was found that many studies have indicated clinical and pathologic characteristics of patients as explanatory variables with respect to survival (12). Age of diagnosis was an independent covariate for survival in patients in this study and better prognosis observed in> 45 year age group . The reasons for this are likely to include a combination of better general health, more effective response to treatment and earlier diagnosis in younger people . Previous report indicated better survival in young patients (13). On the other hand, some other studies do not have the same report (14, 15). The present results showed that patients gender had no significant impact on survival rate (p = 0.48). Median survival time for man and women was (10.01.03) and (12.02.83), respectively . Sex was not an independent prognostic factor in either chinese patients (p = 0.23) or white patients living in the united states (p = 0.28). Overall survival was significantly worse only in male white compared with chinese patients (median survival time, 12.4 versus 14.5 months, respectively; p <0.01), (8). However, some studies have found better survival rate for women (10). The scc cancer is a significant factor that had impact on the survival probability of patients in univariate analysis, which is similar to some other studies . This result reinforced scc and ac are distinct malignancies of the esophagus, with different risk factors and different natural histories . In this study patient with scc histology had a better survival, although in previous studies acs generally have a slightly better prognosis (outlook) in overall(12). Stage of diagnosis was strongly associated with prognosis in our study, which is a finding repeated in several other studies (4, 10). Overall, the five - year survival rate of people with esophageal cancer is about 17% . However, survival rates depend on several factors, including the stage (or extent) of the cancer at the time of diagnosis . Fortunately, new treatment approaches have lowered the death rate from esophagus cancer, which at one time killed 95% of those diagnosed with the disease . Today, about 16% of all esophageal cancer patients will survive five years or more (13). As with most types of cancer, survival odds increase when the disease is caught and treated early (16). The five - year survival rate of people with cancer located only in the esophagus is about 41.2% . The five - year rate for those with disease that has spread regionally is 14.3%, if it has spread to distant organs, about 8.2% (table 2). However, medical centers now report that patients undergoing surgery alone have median survival rates between 13 and 19 months, 2-year survival rates between 35% and 42%, and 5-year survival rates of 15% to 24% (17). In present study median of survival and 4-year survival rate in this cases was (123.86 month) and (21.3), respectively . In patients that radiation therapy was the only optionfor treatment the mean survival rate was (51.06 month). Radiation therapy has been used in the past as a single - modality approach with curative intent . However, except for those with very early - stage disease, radiation has had little impact on long - term survival . Chemotherapy has been given preoperatively, postoperatively, or both median survival rates were (50.548 month). Multimodality treatment approaches have evolved over recent years in response to the frequent loco regional and distant recurrences identified after surgery or radiation therapy alone . There are limitations with the present study in which the survival rate unable to predict future events for a particular person . Meanwhile, it was not possible to consider changes in characteristics after diagnosis, which may have affected survival . They are of limited value because of unavoidable selection bias, in particular in case selection and patient's characteristics . In conclusions, the present study showed that age of diagnosis, histological grade, and location of tumor was prognostic factors for survival in patients with esophageal cancer . It can be concluded the early detection of patients at younger age and in primary stages and histological grade may have positive effect on patients with esophageal cancer and be important to increase the survival time.
Perinatal depression is a major public health problem, affecting up to a quarter of all pregnant women in rural countries . It is associated with profound physical and emotional changes and associated risks for the onset or exacerbation of several mental disorders such as depression . In addition to its effects on the mother's well - being, perinatal depression is associated with adverse outcomes of pregnancy . Relevant effective interventions are being developed, but their effectiveness in low- and middle - income countries may be limited by various factors . Exercise can provide a viable treatment options for depressed patients, whether as an alternative treatment strategy to conventional treatments or as a supplemental strategy, either in the short - term or long - term . Research to date by low numbers to support the use of exercise in some form as a positive treatment option for individuals suffering from depression . All married women aged 1740 years with low socioeconomic status in their second trimester of pregnancy, who had depression, were considered . Women with a diagnosed medical condition, pregnancy - related illness, significant physical or intellectual disability, post par tumor other form of psychosis, and severe depressions were excluded . During the pre - intervention assessment, both groups were asked to attend post - intervention assessment 4 and 8 weeks following the start of the program to complete the questionnaire . Subjects in this group had 4 weeks exercise program, cycle 4 sessions a week for 4060 min / session . The exercise in each session were including: 10 min heating with walking, stretching and flexibility, and three sets of moderate intensity cycling in 6 min with 6065% of maximum heart rate . Subjects in the control group were given a patient handout which is in gujarati explaining about the procedures to do as home based program . The hand out consist of five criteria such as stay active, do something that you think is fun each day, spend time with people who help or support you, relaxing and set simple goals . Inter group analysis was done using wilcoxon - mann whitney test and intra group analysis was done using one - way anova . During the pre - intervention assessment, subjects have undergone kuppuswamy's socioeconomic status scale and the physical health questionnaire-9 . Both groups were asked to attend post - intervention assessment 4 and 8 weeks following the start of the program to complete the questionnaire . Subjects in this group had 4 weeks exercise program, cycle 4 sessions a week for 4060 min / session . The exercise in each session were including: 10 min heating with walking, stretching and flexibility, and three sets of moderate intensity cycling in 6 min with 6065% of maximum heart rate . Subjects in the control group were given a patient handout which is in gujarati explaining about the procedures to do as home based program . The hand out consist of five criteria such as stay active, do something that you think is fun each day, spend time with people who help or support you, relaxing and set simple goals . Inter group analysis was done using wilcoxon - mann whitney test and intra group analysis was done using one - way anova . Of 146 subjects, 80 (55%) met the inclusion criteria and 60 (41%) agreed to enroll . Four subjects were excluded from the analysis in the exercise group (group 1) and 2 subjects were excluded from analysis in the control group (group 2). No significance differences between groups were found at baseline on outcome variable such as depression level . Analysis of depression level shows significant changes within and between exercise and control group . At baseline, the mean depression score was 15.96 (3.09) and 16.23 (3.38) for the exercise group and the control group, respectively . After the 4-weeks intervention, the mean score fell to 6.10 (2.44) for the exercise group (43.54%) and to 10.60 (2.74) for the control group (21.25%). At the 8 weeks follow - up, the mean score was 1.34 (1.09) for the exercise group (72.81%) and 5.03 (1.50) for the control group (42.50%). Exercise has been shown in a number of studies to prove beneficial in the treatment of depressive symptoms especially in perinatal and post natal conditions . Bartholomew showed that a single incidence of exercise can have a positive effect on mood . Maintaining the intensity and frequency of exercise recommended by most public health agencies were sufficient to provide a significant reduction in major depressive disorder (mdd) symptoms . The additional benefits of exercise to individuals suffering from depression include stress reduction, better attitude, improved outlook, self - confidence, and mental well - being . Our intervention, the physical exercises was developed to provide a culturally appropriate, feasible, and evidence - based physical therapy intervention for women living in very poor communities in rural india . These results suggest that supervised physical exercise provides better improvement in depression status in perinatal subjects.
Depending on the location of the perforation, signs of peritoneal irritation may be evident in cases of intra - peritoneal free perforation but hidden in cases of retroperitoneal perforation . The abscess or colonic gas may spread via various anatomic pathway when perforation of diverticulitis in the retroperitoneum occurs, and diverse atypical clinical symptoms may be present . Thus, the atypical manifestation of retroperitoneal perforation of diverticulitis can cause difficulties when making the diagnosis . The delayed diagnosis and treatment could result in high morbidity and mortality . Pneumomediastinum is initiated by many precipitating factors or diseases . However, it is rarely associated with colonic perforation, particularly in patients with no history of colonoscopic procedure [3 - 6]. Here a 59-year - old man was referred to the emergency department because of persistent abdominal and left flank pain . The left flank pain had started 30 days before this visit as an acute nephrolithiasis . He had visited the local clinic and presumptive impression was left ureter stone because left costovertebral angle (cva) tenderness was apparent although radiologic study was not performed . However, the left flank pain worsened and was accompanied by abdominal pain 1 week ago . On physical examination, the patient looked ill and had a temperature of 38.2, the blood pressure was 90/40 mmhg, the pulse rate was 101 beats / min, and the respiratory rate was 24 breaths / min . The abdomen was slightly distended and tender over the lower abdomen, without signs of generalized peritoneal irritation . Laboratory results were within the normal range, except for the white blood cell count of 21,000/l . Chest x - ray showed scanty bilateral pleural effusion without pneumomediastinum or free air in the peritoneal cavity (fig . However, a computed tomography scan showed an infiltrative mass in the descending colon involving the left para - renal space . In addition, it showed massive air bubbles in the left retroperitoneum with diffuse infiltrates into the soft tissue (fig . An emergency operation was performed because of suspected diverticulitis or colon cancer perforation with abscess formation . During the operation, a colonic diverticulitis perforation over the posterior wall of the sigmoid descending junction and a retroperitoneal abscess with necrosis of the parietal peritoneum were found . A segmental resection of the diseased colon and end colostomy were performed (hartmann's procedure). Post - operative culture of the drain revealed enterococcus faecalis, which were sensitive to various antibiotics including penicillin, vancomycin, erythromycin, ciprofloxacin, and gentamicin . Thus, we added vancomycin instead of metronidazole to the previous empirical antibiotics (1st generation cephalosporin, gentamicin, metronidazole) on the 4th post - operative day . However, the condition of the patient deteriorated progressively after operation . Colonic diverticulosis is a common disease, and its incidence increases with aging . Among the patients with colonic diverticulosis, approximately 20% may develop diverticulitis as a result of infection and inflammation of the diverticuli . The perforation of diverticulitis is one of the most serious complications that require an urgent operation . The perforated lesion is usually covered and leads to abscess formation, whereas free perforation into the peritoneal cavity leading to diffuse peritonitis is relatively rare . In cases of free perforation, the diagnosis of diverticulitis perforation may be difficult depending on the location of the perforation, e.g., perforation into the retroperitoneal space . In such cases, its manifestation can be presented with various conditions, including thrombophlebitis of the leg, inguinal abscess, hip and buttock pain, subcutaneous emphysema, and shortness of breath [1,3 - 6]. These various pathologic conditions caused by diverticulitis perforation can be explained by considering the anatomy . Meyers reported that if gas is present in the para - renal space, which can be caused by sigmoid diverticulitis perforation, its progression from the posterior para - renal space through the diaphragm hiatus results in pneumomediastinum and cervical subcutaneous emphysema . Reported that the soft tissue compartment of the neck, thorax, and abdomen contains four regions defined as the subcutaneous tissue, prevertebral tissue, visceral space, and previsceral space . The visceral space extends from the neck through the mediastinum to the retroperitoneum, forming an anatomic connection between these areas . Therefore, an abscess or colonic luminal gas caused by diverticulitis perforation into the retroperitoneal space can spread to the other areas, resulting in unusual manifestations, similar to those observed in our case . The unusual complications of diverticulitis perforation have been reported, e.g., subcutaneous emphysema, cellulitis, necrotising fasciitis, pneumopericardium, pericarditis, pneumothorax, and pneumomediastinum [1,3 - 6]. It can be caused by traumatic injury, intrathoracic infections, excessive coughing, sneezing, and in rare cases, colonic perforation, which is associated with a complicated colonoscopic procedure . To the best of our knowledge, there have been 5 cases of pneumomediastinum as a sequence of diverticulitis perforation in english literature (table 1). In these cases, the main symptoms were abdominal pain and fever, which were often accompanied with subcutaneous emphysema [3 - 6]. The symptoms and clinical signs of retroperitoneal diverticulitis perforation may be unclear and nonspecific at an early stage, and complications may occur without any previous symptoms of diverticulitis . Therefore, making the correct diagnosis could be delayed and difficult, like in our case . In concusion, pneumomediastinum caused by retroperitoneal diverticulitis perforation is a very rare manifestation, and the symptoms are unclear . This leads to a delayed diagnosis, which can cause a life - threatening condition, particularly in patients without signs of peritonitis . It is necessary to consider the possibility of a colonic origin in patients presenting with abdominal pain and showing findings of pneumomediastinum for the prompt management of this condition.
Breast conserving therapy (bct) is currently considered as the standard management of early breast cancer . Reduced risk of local recurrence in patients administered a boost dose of 16 gy to the tumor bed in addition to 50 gy delivered to the whole breast, was confirmed in a large randomized trial [1, 2]. Boost options include interstitial brachytherapy (bt), electron or photon therapy, all following or preceding whole breast radiotherapy (wbrt) using external beam [1, 3, 4, 5, 6, 7]. In all above approaches, the extent of boost tumor volume may be incorrect if the tumor bed is determined using clinical parameters (e.g., palpation, pre - operative mammography, scar position, operative and pathology reports, or surgical clips placed at the excision site boundaries) [8, 9, 10]. The direct visualization of the operative site during surgery allows for decreasing the risk of geographical miss in determining the target volume . Pulsed - dose - rate (pdr) treatment is a bt modality combining the physical advantages of high - dose - rate (hdr) technology (isodose optimization, radiation safety) with radiobiological advantages of conventional low - dose - rate (ldr) bt . Despite its favorable radiobiological features, pdr - bt has rarely been used as a component of bct . Here, study group included 96 consecutive patients with microscopically confirmed early breast cancer, 17 of whom, with initial t1 - 3 or n1 - 2 tumor, received induction chemotherapy . All patients underwent bct between may 2002 and october 2008, including peri - operative pdr - bt boost following intra - operative bt tube placement to the primary tumor excision site (table 1) and were technically suitable for bt . Breast carcinoma was primarily diagnosed using excisional or tru - cut biopsy in 59 women (61.5%) and by fine needle aspiration biopsy in the remaining 37 patients (38.5%). Flexible tubes were implanted during breast conserving surgery (bcs) including primary tumor excision or re - excision, with immediate tumor cavity reconstruction using surrounding breast tissue in all but one case . The bt implant covered the tumor excision site and the 1 - 2 cm margin of normal breast whenever possible . The number of tubes ranged between 4 and 17 (median: 9). In most of patients, in order to guide needles, the standardized templates for a triangular array with a space of 10 - 14 mm were used . On the following day, 2d radiographic verifications of tube placement with the skin markers were taken, digitized, and entered into a bt planning system (plato, version 13.7 or 14.1, nucletron, an elekta company, elekta ab, stockholm, sweden). The skin dose was reduced by keeping a distance of at least 10 mm from the first dwell position of the stepping source . A total dose of 15 gy (1 gy per pulse repeated every hour) was delivered . Brachytherapy was followed by external beam radiotherapy (ebrt) to the entire breast after the final histology of the excised tissue had been obtained . Patient and treatment characteristics (n = 96) invasive and/or in situ ductal carcinoma present at an linked margin including three patients with invasive ductal carcinoma; an adequate margins in two of them was unachievable due to tumor adjunction to the chest wall, the third one underwent subsequent mastectomy v100 the percent volume of the post - implant receiving 100% of the prescribed dose, cc cm follow - up data including cosmetic outcome assessed by a patient were obtained through personal contact or phone interview . Classification of side effects was performed using the common terminology criteria for adverse events version 4.0, and late toxicity was reported if it occurred at least 6 months after bt . Statistical analysis was performed with spss software (version 13.0, ibm, usa). Time to event endpoints was estimated using kaplan - meier method from the date of brachytherapy to the date of any local, regional or distant relapse, or death from breast cancer, whichever occurred first, or to the date of last visit in case of no events . On the average, tube implantation prolonged time of surgery by no more than 20 minutes . In three cases with deep located tumor, to construct the deep plane close to or upon the pectoralis minor muscle, tubes were implanted before excision cavity closing . The average volume for the prescribed dose (v100) was 34.1 cc (range: 10.8 - 95.6 cc), and the median v100 for 93 patients with invasive tumor was 31.2 cc . At the time of the analysis, data regarding another dose - volume parameters were available in 36 patients (38.7% of all patients with invasive breast cancer). In this subgroup, the median volume of tissue receiving 150% (v150) of the prescribed dose was 10.7 cc (range: 2.87 - 33.44 cc), and the median dose homogeneity index [dhi], defined as 1-(v150/v100), was 0.72 (range: 0.53 - 0.78). In 91 patients, bt has started the day after the implant placement, and in five patients bt was delayed by 1 - 2 days . These included eight cases with multiple adverse prognostic pathological factors diagnosed postoperatively, implying the superiority of mastectomy, one with the final diagnosis of lobular carcinoma in situ, and two with no malignant tumor; all originally were diagnosed with fine needle aspiration as carcinoma . The remaining 85 patients received wbrt, including one with massive axillary lymph node involvement, in whom breast irradiation was preceded by chemotherapy . Except this case, the break between bt and wbrt ranged from 8 to 31 days (median: 12 days). No intense bleeding during surgery or at tube removal was observed and neither there were wound healing problems or significant skin reactions related to the implant . One patient with re - excision experienced temporary peri - operative breast infection requiring antibiotic administration . One patient with large - sized breast underwent subsequent surgical intervention due to grade 3 fat necrosis . In this case, wbrt was delayed up to 31 days after bt . After median follow - up of 12 years (range: 7 - 14), one case of true local recurrence and one regional nodal recurrence were observed (1.2% each). Four patients (4.7%) developed contralateral breast carcinoma (of another histology in two patients or another histological grade in two patients). Six years after bct, one patient presented with angiosarcoma within the irradiated breast outside of the primary breast cancer . Another seven patients (8.2%) developed second cancer including lung, ovarian, colon, skin cancer, and lymphoma . Apart from a case with angiosarcoma and a case with disseminated ovarian cancer, six relapsed breast cancer patients died due to cancer dissemination . The actuarial 5- and 10-year disease - free survival was 90% (95% ci: 84 - 96%), and 87% (95% ci: 80 - 94%), respectively, for the whole cohort of 93 invasive breast cancer patients, and 91% (95% ci: 84 - 97%) and 89% (95% ci: 82 - 96%), respectively, for patients who completed bct . In 58 out of 64 (91%) assessable patients, a good or excellent self - assessed cosmetic result was obtained . One patient with fair cosmetic effect had no tumor excision site reconstruction during bcs, and another one, due to centrally located tumor, required the nipple - areolar complex excision . Intra - operative irradiation using electron beams, photon beams, or bt tube implantation provides a high precision boost, thus minimizing the risk of a geographical miss we demonstrated the feasibility, good tolerance, and efficacy of the peri - operative bt using pdr . Interstitial pdr - bt boost seems to be particularly suitable component of bct, owing to its hypothetical favorable cosmetic outcomes . High local control and satisfactory cosmesis with pdr boost following the whole breast ebrt in breast cancer was reported by other authors [4, 5]. The rationale for a bt boost is the delivery of a high dose to the tumor bed with reduced exposure of the skin, lung, and subcutaneous tissue . The important advantages of intra - operative implant include reduced risk of geographical miss, shortening the treatment time, and avoidance of another anesthesia . An apparent limitation of this approach is the lack of the full pathology assessment, especially regarding the margin status at the time of bt . In this series, 28 patients (29%) underwent a re - excision, and in the breast cancer group the tumor resection margins were tumor - free in 85% of patients . Among cases with positive or close margin, positive pathologic margin has been considered a major risk factor for ipsilateral breast tumor recurrence (ibtr), although the use of higher boost dose in these cases is debatable [16, 17]. A substantial risk of residual disease was reported for breast cancer patients with <2 mm margin of excision . In the large retrospective study of 8485 early breast cancer patients with 5% ibtr incidence at 10-years, the invasive carcinoma margin status did not influence the risk of local relapse . In this cohort, 9% of patients underwent a re - excision, and ibtr - free interval was longer for patients who received a rt boost or systemic therapy . A multidisciplinary consensus from 2014 defined an adequate breast cancer margin as no ink on tumor . The role of surgical resection margins after breast - conserving surgery is summarized in recently published senonetwork recommendations . This document proposes standards for investigating resection margins and recommends in patients with positive margins re - excision or mastectomy, or increasing the boost dose during radiotherapy . In case of negative margins, boost administration and its dose depend on the estimated risk of local recurrence, which is linked to demographic and pathological tumor features as well as the width of surgical margin . In this series, diagnosis was established using excisional or tru - cut biopsy in the majority of patients; nevertheless, in 11.4% of patients subsequent wbrt was omitted due to the postoperative pathology findings . All these patients were primarily diagnosed by fine needle aspiration cytology used in the first period of this study . Thus, obtaining the definite diagnosis by a tru - cut biopsy seems to be essential in all patients considered for this strategy . The optimal total and boost doses as well as bt dose rate, have not yet been determined in bct . In this series, 15 gy boost dose and 1 gy / pulse / h, followed by 50 gy or 40.05 gy and 42.5 gy (2 gy or 2.67 and 2.5 gy daily dose, respectively) to the whole breast were used . Others administered 20 - 25 gy as the pdr boost following breast 50 gy ebrt [4, 5]. These authors found that the 25 gy boost dose is associated with a significantly higher rate of late toxicity compared to 20 gy . Others reported poorer cosmetic outcome in patients boosted with dose rates above 1 gy / h . Until recently, in patients managed with bct, the standard wbrt dose was 50 gy . The boost dose was used in 43 - 75% of patients enrolled in three out of four randomized trials of hypofractionated radiotherapy as a part of bct in early breast cancer patients . Immediate catheter placement reduces the target volume; however, its determination remains subjective . In our series, this may allow for better cosmetic outcome but at the expense of potentially increased risk of local recurrence ., in a series of patients with high risk of recurrence, reported the mean pdr boost volume of 57 cc . In the study by resch et al ., in which 60% of patients underwent quadrantectomy, 18% wide excision and 22% tumorectomy, the average volume for the prescribed dose was 83 cc . Of note, in that study, the type of surgery did not impact the local control . Notably, excellent local control (1.6% true local recurrence at 10 years for patients after quadrantectomy, 0% for wide excision, and 2.2% for tumorectomy) was accompanied by a fair cosmetic effect (excellent or good cosmetic effect in only 38% of patients). In this series, the dhi was available for two patients, and in both cases, it was 0.68 (v150 of 10.7 cc and 17.8 cc). The reported incidence of fat necrosis including symptomatic / clinically overt cases varies considerably in particular studies, and is influenced by differences in patient characteristics, treatment, duration of follow - up, and diagnostic criteria for a diagnosis of fat necrosis . No late fat necrosis was reported in the two above mentioned studies applying pdr bt boost at a dose of 15 - 25 gy following whole breast irradiation [4, 5]. The median follow - up in these series was 30 months and 60.9 months . In another small series with patients boosted with pdr bt with the median dose of 12.3 gy (range: 12.0 - 20.3 gy) (median v100 of 55.2 cc, median dhi: 0.82), in addition to 50.4 gy wbrt, the incidence of mammographically evident signs of fat necrosis was 9.0% at the median follow - up of 37.5 months, but no patient needed surgical intervention . After a median follow - up of 46 months, fat necrosis in one case (1.3%) was observed in a series with hdr - bt (median v100 of 94.49 cc) performed immediately after completing wbrt . The late toxicity data in patients treated with multicatheter interstitial hdr - bt as a form of accelerated partial breast irradiation (apbi), suggest that v150 and v200, as well as anthracycline - based chemotherapy administered after apbi may be associated with an increased risk of fat necrosis . In another apbi study, acute breast infection and anthracycline - based chemotherapy, number of catheters, v100, v150, v200, and integrated reference air - kerma were associated with fat necrosis . Of these, v150 was independent treatment - related parameter . In these studies, the mean v100 was 176 cc and 239 cc, respectively . Our series includes 17 breast cancer patients (17.7%) who were administered preoperative chemotherapy, an increasingly used strategy . In this group, secondary angiosarcoma may develop in a lymphedematous arm, in the irradiated chest wall after radical mastectomy, and following bct . This event is rare but associated with poor prognosis . In a large retrospective series of 18,115 breast cancers treated with bct, including 50 gy whole breast irradiation and a boost dose of 15 - 25 gy, post - irradiation angiosarcoma was diagnosed in only nine cases, after the median latency period of approximately 74 months . Adjuvant radiotherapy, an indispensable part of bct, is associated with the risk of second malignancy including all sarcomas and angiosarcoma . Beside radiotherapy, partial mastectomies and lymph node dissections were found to be independent risk factors for the development of angiosarcoma in breast cancer patients . Post - irradiation breast angiosarcoma, the most frequent second type of sarcoma after primary breast cancer, has been paradoxically increasingly reported since currently most women with breast cancer have long - term survival . In addition, the increasing use of intensity modulated radiation therapy (imrt) and volumetric modulated arc therapy (vmat), might be associated with a higher risk of mutagenesis . Due to the higher number of fields and monitor units, these newer techniques have been shown to have greater out - of - beam doses including higher low dose exposure of the normal structures . Whether angiosarcoma is induced by radiation or persistent edema, or our patient with ypt2n1 breast cancer underwent axillary dissection and received irradiation at a standard dose of 50 gy in 25 fractions with tangential fields to the breast and the supraclavicular region . Thus, despite the lack of clinical lymphedema, she might have had some minimal subclinical lymph stasis involving the breast . The addition of neoadjuvant chemotherapy consisting of 6 cycles of doxorubicin and docetaxel, might have also contributed to secondary malignancy in this patient . Peri - operative pdr - bt with tube implantation at the time of surgery seems to be a safe and convenient boost method allowing for good local control and satisfactory cosmetic effect . The direct visualization of the operative site during surgery allows for precise defining of the tumor bed and decreases the risk of geographical miss in determining the target volume . However, in a proportion of patients, the treatment plan should be verified after the final histology is obtained.
Plant researchers took efforts to elucidate biological roles and functioning mechanisms of phytohormones in various plant responses (1). And thus, phytohormones had been revealed to mediate a whole range of developmental processes, as well as to interact with environmental factors by extensive physiological and genetic studies . To summarize the advance made on phytohormone research in recent decades, an arabidopsis hormone database (ahd, http://ahd.cbi.pku.edu.cn) the database includes a large collection of arabidopsis hormone related genes (ahrgs), which are defined as genes involving in the biosynthesis, metabolism, transport, perception or signaling pathways of eight types of phytohormones . The ahd also integrates detailed gene information (largely manually curated) and a phenotype ontology that is developed to precisely describe myriad hormone - regulated morphological processes with standardized vocabularies in the model organism arabidopsis . Free and friendly interface is available for online access to the database, which benefits searching particular hormone related gene or retrieve bulk data by hormone category or specific phenotypic trait . Meanwhile, cross - links to ppi and kegg were also provided for molecular biochemical studies . In the past 2 years many new genes have been identified to function in plant hormone production or signaling pathways . For example the discovery of etp1/2 that regulate ein2 proteolysis filled the gap of ethylene signaling pathway (3). Meanwhile, unequivocal evidence had been accumulated to solve some long - sought questions or clarify long - standing controversies, for instance, the recent identification of pyr / pyl / pcar proteins as authentic aba receptors (4). Besides, wide - range interactions among various components lead to complicated modes of phytohormones action, thus increasing efforts using systems biology approaches have been employed in order to uncover the underlying mechanism (5). As a result, phytohormone studies have been expanded to a much wider scope . A set of microarray profiling data sets have been released by atgenexpress group (68), including the data sets derived from different developmental stages or various treatments of arabidopsis, providing a systematic view of transcriptional regulation in response to environmental or developmental factors . Meanwhile, next generation sequencing has also enabled us to analyse plant gene functions from different perspectives . On one hand, sequencing information helps us understand features of genomic sequences as well as epigenetic regulations of plants such as methylation or microrna binding on particular loci (9,10); on the other hand, as more plant species are in the sequencing pipeline, comparative genomic approaches make evolutionary studies of plant pathways available . For example the orthologs of abi3 proteins in the moss physcomitrella patens were found to be functional in drought tolerance responses (11), indicating that aba regulation is at least partly conserved between early land plants and seed plants . These approaches are highly relied on the information about genetic and biochemical interactions among various genes or components, and often utilize mathematical methods to integrate and model verified experimental data sets, leading to conceptual understanding of complex regulatory networks . One example is a recently published work using mathematical modeling to illustrate how polaris gene functions and interacts with auxin, ethylene and cytokinin in arabidopsis (12). In an attempt to cover the above - mentioned advances and extending the functionality of the previous ahd, we have updated our database from its first version to ahd2.0 . In this update version, we made tremendous modifications and improvements to the original database and added several new features to cater to nowadays interests . First, we manually updated our collection of ahrg genes based on most recent publications (the latest 2-year literatures) as well as corrected information (e.g. The arguable aba receptors). Besides, elaborate schematic diagrams of phytohormone biosynthesis and signaling pathways are constructed and provided in ahd2.0, which represents the first effort to integrate a large volume of data sets into a comprehensive regulatory network for each hormone; second, we integrated orthologs of sequenced plants in orthomcl - db (13) into each gene in the database for comparative genomic or evolution studies of phytohormone related genes; third, we predicted microrna splicing site of each gene . The sequence of each binding site is now also available for performing further analysis such as primer design; fourth, we provided the genetic relationship of these phytohormone related genes manually curated based on published literatures; fifth, for the convenience of in - time bioinformatics analysis, we provided links to a powerful online analysis platform weblab, which was recently developed by us . It allows users to readily perform various sequence analysis with these phytohormone related genes retrieved from the database; finally, we provided links to other protein databases as well as more expression profiling information that would facilitate users for a more systematic and integrative analysis for phytohormone research . Based on these newly developed features, ahd2.0 would greatly intensify and expand its capability in the systematic studies of plant hormone responses . Compared with ahd, gene entries have increased from 1026 to 1318 (figure 1a). The increased entries are all manually curated based on the recent 2-year literatures from september 2008 to august 2010 . These newly added gene entries were categorized by hormones, while the mutants related to those genes were classified by their phenotype ontology that was developed in ahd . In ahd, a number of genes were considered as ahrgs merely based on annotation from gene ontology . While in ahd2.0, with a large number of genetic studies using mutants and transgenically overexpressing or silencing lines had been incorporated to some of these genes, the number of mutants (or genes with verified genetic evidence) in the database was increased by 76% . Histogram illuminates the comparisons of gene number (a) and mutant number (b) between the two versions of the database . Color in blue represents the numbers in ahd and color in red represents the numbers in ahd2.0 . Histogram illuminates the comparisons of gene number (a) and mutant number (b) between the two versions of the database . Color in blue represents the numbers in ahd and color in red represents the numbers in ahd2.0 . Besides, we modified existed information based on the most recent publications . For instance, gcr2 and fca were classified into aba receptors in ahd, but in ahd2.0 these genes have been re - classified into aba signaling category due to the recent suspicion . Meanwhile gtg1/2 and pyr / pyl / pcar were added into ahd2.0 and were classified into aba receptors based on current studies (4,14). For epigenetic study users, we now provide predicted microrna splicing site of each gene . Prediction of the potential interactions between micrornas and genes utilized rnahybrid method as described (15). In sum, we have predicted 954 microrna splicing site targeting on hormone related genes . All details were given in the mirna interaction information for this gene section with the name of mirnas and their target genes, the length of mature mirnas and their target genes and their binding site (figure 2d). Detailed information of a given mirna is available in the cross - link to mirbase from each i d of the mirna . The information of an updated hormone - related gene that contains seven sections, including (a) response hormone(s) and basic gene information retrieved from tair, go and kegg, (b) associated mutants, (c) expression data from microarray experiements, (d) predicted mirna splicing site, (e) genetic interactions related to the gene, (f) ortholog groups annotation for the gene and (g) cross - links to other protein databases . The information of an updated hormone - related gene that contains seven sections, including (a) response hormone(s) and basic gene information retrieved from tair, go and kegg, (b) associated mutants, (c) expression data from microarray experiements, (d) predicted mirna splicing site, (e) genetic interactions related to the gene, (f) ortholog groups annotation for the gene and (g) cross - links to other protein databases . The interactions are manually curated based on literatures . In gene interaction information for this gene section we provide information including related genes, genetic relationships (represented by positive / negative regulation), regulation type, enzyme activity (if possible) and the accesses to literature evidence from pubmed i d for each interacting gene (figure 2e). Nodes to describe genes and edges to indicate their reciprocal interactions are used for modeling a regulatory network . In general, we have found a total of 911 nodes and 2608 edges derived from arhgs listed in ahd2.0 . In addition, a quick description with the schematic pathways of all eight hormones is added in the ahd2.0 introduction . By integrating all available information from literatures, we constructed the schematic diagrams for each type of phytohormones, including their biosynthesis, metabolism, transport (if applicable), perception and signal transduction pathways . The schematic diagrams of these pathways are now available from the introduction page of the database and can also be found in the left bar of the database . By clicking the name of each hormone in the table for easy access to comparative or evolution studies, we also provide ortholog groups in orthomcl - db (13) for each gene . Sequence of ortholog groups can be retrieved from the i d shown in the section ortholog groups annotation for this gene from the cross link to orthomcl - db (figure 2f). In ahd2.0, links to weblab (16) are provided in the sequence page of each gene . Weblab is a data - centric knowledge - sharing bioinformatics platform in which 279 local tools and web - services and grid - services are integrated . This platform will allow users to benefit from on - time analysis when sequence is retrieved . With submitted sequence to weblab, users can help themselves to analysis such as sequence alignment, protein 2d and 3d structure prediction and motif search . Besides, registered users can take benefit from saving and sharing data, which is our highly recommended procedure . In ahd2.0, cross - links to other protein databases are provided, which include pfam, superfamily, prosite, gene3d, prints, panther, pir, hamap, tigrfam, smart, prosite and prodom (figure 2 g). We believe that this protein information will facilitate users in predicting gene functions in the network based on domain analysis . Besides, we have integrated more expression profiling information in ahd2.0 derived from atgenexpress (68), including different developmental stages and abiotic - stress treatment profiling data sets (figure 2c). These data sets would facilitate researches to study transcriptional regulation of hormone related genes in various plant responses . To make it user friendly, the normalized expression level was shown in log2 transformation and the graphic view of gene expression data was only displayed for those genes influenced> 2-fold in at least one experiment within one data set . Compared with ahd, gene entries have increased from 1026 to 1318 (figure 1a). The increased entries are all manually curated based on the recent 2-year literatures from september 2008 to august 2010 . These newly added gene entries were categorized by hormones, while the mutants related to those genes were classified by their phenotype ontology that was developed in ahd . In ahd, a number of genes were considered as ahrgs merely based on annotation from gene ontology . While in ahd2.0, with a large number of genetic studies using mutants and transgenically overexpressing or silencing lines had been incorporated to some of these genes, the number of mutants (or genes with verified genetic evidence) in the database was increased by 76% . Histogram illuminates the comparisons of gene number (a) and mutant number (b) between the two versions of the database . Color in blue represents the numbers in ahd and color in red represents the numbers in ahd2.0 . Histogram illuminates the comparisons of gene number (a) and mutant number (b) between the two versions of the database . Color in blue represents the numbers in ahd and color in red represents the numbers in ahd2.0 . Besides, we modified existed information based on the most recent publications . For instance, gcr2 and fca were classified into aba receptors in ahd, but in ahd2.0 these genes have been re - classified into aba signaling category due to the recent suspicion . Meanwhile gtg1/2 and pyr / pyl / pcar were added into ahd2.0 and were classified into aba receptors based on current studies (4,14). For epigenetic study users, we now provide predicted microrna splicing site of each gene . Prediction of the potential interactions between micrornas and genes utilized rnahybrid method as described (15). In sum all details were given in the mirna interaction information for this gene section with the name of mirnas and their target genes, the length of mature mirnas and their target genes and their binding site (figure 2d). Detailed information of a given mirna is available in the cross - link to mirbase from each i d of the mirna . The information of an updated hormone - related gene that contains seven sections, including (a) response hormone(s) and basic gene information retrieved from tair, go and kegg, (b) associated mutants, (c) expression data from microarray experiements, (d) predicted mirna splicing site, (e) genetic interactions related to the gene, (f) ortholog groups annotation for the gene and (g) cross - links to other protein databases . The information of an updated hormone - related gene that contains seven sections, including (a) response hormone(s) and basic gene information retrieved from tair, go and kegg, (b) associated mutants, (c) expression data from microarray experiements, (d) predicted mirna splicing site, (e) genetic interactions related to the gene, (f) ortholog groups annotation for the gene and (g) cross - links to other protein databases . The interactions are manually curated based on literatures . In gene interaction information for this gene section we provide information including related genes, genetic relationships (represented by positive / negative regulation), regulation type, enzyme activity (if possible) and the accesses to literature evidence from pubmed i d for each interacting gene (figure 2e). Nodes to describe genes and edges to indicate their reciprocal interactions are used for modeling a regulatory network . In general, we have found a total of 911 nodes and 2608 edges derived from arhgs listed in ahd2.0 . In addition, a quick description with the schematic pathways of all eight hormones is added in the ahd2.0 introduction . By integrating all available information from literatures, we constructed the schematic diagrams for each type of phytohormones, including their biosynthesis, metabolism, transport (if applicable), perception and signal transduction pathways . The schematic diagrams of these pathways are now available from the introduction page of the database and can also be found in the left bar of the database . By clicking the name of each hormone in the table for easy access to comparative or evolution studies, we also provide ortholog groups in orthomcl - db (13) for each gene . Sequence of ortholog groups can be retrieved from the i d shown in the section ortholog groups annotation for this gene from the cross link to orthomcl - db (figure 2f). In ahd2.0, links to weblab (16) are provided in the sequence page of each gene . Weblab is a data - centric knowledge - sharing bioinformatics platform in which 279 local tools and web - services and grid - services are integrated . This platform will allow users to benefit from on - time analysis when sequence is retrieved . With submitted sequence to weblab, users can help themselves to analysis such as sequence alignment, protein 2d and 3d structure prediction and motif search . Besides, registered users can take benefit from saving and sharing data, which is our highly recommended procedure . In ahd2.0, cross - links to other protein databases are provided, which include pfam, superfamily, prosite, gene3d, prints, panther, pir, hamap, tigrfam, smart, prosite and prodom (figure 2 g). We believe that this protein information will facilitate users in predicting gene functions in the network based on domain analysis . Besides, we have integrated more expression profiling information in ahd2.0 derived from atgenexpress (68), including different developmental stages and abiotic - stress treatment profiling data sets (figure 2c). These data sets would facilitate researches to study transcriptional regulation of hormone related genes in various plant responses . To make it user friendly, the normalized expression level was shown in log2 transformation and the graphic view of gene expression data was only displayed for those genes influenced> 2-fold in at least one experiment within one data set . We have updated our previous version of ahd and renamed it as ahd2.0 . With the tremendous expansion of gene collection and correction, as well as newly developed features and web tools, we believe that ahd2.0 would be more efficient and powerful for the systems biology studies of plant hormone functions and mechanisms . These kinds of integrative research would allow a better understanding of the phytohormone regulatory network in myriad plant responses and eventually lead to the proficient manipulation of phytohormone action to improve plant fitness, defense and crop yield . Comparative or evolutionary studies are powerful methods in uncovering essential mechanisms in plant responses due to the fact that each complex network is originated from simple state and is fixed by nature selection (17). Analyzing function of ortholog genes in ancestors or related species may indicate the origin, adjustment as well as function in particular responses of a pathway . Ahd2.0 will benefit users in searching orthologs of hormone related genes and performing necessary analysis from quick access to weblab (figure 3). Based on ortholog groups retrieved from ahd2.0, multiple sequence alignment can be performed using clustal software in weblab (a). It reveals that in lower plant such as the moss physcomitrella patens (with the i d 175000002 and 120199), although ortholog proteins of atrga1 had been evolved, della motif is lacked in the moss . Della motif can be visualized by using weblogo in weblab (b). And phylogenetic tree can be constructed by fneighbor in weblab based on n - j algorism (c), which indicates systematic relationship of these ortholog genes . A case for the application of weblab . Based on ortholog groups retrieved from ahd2.0, multiple sequence alignment can be performed using clustal software in weblab (a). It reveals that in lower plant such as the moss physcomitrella patens (with the i d 175000002 and 120199), although ortholog proteins of atrga1 had been evolved, della motif is lacked in the moss . Della motif can be visualized by using weblogo in weblab (b). And phylogenetic tree can be constructed by fneighbor in weblab based on n - j algorism (c), which indicates systematic relationship of these ortholog genes . Second, statistic methods can be used in analyzing information retrieved from ahd2.0 . Take interactions between microrna and hormone related genes as an example . Based on on - line information, the majority of genes interacted with microrna are related to auxin and abscisic acid (figure 4a). Interestingly, our database and analysis revealed that 88% ethylene related genes have interactions with microrna (figure 4b), indicating that ethylene response pathways are probably extensively under small rna regulation, which is supported by previous findings (1820). Auxin and abscisic acid related genes predicted to have more mirna targets, as shown in (a). The percentages of genes predicted to have mirna binding sites in each hormone category are shown in (b), in which a red line marked out the average percentage of 72.38% auxin and abscisic acid related genes predicted to have more mirna targets, as shown in (a). The percentages of genes predicted to have mirna binding sites in each hormone category are shown in (b), in which a red line marked out the average percentage of 72.38% . In another application, genetic interactions can be used as important resources in systems biology study . Detailed schematic diagrams illustrating biosynthesis, perception as well as transduction process of phytohormones would enable phytohormone researchers to get more comprehensive view of phytohormone actions . Meanwhile, these diagram model (nodes with connections by arrowhead or perpendicular lines) can be changed into boolean networks through a simple transformation, which may facilitate for mathematical modeling by employing boolean rules and binary values as well (21,22). Based on 911 nodes and 2608 edges provided in ahd2.0, a combination of genetic interactions may enable us to draw a relatively complete regulatory network of phytohormones, which may assist systematic users performing theoretical analysis (figure 5). It should be pointed out that interactions among hormones are too intricate to be concluded by simple up or down regulations, because they are under the influence of tissue and treatment specificity . For example for the reason that some of the aba related genes are seed - specific genes, the relationship of ethylene and aba is different in drought tolerance and seed germination (23). The addition of more expression profiling data sets and the powerful on - line analysis platform are believed to be able to facilitate detailed researches . As for systems biology, conclusions could be heuristically derived when data sets from different angles are perfectly integrated . Figure 5.boolean network of phytohormone . Different color of the node represents hormone category the gene (node) belongs to, (auxin: orange, gibberellin: grey, cytokinin: yellow, abscisic acid: purple, ethylene: light green, jasmonic acid: red, salicylic acid: blue, brassinosteroid: dark green). Different color of the node represents hormone category the gene (node) belongs to, (auxin: orange, gibberellin: grey, cytokinin: yellow, abscisic acid: purple, ethylene: light green, jasmonic acid: red, salicylic acid: blue, brassinosteroid: dark green). To date, information in ahd2.0 mainly focuses on arabidopsis genes, although ortholog groups are provided to assist phytohormone studies in other plants . In the future, we aim to include more characteristics of phenotype ontology for other plant species and to integrate physiological and/or genetic studies from other species . The ultimate aim of our database is to provide an informative resource and analysis platform for phytohormone studies, not only in arabidopsis, but also in other non - model plant species as well . We believe that the new version of ahd will make a broader impact on plant biology community and cater to the demand of more users . Users are also welcome to help us in improving this database via our online platform (http://ahd.cbi.pku.edu.cn/help.php). All the useful information will be appreciated and carefully considered to be integrated into our database . National natural science foundation of china (30625003 and 30730011 to h.g . ); ministry of science and technology of china (2009cb119101 to h.g . ); ministry of education of china (ed20060047 to h.g . ). Funding for open access charge:
There are a number of open issues in the hfd mouse modeling of t2dm: will these mouse models be useful in preclinical investigations?what about the differences identified between wild type mouse strains?why some protocols do not elicit dcm?can the research community agree upon a common hfd feeding protocol? Comparisons between papers, genetic manipulations, and therapies will be much easier and more informative if a specific diet, length of diet, and analysis protocols can be agreed upon . Why some protocols do not elicit dcm? Can the research community agree upon a common hfd feeding protocol? Comparisons between papers, genetic manipulations, and therapies will be much easier and more informative if a specific diet, length of diet, and analysis protocols can be agreed upon . An updated murine hfd review is urgently required as 223 references for january 2016 were found on pubmed using high fat diet and mouse as the search term (too many to read all). The body of this review will therefore be focused upon a researcher's point of view . The goal is that researchers will use this paper to plan their hfd protocols and to create some consistency in future experimental protocols and analysis techniques . Due to differences in the literature, we will use the following convention: obesity leads to metabolic syndrome which can progress to t2 dm . In humans obesity is defined by a body mass index (bmi = weight / height) of over 30 kg / m . Progression to metabolic syndrome is diagnosed when a patient has 3 of the following 5 pathologies: obesity, hypertension, fasting hyperglycemia, elevated serum triglycerides, and decreased high - density lipoprotein . After years of the associated hyperinsulinemia the pancreas may falter and the patient will then also suffer from hypoinsulinemia . The pancreas fails to produce insulin and therefore the patient becomes hyperglycemic, but never hyperinsulinemic . Examples of knowledge and therapies which have progressed from basic knowledge to preclinical trials in mouse models are numerous (and some examples are reviewed in [35]). In the obesity and t2 dm fields, mouse models have proven invaluable in the basic science of the diseases by identifying the roles of inflammation, insulin resistance, fat content of the diet, pampk, exercise, and potential treatments . In addition, and very importantly, what has been learned from the mouse models has faithfully been carried over into the human patients . These physiological similarities between the two species are due to the genetic homology between the two species . One of the largest problems or perhaps one of the most advantageous aspects of mouse models for t2 dm are the different pathologic responses between mouse strains . Multiple publications have identified strain specific differences in hfd susceptibility (a few recent examples are [813]). The advantageous aspects of these differences lie in the pursuit of mechanisms which cause a mouse strain to be resistant to hfd pathologies and therefore provide avenues to identify targets for future therapies . However, researchers must also be aware of these phenotypic differences when making comparisons with earlier publications which have utilized different mouse strains or even different sources for a mouse strain . In the hfd field many manuscripts have identified dcm after a relatively short diet intervention [1417] while other experiments did not identify dcm after an 8-month hfd protocol or 6-month hfd protocol . Some of these disparities are easily identified to lie in specific hfd formulations or specific mouse strains being utilized or even to know which characteristics of dcm are being considered for comparison . However, some of the disparities are not so easily identified and having standardized hfd protocols will aid in identifying where the differences are arising and if they are worth pursuing for increased t2 dm mechanistic knowledge . For further insights into dcm the reader is revered to a recent review article concerning dcm in both t1 dm and t2 dm mouse models . Of further interest to researchers is that mice subjected to hfd dependably model other human conditions in addition to t2 dm . Among these other diseases are those usually related to t2 dm but not occurring in all patients or mice . Nonalcoholic fatty liver disease (nafld) and the progression of this disease to nonalcoholic steatohepatitis (nash) and hepatocellular carcinoma (hcc) are common sequela of obesity and t2 dm . These conditions are well modeled with murine long - term hfd strategies (reviewed in). Hfd has also been utilized to model chronic inflammation, which is an important pathogenic mechanism of t2 dm and many other diseases including aging . The hfd - mediated chronic inflammation is marked by increased tnf-, il-1, and il-6 in the circulation . Among the additional inflammatory diseases investigated using murine hfd are wound healing [24, 25], prostate disease, dysbiosis [2729], aging, and alzheimer's . The hfd mice also provide very good models for investigating exercise benefits and muscle regeneration . Hfd has also been shown to decrease skeletal muscle regeneration, likely through inflammation and lipotoxicity in the muscle stem cells known as satellite cells (reviewed in). Furthermore, impaired leptin signaling which occurs in hfd fed mice also decreases satellite cell proliferation . Insulin resistance occurs in many tissues, of primary importance in the skeletal muscle, liver, and heart . Using acute insulin challenge protocols described below, individual the thrifty genome theory posits that slight insulin resistance may have had some advantage when our ancestors (and the ancestors of mice in the wild) underwent times of food scarcity . This transient insulin resistant after a large meal would cause increased calorie storage for the following times of food scarcity . However, now during times of constant food overabundance in many cultures, and for the mice, the transient and slight insulin resistance has become chronic and leads to pathologies . Although recent reports do not support the theory [3840], there must be an evolutionary reason for the maintenance of loci associated with t2 dm . This is a very important idea when considering insulin sensitizing agents as therapy, which, if this theory is valid, would cause further cardiac damage as has been seen with rosiglitazone treatment [41, 42]. In either case, hfd in mice a number of recent publications and reviews have addressed the utility of the mouse model hfd protocol . These reviews have excellent details and i will therefore refer the reader to these details at appropriate sections, instead of being repetitive . Discuss the cardiac disease phenotype of murine hfd feeding and present an important time course of phenotypic progression . Islam and loots very competently present the similarities between the murine hfd model and humans suffering from t2 dm . Another review manuscript describes the use of hfd mice to identify factors involved in obesity resistance or sensitivity . In addition, a recent chapter has described the utility of mouse models for t2 dm drug discovery . In addition, a thorough discussion of insulin resistance in multiple tissues was recently published . Additional publications describe genetically engineered t2 dm mouse models, which have recently been reviewed . However, due to the many genes identified which cause t2dm more than 50 genes having been identified by human gwas studies -none of these murine genetic models can model disease etiology of more than a few patients . Many of these mice faithfully recapitulate some of the t2 dm phenotypes and can therefore be useful for preclinical trials and can be used to investigate specific portions of the phenotype . However, they do not model the disease etiology of the majority of patients . Most t2 dm humans have become sick due to their diets, not their genetics (cdc, http://www.cdc.gov/diabetes/data/statistics/faqs.html). There are many important considerations for designing a murine hfd protocol (table 1). As discussed, which mouse strain to use is also highly important to consider when designing a hfd protocol . We investigated many manuscripts and identified many different durations for diet intervention; the most commonly utilized times are listed in table 2 . Clearly this wide array of times (and diet specifics and age at start) makes interstudy comparisons very difficult . Such an early adaptation phase was noted in the hfd fed wild type mrl mouse strain after 2 and 3 weeks of hfd . The mrl mice became slightly hyperglycemic at these early weeks, which they fully recovered from by 7 weeks of diet . The earliest hfd effects we found in the literature were after 3 days of diet as an increase of pancreatic -cell proliferation . These authors also followed a time course through 11 weeks of hfd which revealed important differences between early (1 week) and late (11 weeks) results in intraperitoneal gtt and the dynamics of disease progression . Insulin tolerance also progressively worsened over the time course, while hyperinsulinemia was only apparent at 11 weeks of diet and was not seen at 1 or 5 weeks of diet . This is a great illustration that the length of time of hfd must be carefully considered depending upon which variables one is interested in investigating . Furthermore, the above - mentioned early adaptation phase seen in the mrl mice did not occur in the hfd fed c57bl/6 mice, again stressing the importance of choosing the correct mouse strain depending upon the desired investigations . Others have identified adipocyte baseline differences that is in wild type mice fed a normal diet between male and female mice in glucose metabolism . In a highly comprehensive study morselli et al . Identified that due to hfd changes in the c57bl/6 male hypothalamus the male mice were more susceptible to hfd - mediated chronic inflammation, while the female mice gained as much weight; they did not suffer from the pathogenic inflammation . Similarly, it has been identified that multiple fat deposits in c57bl/6 t female mice and castrated male mice are more insulin sensitive than those from intact male mice . It is well known that age - related chronic inflammation correlates strongly with insulin resistance . As t2 dm is still considered a disease of the elderly it is fully appropriate to analyze older mice . However, unless one is contrasting age, comparisons with younger mice must be made with caution . A great example of the variable responses of different mouse strains to a hfd was recently published . The authors compared the c57bl/6 mouse strain, containing a typical th1 leaning immune system, to the balb / c mouse strain which is a th2 leaning strain . The authors identified that the c57bl/6 mice were more susceptible to adiposity, liver inflammation, and liver fibrosis . Furthermore, slight genetic differences due to genetic drift result in significant differences between mouse substrains [13, 65, 66]. These strains were physically separated in 1951 and kept in different facilities since then . As no selection pressure was applied the current genetic differences are purely due to genetic drift . The strains are different in the severity of hfd - induced characteristics; the c57bl/6n strain has a milder phenotype . Another advanced genetic analysis was conducted on some of the bxd mouse strains (c57bl/6j and dba/2j intercrosses for 20 plus generations,). These authors identified strains that were susceptible and resistant to hippocampal dysfunction elicited by hfd . The advantage of this strategy is that genome wide association studies and additional genetic analyses will be very informative due to the complex but known sequences . A further caution must be mentioned for the variations in diet compositions particularly important in light of continued data that the type of the fat is critically important to provide protection or pathology . The complete review of diets is beyond the scope of this review; here are a few important points to consider . In the united states there are a few main suppliers of main suppliers of lab diets: teklad (http://envigo.com/), labdiet / purina (http://labdiet.com/), research diets (http://researchdiets.com/), and bio - serve (http://bio-serve.com/). In addition, teklad provides many fat additives to custom make diets with specific lipid types . Not only are the amount and type of fat important but also the grams of carbohydrate in each formulation are highly important to consider . As mentioned before a true diets high in saturated fatty acids are more obesogenic than mono- and polyunsaturated as the saturated fatty acids are inefficiently used for energy production and are therefore more readily stored . Furthermore, long - chain fatty acids (c14:0c24:0) are more obesogenic than short- and medium - chain because they are transported into the mitochondria less readily and are therefore more likely to be stored . A very interesting study regarding diet composition came from the laboratory of ezaki . These scientists balanced carbohydrate with different lipid percentages in an isocaloric diet and demonstrated a clear correlation between lipid content and hyperglycemia . As calories from lipid increased and carbohydrate calories decreased to keep total calories constant, the glucose tolerance of the mice deteriorated . Similarly, maiolo et al . Recently published comparisons of a hfd versus high sugar diet versus a diet of high fat and high sugar . The results demonstrate that the combined high fat and sugar diet presented with most severe symptoms, including hyperglycemia, hypercholesterolemia, and higher levels of inflammatory mediators and lower levels of regulatory t cells . As we are all trying to improve patient lives the diet that best approximates the true western diet (high fat and high sugar) is arguably the best diet to use, unless diet specifics are being investigated . Many researchers use a straightforward hfd in their protocols, but some utilize additional stressors to model human t2 dm . An additional stressor is a low dose streptozotocin injection to boost the disease severity and model a later more progressed stage of disease after hyperinsulinemia when hypoinsulinemia has appeared [54, 73, 74]. The dose of streptozotocin does not cause any phenotypes in chow fed mice and does not always cause dcm in hfd mice . Additionally, to compensate for the additional calories provided by the hfd, some scientists have calorie that matched the amount of hfd given to their mouse cohort [75, 76]. In this way the hfd mice are only allowed to consume the same amount of calories as the cd cohort consumed in the previous 24 hours . These variations provide very useful protocols to investigate different and specific aspects of t2 dm . Although there has been speculation that sucrose with or without fat should also cause t2 dm, this has not been found . In humans there is retrospective epidemiological evidence that sucrose is not causative to t2 dm . In mice, where the experiment can be conducted with all of the proper controls, sucrose did not alter the progression to t2 dm with or without a hfd component . Therefore a straightforward hfd may be the optimum diet to model early t2 dm in mice with an additional streptozotocin injection to model the later stages of disease when hypoinsulinemia is also present . Type 2 diabetes mellitus (t2 dm) and the murine hfd model are complex diseases with complex and overlapping pathogenic mechanisms (figure 1). The overlapping nature of these mechanisms clearly causes researchers and clinicians anxiety when thinking about prevention and treatment strategies . It is therefore clear that once t2 dm is present, additional therapeutics beyond glucose control are required . Targets for the additional therapeutics require the consideration of many t2 dm and hfd pathogenic mechanisms . It appears that endothelial cells are highly sensitive to chronic hyperglycemia most possibly due to their constant proximity to the circulation . Generally it may be that the body's fuel storage cells (primarily adipose and liver cells) adapt to hyperglycemia through decreasing levels of insulin sensitivity . It may be that what is viewed as the most pathogenic portion of the disease is actually a beneficial adaptation to hyperglycemia . This view is supported through evolutionary history during which humans were subjected to periods of feast or famine and during times of feast the extra calories needed to be stored in glycogen and fat deposits not utilized immediately for atp production . By temporarily shifting the insulin receptors to resistance the storage pathway would be enhanced . However, now we (humans and mice on a chronic hfd) no longer have the famine times and the resulting chronic hyperglycemia is toxic at many levels . At the onset of a hfd the murine adipose tissue adequately expands and stores the excess calories . However, during chronic hfd the adipose tissue can no longer store the excess calories and the excess lipids are deposited into other organs . This can lead to disruption of normal cellular processes and then also to frank lipotoxicity where the excessive lipid molecules damage cellular molecules . Lipotoxicity occurs in multiple tissues post - hfd protocols: heart, skeletal muscle, liver pancreas, and kidneys . The definition of this pathology can be considered an imbalance in lipid uptake and disposal leading to an accumulation of lipid intermediates in nonadipose cells, which causes impaired cellular function . Excessive lipids appear to cause stress responses often leading to apoptosis and replacement of functioning cells with fibroblasts and extracellular matrix . It is currently thought that healthy individuals with a varied diet and adequate exercise can adapt to their caloric source, such that eating a few hfd meals in a row will not produce any pathology . However, after chronic hfd mice and individuals become metabolically inflexible . For example, cardiac tissue often responds to hfd by increasing its carbohydrate utilizing proteins to extract as much energy from glucose as possible [16, 18]. However when the diet then switches to a normal diet the heart draws too many calories from glucose causing a pathologic excessive caloric intake . Inflammation is a central pathogenic mediator in all aspects of t2 dm and murine hfd - induced pathologies including the cardiovascular system [8082]. Immune response time courses are also required as it has been shown that continued immune responses are pathogenic in many disease models, including t2 dm . As inflammation is also associated with many age - related diseases and conditions it becomes doubly critical in the older population historically affected by t2 dm . Demonstrated that mice with a th2 leaning immune response (balb / c) are spared many of the hfd - induced pathologies, while mice leaning towards a th1 immune response are susceptible to many hfd pathologies . Infiltrating macrophages are known to be involved in pathogenesis . A major contributor to hfd - induced dcm extensive recent publications describe decreased autophagy [89, 90] and in particular mitophagy as pathogenic in t2 dm and hfd mouse models . Increasing autophagy can alleviate many of the hfd - mediated pathologies [92, 93]. This is clearly an ongoing and interesting research area in the hfd mice . In diabetic humans hyperglycemia is thought to cause mitochondrial dysfunction and fragmentation, thereby linking hyperglycemia, mitochondrial dysfunction, and temporary insulin resistance characteristics in cardiomyocytes before obvious pathology . There is significant new data that the insulin signaling cascade directly impacts cardiac mitochondrial fission and fusion (reviewed in). This data arises from a number of observations in knock - out mouse models and cultured cardiomyocytes in which disrupted mitochondrial morphology and function precede t2 dm . The beneficial effects of metformin on increasing mitochondrial function and volume are also consistent with mitochondrial pathology being pathogenic for t2 dm . Common human t2 dm and mouse hfd pathologies are as follows: weight gain.hyperglycemia.hyperinsulinemia followed by hypoinsulinemia.insulin resistance.increased fasting leptin levels.decreased fasting adiponectin levels.inflammation with increases in inflammatory cytokines and reactive oxygen species.fatty lipid droplet accumulation in multiple tissues.fibrosis in multiple tissues.increases in blood pressure, but not always seen in the mouse models . There are many assays to quantify the extent of these pathologies in the hfd mouse model (table 1). All of these assays have been described in detail, so i will just highlight some of the important considerations that may be overlooked by busy investigators and direct you to the pertinent references . Based upon my own limited experience many of these assays should be done weekly . We have noticed an adaptation phase in the first few weeks of hfd, which we now wish we had analyzed more closely instead of beginning another cohort of animals through the long and costly procedure . Therefore, all of the nonterminal procedures, weight gain, blood glucose, insulin, adiponectin, echocardiography, dexa, and blood pressure (noninvasive tail cuff), should be conducted weekly . Terminal assays that should be considered at these intervening dates are liver, skeletal muscle, and adipose deposits for weighing, histology, and immunoblot analysis . Interesting results would include identifying changes in molecules used by the tissues in attempts at adaptation . For example changes in enzymes and molecules controlling glucose and lipid metabolic rates would be of interest to investigate early in the hfd protocol . A recent chapter from baribault describes the gtt, itt, insulin secretion tests, nonesterified free fatty acid quantification, body composition, and terminal harvesting techniques . As this chapter appears in methods in molecular biology and contains protocol details we will not repeat these descriptions here . Although many of these tests appear straightforward some additional considerations are important . Animal mass can be reported as absolute or relative to cd mice or relative to beginning masses . Perhaps the most accurate is comparing the ratio of the animal's final weight to the animal's own beginning weight, such that the animal serves as its own control . However, using this method may not be the optimum method to compare between sexes or mouse strains where an absolute change would be optimal . Another consideration for many of these tests is the time of day performed and if any fasting is required . As mice are nocturnal many of their diurnal cycling hormones and activities would be opposite of those found in humans . Therefore, as with all biological experiments the time of day must be kept consistent . Multiple publications discuss the appropriate length of fasting to achieve the most accurate data for various tests . Fasting is required to minimize variation and therefore minimize the number of animals needed to uncover differences . Fasting is usually used with the basal blood glucose measurements, gtt, itt, and acute itt . These authors empirically identified 6 hours of fasting and 2 g / kg of oral glucose as the most informative assay parameters . The most often used insulin dose is 1 mu / g of humalog (e. l. lilly, indianapolis) for itt [4, 12]. These experiments require additional equipment and are not high - throughput . In brief, for a hyperinsulinemic euglycemic clamp, the animals receive a basal insulin delivery (3 mu / min / kg) which is a commonly used dose for rats and the amount of glucose required to keep the blood sugar in a certain range is quantified . These studies quantify insulin sensitivity better because they circumvent the counterregulatory responses associated with itt . Clamp techniques are often used in conjunction with labeled glucose so that individual tissues can be analyzed for glucose uptake . The most high - throughput method is simply weighing some of the various fat masses and comparing this to the animal's body mass, both measurements should be determined anyway as part of the harvesting protocol . The next upgrade is to utilize dexa measurements on all or a consistent portion of the mouse . This technique has the added bonus that it is not a terminal procedure and a time course can be constructed for each animal . This also benefits from being amenable to establish a time course, although it is the furthest from high - throughput of the three methods and may also be expensive . Human type 2 diabetes mellitus is truly a systemic, multiorgan disease and each of these organs is also negatively affected in mouse models on a hfd . The mouse organs affected each are being investigated to gain pathologic and therapeutic knowledge into the disease . The ultimate goal is to identify therapeutics which reduce all systemic and organ pathologies . In this section additional, tissue - specific analysis methods will also be listed and referenced or discussed . Extensive data identifies that adipose inflammation, including secretion of adipokines and activation of adipose resident macrophages, is pathogenic for all other tissues and organs . It is indicated that visceral white fat is the most pathogenic type because it secretes large amounts of adipocytokines such as leptin, tnf, il6, and adiponectin that can disrupt homeostasis when dysregulated due to hfd . As the visceral fat undergoes hypertrophy and perhaps hyperplasia it secretes more of these potentially unhealthy adipocytokines . Subcutaneous adipose tissue also secretes the adipocytokines but in smaller quantities so this adipose site is not as frequently studied . The adipocytokines are reliably measured by serum or tissue extract enzyme - linked immunosorbent assays (elisas). The weights of the various adipose deposits are also very important to obtain at harvest time . Relatively small amounts (0.1 mg) of brown adipose tissue were transplanted into the visceral cavity of hfd recipients . Eight weeks after surgery the recipients had significant improvements in glucose tolerance and at 12 weeks the recipients had significant improvements in insulin tolerance tests . A detailed review of brown adipose tissue physiology assessment has recently been published and will therefore not be repeated here . Adipocytes usually respond to insulin by dividing, increasing glucose uptake, and inhibiting lipolysis . When the cells become insulin resistant due to hfd the body is subjected to increased levels of hyperglycemia and lipotoxicity because of the excessive lipolysis . As skeletal muscle skeletal muscle also provides one of the best methods to cure t2 dm through increasing exercise and disposing of glucose in an insulin independent manner, thus by passing the pathology and reestablishing homeostasis . For these reasons investigating and treating the skeletal muscles of hfd mice are critically important . Skeletal muscle of hfd mice succumbs to many of the above - mentioned pathologies: insulin resistance, lipotoxicity from excessive lipid storage, and inflammation . Insulin resistance can be measured with an acute itt, fifteen minutes after an insulin bolus (1 mu / g); skeletal muscle is harvested and immunoblots are used to determine the levels of phosphorylated pakt at t and s . Insulin resistant tissues produce less pakt and this appears to be a linear relationship so that the degrees of insulin resistance can be quantified . Other downstream targets of the insulin receptors can also be monitored for insulin signaling, although specificity of insulin signaling must be considered during data analysis . The gold standard is perhaps by electron microscopy so that lipid droplet location can also be determined . Lipid droplets in the mitochondria [12, 16] versus cytoplasmically located would reveal specific pathologic mechanisms . Inflammation and specific cells of the invading immune system can be measured by histology or flow cytometry . The liver of hfd mice is negatively affected in a number of ways [21, 103]. As with many hfd - elicited diseases the pathology usually begins with patchy steatosis / fatty droplet accumulation (nonalcoholic fatty liver disease (nafld)), inflammation (pathogenic cytokines and ros release), fibrosis, hepatocyte hypertrophy and hyperplasia, more global steatosis (nonalcoholic steatohepatitis (nash)), liver mass gain, cirrhosis, and finally hepatocellular carcinoma (hcc). The usefulness of a long - term hfd to model the steps of this disease progression is discussed in a review by kanuri and bergheim . Therefore, in an insulin resistant animal or t2 dm patient the liver actually makes and secretes more glucose than normal . In an organism that is however, due to selective insulin resistance this signaling pathway is not inhibited during times of systemic insulin resistance and therefore the liver inappropriately produces lipids in response to the hyperinsulinemia in the later stages of disease . In addition, a large proportion of a health body's readily available lipid is stored in the liver; the liver is therefore highly susceptible to lipotoxicity . Of interest requires a long hfd protocol to be established; at 60 weeks of hfd still only 54% of the mice displayed hcc . However, this hfd model is still very useful in liver pathology and treatment investigations . For example, long - term (60 weeks) hfd feeding establishes nafld, including the hcc, in just over half of the hfd c57bl/6 mice . The group went on to use this hfd mouse model, at multiple hfd durations, to demonstrate the preclinical efficacy of metformin treatment in reducing hcc if the treatment starts coincident with the diet, but not if metformin treatment is begun after nafld has begun to develop . These authors also compared various time points of diet duration and treatment to elucidate the mechanisms behind metformin cancer reduction . Histology is very useful to identify the stage of disease progression and to quantify the degree of pathology . The liver is also well studied by the acute itt to quantify its level of insulin resistance . As many t2 dm patients die of cardiomyopathy without vessel disease or hypertension, investigating diabetic cardiomyopathies multiple studies have investigated the effects of hfd upon cardiac function, remodeling, and metabolism . Mice fed a hfd for 10 weeks develop myocardial insulin resistance evidenced by a downregulation of insulin receptor activity, downregulation of akt signaling, and increased fatty acid oxidation . It is exacerbated in chronic (hfd) situations and obesity when the body is overwhelmed by free fatty acids (ffa). Even though the heart metabolizes lipids for 6080% of its total basal energy it will store excess ffa and metabolites . Abnormal accumulation of nonoxidative lipid derivatives leads to increased apoptotic signaling, oxidative stress, and broad cellular dysfunction in the heart . Overwhelming evidence indicates that the type of ingested fat is critically important for its place in the pathogenic or protective spectrum (reviewed in). This is especially evident in the effects of particular fats in the diets on cardiac function . For example, a diet high in saturated fats is protective in muscular dystrophy mediated cardiomyopathy in hamsters . Rodent hfd models of t2 dm also reproduce the obesity paradox, initially identified in humans . The situation is labeled a paradox because obese patients are better at overcoming long - term damage after myocardial infarctions . In rats subjected to pressure - overload hypertrophy, those receiving hfd furthermore, mice on a hfd have less postinfarct myocardial remodeling and dysfunction compared to normal diet controls . The similarities between the human diseases and the rodent models facilitate investigations into the mechanisms of this paradox and identify if it can be leveraged for human therapeutics . For example, in a 6-month hfd study the investigators did not find any cardiac pathology by echocardiography . Alternatively for example, in a 12-week hfd study we identified cardiomyocyte hypertrophy, unsuccessful metabolic adaptations, and lipid droplet accumulation . These results indicate that caution must be used when designing hfd experiments, especially for diabetic cardiomyopathy assessments . It may be that in these long - term experiments the hearts have compensated fully and have established a new homeostasis and the pathology must be evaluated over a time course . This is an interesting point requiring further study, because such compensatory mechanisms may reveal therapeutic targets . These authors compared 8, 12, and 16 months of hfd on the c57bl/6 male hearts . They identified cardiac remodeling at all time points, which became more severe as the diet duration was extended . In the hfd group they identified functional pathology, but only after dobutamine pharmacological stress . There are a number of possible reasons for the discrepancies regarding hfd - induced dcm . Some of the reasons are easily identified: different diets (fat type is highly important, w. c. stanley), diet duration, different assays, and age of animals . Some of the harder to identify reasons are genetic drift, seasonal variation, and diurnal variation in time of assays . When designing a hfd protocol these variations each must be considered and minimized as much as possible . Cardiac pathology assessments are critically important to obtain due to the large number of t2 dm patients dying from heart failure . Blood pressure values can be quantified by either terminal (pressure volume loops (pv loops)) or survival (tail cuff) techniques . The tail cuff procedure benefits from being survival, relatively high - throughput, and inexpensive while the pv loops benefit from being more accurate and accepted by reviewers . The best possible scenario would be to perform tail cuff weekly throughout the hfd protocol and hypertrophy can occur in two distinct or overlapping characteristics at the organ level and/or cellular level . The organ level hypertrophy can be assessed with echo and heart weight to tibia length and the cellular hypertrophy can be assessed by histology methods and imagej . The hyperglycemia caused by the hfd signals the pancreas to secrete more and more insulin . Eventually the pancreas falters and hyperinsulinemia is followed by hypoinsulinemia . These changes in insulin levels are seen in patients and in mice fed a hfd . Of course, because t2 dm is a systemic disease, all cells and tissues are affected by the disease - defining hyperglycemia and lipotoxicity . Principal among these cell types would be endothelial cells, neurons, and beta cells of the pancreas . The hfd mice have proven invaluable not only for the identification of molecular pathways affected by hfd, but also in preclinical protocols to test potential therapies to reverse the condition and its many associated pathologies . As the research community identifies the most human - like hfd model the strength of using this model for preclinical protocols can only become better . Recently resveratrol (rsv, red grape extract) has come into the news as being antiaging and potentially inhibiting t2 dm symptoms . When given for the 12 weeks of hfd, rsv produced only a slight reduction of body weight and blood glucose but had a significant reduction on renal fibrosis and renal dysfunction . Other pharmacologies that have been tested in the hfd protocol include metformin, ezetimibe, acarbose, and atorvastatin . As the research community approaches the ideal hfd protocol, consistent protocols will be undeniably helpful . As stated previously this would enable ease when comparing study to study, especially important for the preclinical therapeutic trials . I will now go out on a limb and steel myself for the upcoming onslaught of detractors . I propose this following standard protocol: using c57bl/6j.feeding from 4 to 20 weeks old.using both male and female mice.using a hfd plus high fructose to mimic the human diet best . Feeding from 4 to 20 weeks old . Using both male and female mice . Using a hfd plus high fructose to mimic the human diet best . It can be also modified to study specific mouse strains, exercise, age, length of diet, and so forth . For example, it would be of great interest to compare young and old mice in a hfd protocol . Although older mice do not have the high levels of chronic inflammation as found in the aged human population, it would be of interest to establish if the older mice develop t2 dm symptoms at a shorter hfd time period.
Psychiatric and psychological morbidity has been reported in patients with genital warts . Even though, genital warts are generally not associated with severe symptoms, they have profound adverse impact on quality of life of patients . If patient with genital warts, complains of severe and unusual symptoms are often considered psychological . We report a case of giant genital warts where the patient complained of formication (insect - crawling sensation) which was subsequently found to be due to maggot infestation in the warts . A 23-year - old married woman presented with large exophytic growth arising from perineum, vulva, introitus of the vagina, and inner aspect of thighs for past 4 months [figure 1a]. Patient developed severe itching and formication in the lesions for 1 week, though she had never seen or recovered any insect . Sexual history of the patient and her husband was unremarkable . In view of giant genital wart, serum enzyme - linked immuno sorbent assay (elisa) for hiv and venereal disease research laboratory (vdrl) test for syphilis was done for both husband and the wife but were negative . (c) three months after complete excision using a radiofrequency device cutaneous examination revealed an approximately 10 5 cm hyperpigmented, pedunculated growth with verrucous surface over both the labial folds symmetrically, completely obliterating the introitus . On per speculum examination, a diagnosis of giant condylomata acuminata was made and it was thought that patient's unusual complaint about the the hpv viral genotyping using linear array (roche) showed hpv 6 and 11 . Histopathology from the warts did not show any evidence of bushke lwenstein tumor or squamous cell carcinoma . On histopathological examination hyperkeratosis, papillomatosis, koilocytosis and dilated and congested capillaries in the dermis were found . Within few days of presentation, she started complaining of paroxysms of severe perineal scratching consequent to increased sensation of crawling insects . But thinking that the disease was causing immense psychological stress hence leading to the strange symptoms, serial excision of the warts with a radiofrequency device (ellman, oceanside, ny, usa) was planned . During the procedure, local anaesthetic (2% lignocaine with adrenaline) was infiltrated in the lesion and the procedure was started . Suddenly, perhaps due to irritation caused by radiofrequency current, multiple live maggots were seen coming out of verrucous masses [figure 1b]. The procedure was abandoned and thick white petrolatum was applied to the area . Approximately 20 live maggots were then removed with the help of forceps [figure 2] and few of them were sent for entomological examination . The patient was given intravenous antibiotics (ceftriaxone and amikacin) in view of some denuded area produced due to radiofrequency excision of few warts . A magnetic resonance imaging (mri) of abdomen and pelvis was done to rule out deeper soft tissue infestation and damage by the maggots which turned out to be normal . Patient's symptoms completely resolved with few more sessions of extraction of maggots over the next 2 days and subsequent examination did not reveal any maggots . Some of the removed maggots later, external genital warts were completely excised with radiofrequency [figure 1c]. There was no recurrence of excised warts in 4-month post - operative follow - up . Warts inside the vagina are being treated with imiquimod and at the time of writing this report, they have reduced substantially in size and this treatment is being continued . Myiasis is a parasitic infestation of human or animal skin, necrotic tissues and natural cavities by fly larvae or pupa . Human myiasis is caused by fly larvae capable of penetrating body orifices as well as healthy or necrotic tissue . Patton divided myiasis - causing flies into three parasitologic categories: obligatory, facultative, and accidental . Cochliomyia hominivorax, chrysomya bezziana, and wohlfahrtia magnifica are the most common flies worldwide that cause human wound myiasis . Chrysomia bezziana is the most common cause of cutaneous myiasis in india but usually infests wounds and mucous membranes . Predisposing factors for human wound myiasis include open wounds, poor hygiene, advanced age, psychiatric illness, diabetes, vascular occlusive disease, and physical handicap . The clinical manifestations of wound myiasis vary according to the affected body area and the extent of the infestation . Patients may present with fever, pain, bleeding from the infested site or may be complicated by secondary infection and tetanus . As far as ascertained, there is only one report in literature on myiasis in genital warts from brazil in which the patient was pregnant and after removal of maggots the pregnancy ended in spontaneous abortion . Myiasis in our case was unusual in view of its site and its occurrence in the absence of the usual risk factors . Due to rarity of condition, and our unfamiliarity we did not consider infestation by maggots though patient complained of crawling sensation and severe itching in the genital warts . The lesson learnt is to examine and investigate the patient thoroughly before dismissing patient's complaints as irrelevant, psychogenic or functional, however unusual and unexplainable they are for the disease . The simplest treatment for myiasis is application of an occlusive agent such as white soft paraffin, wax, glue, adhesive tape or chewing gum followed by physical removal of maggots.
A packet was sent to the clinical microbiology laboratories of the 77 hospitals in the area . It included a letter describing the project, a questionnaire on hospital characteristics and laboratory testing methods, and a request for existing antibiograms from the most recent period for which completed data were available . The numbers of isolates tested and number of susceptible isolates were added for each antimicrobial agent from all hospitals for each region (table a1) and for all regions combined . Data from 10 hospitals were excluded, 7 because the aggregated antibiograms did not include the number of isolates tested and 3 because the antibiogram data predated january 2001 . Thirty - one hospitals that were included in the final analysis represented the 4 regions as follows: 16 (42%) of 38 hospitals in the central region, 6 (43%) of 14 in the west, 4 (40%) of 10 in the south, and 5 (33%) of 15 in the southwest . Of the hospitals included, 16% did not send a cumulative antibiogram but instead sent their data as a monthly report for a period from 3 months to 1 year . The proposed guidelines for analyzing and presenting cumulative antimicrobial susceptibility data were published by the clinical and laboratory standards institute (formerly nccls) in 2002 . The m39-a document provides a standardized means of data extraction for all drugs tested and outlines the most appropriate way to present the data (2). In our discussions with laboratory personnel, we found that many laboratories are unaware of these guidelines, and laboratories that use the document find that adhering to all recommendations is difficult . Many laboratories lack a microbiology supervisor with insight into the clinical relevance of the results they generate . For example, a laboratory reported 4% vancomycin resistance in streptococcus pneumoniae, but the laboratory staff was not able to explain this finding or recognize the clinical implications . Also 2 of the hospitals reported 2 vancomycin - intermediate staphylococcus aureus in their antibiogram . However, the isolates were not available for verification, and the laboratory staff was not aware of the implications of this finding . The staff did not know that such findings should be reported to the illinois department of public health and the centers for disease control and prevention . In all regions, coagulase - negative staphylococci, pseudomonas aeruginosa, and enterococcus faecalis were among the 5 most frequently reported species (tables a1 and a2). The 10 most frequently reported species in our study are generally comparable to those found in the sentry survey conducted by pfaller et al . Isolates tested in the central, west, south, and southwest regions, 27%, 53%, 34%, and 42%, respectively, were resistant to methicillin . Of the hospitals that reported speciation of enterococci, e. faecalis was susceptible to vancomycin at 91%99% . However, hospitals from the southwest area reported enterococci other than e. faecalis as enterococcus spp . Only . The unusually low susceptibility of e. faecium in our study may be attributed to specimen duplication . In the central illinois region, the susceptibility of s. pneumoniae to penicillin was 64%75%, and 52%77% of isolates were susceptible to erythromycin . The susceptibility of common gram - positive bacteria in our study appears to be lower than reported national averages (3). Although antibiogram surveillance and active surveillance yield comparable results (4), national data may not be directly comparable to our findings because national data used for comparison results from active surveillance with different reporting periods . In spite of expertise and resources available in the united states, the use of antimicrobial drugs in day - to - day practice is suboptimal and directly responsible for multidrug resistance in a number of common pathogens . The factor that converts antimicrobial therapy from " empiric " to " rational " is in vitro susceptibility testing and reporting . However, if these tests are either not conducted or conducted poorly, they are not useful clinically and may create a false sense that therapy is rationally guided . Given the differences and shortcomings we reported among laboratories in a region, national recommendations are either unknown or not followed . Use of expertise, cooperation, and collaboration at the regional levels may be the simplest and most useful public health measures to optimize the usefulness of diagnostic microbiology in managing infectious diseases . Antimicrobial drug use guidelines, if they are based on consistent, reproducible, and comparable data between different laboratories, will produce better outcomes . A master antibiogram for a region would allow a tertiary care institution to consider resistance patterns in hospitals referring patients and to select appropriate " presumptive " antimicrobial therapy or change drugs in nonresponding patients . We hope that the concept of " empiric antimicrobial therapy " would be changed to that of " presumptive antimicrobial therapy " based on host factors, common pathogens, and known susceptibility patterns in any given region . This study has helped us identify serious shortcomings in susceptibility testing methods and reporting, and we hope to address these issues through a regional advisory group . Even if following all the recommendations in m39-a are not possible, the second best option may be to have all regional laboratories adhere to the same subset of recommendations . Antimicrobial resistance data generated by this approach will have better day - to - day application than will data generated by large national databases . The data will also be useful in monitoring resistance trends in a region over time and assessing the effects of interventions to reduce antimicrobial resistance . We recognize the shortcomings of the data presented in this article but believe them to be the basis for improvement at a fundamental level.
Midaortic syndrome (mas) is a rare disease characterized by luminal narrowing of the abdominal aorta, and involves stenosis of the major branches in the abdomen . In 1963, the first paper was published describing the narrowing of the descending aorta as mas, unlike takayasu's syndrome that involves the aortic arch and leriche syndrome that involves aortic bifurcation1). Clinical manifestations typically include malignant hypertension, claudication, or abdominal angina in adolescents and young adults . In children, hypertension is an undervalued problem, which can be fatal or lead to a life - threatening emergency if untreated . Hypertension with mas is severe and often unresponsive to several antihypertensive drugs, ultimately requiring surgical intervention . Here, we report a case of a 3-year - old boy presenting with left ventricular hypertrophy, who was diagnosed with idiopathic mas following identification of severely elevated blood pressure (bp). An echocardiogram showed mild left ventricle (lv) hypertrophy, and he was referred to seoul national university children's hospital for further management . Transthoracic echocardiogram revealed mild lv hypertrophy with increased lv mass index (89 g / m or 52 g / m, fig . Even though he did not complain of any symptoms at the time, we administered carvedilol 3.125 mg every 12 hours for lv reverse remodeling . During a routine follow - up echocardiogram 9 months later, we found abnormally increased celiac artery velocity with a diastolic tail (peak velocity of greater than 4 m / sec, fig . He was hospitalized for evaluation of renovascular hypertension and bp management at 4 years of age . On admission the patient's body weight was 21.2 kg (> 97th percentile) and height was 110.1 cm (> 97th percentile). His bp was high (206/113 mmhg in the right arm and 194/104 mmhg in the right leg). A chest x - ray showed no significant cardiomegaly and a 12-lead electrocardiogram revealed lv hypertrophy (fig . Laboratory findings on admission were as followings: serum blood urea nitrogen, 17.0 mg / dl; serum creatinine, 0.53 mg / dl; serum renin activity, 21.0 ng / ml / h (reference value, <6.7 ng / ml / hr); and serum aldosterone, 39.4 ng / dl (reference value, 335 ng / dl). Renal artery doppler ultrasonography showed an intermittent tardus parvus spectrogram in both renal arteries with a smaller right kidney (fig . 3a). Under the impression of renovascular hypertension, we performed computed tomographic (ct) angiography of the abdomen . The ct angiography revealed severe stenosis at the bilateral renal artery, the ostium of the celiac branch, and the ostium of the superior mesenteric artery with diffuse luminal narrowing of the abdominal aorta into the femoral artery (fig . We prescribed labetalol continuous infusion, titrated up to 3 mg / kg / hr and administered concomitant oral antihypertensive medications (amlodipine 2.5 mg twice a day and atenolol 5 mg twice a day) to decrease the bp . Percutaneous transluminal renal angioplasty (ptra) was planned and abdominal aortogram confirmed diffuse luminal narrowing of the abdominal aorta into the femoral artery (fig . Ptra was attempted with a 320 mm savvy balloon catheter via the right femoral artery; however, the effect was unsatisfactory because of the resistance to the balloon dilatation . He was discharged with combinations of 3 oral antihypertensive drugs (amlodipine 2.5 mg twice a day and atenolol 12.5 mg once a day and enalapril 2.5 mg twice a day). At the time of discharge, bp was still high and measured as 134/60 mmhg in the right arm . Since three years after first admission, he has been followed up at the outpatient clinic without any complications and is taking 3 combinations of antihypertensive drugs (amlodipine 5 mg twice a day, atenolol 12.5 mg once a day and enalapril 1.25 mg once a day). We described a 3-year - old boy presenting with lv hypertrophy who was diagnosed with idiopathic mas following identification of severely elevated bp . To the best of our knowledge, mas is prone to be referred to as hypoplasia or coarctation of the descending aorta, which presents with symptoms such as headache, claudication, and postprandial abdominal pain . Severe hypertension has been known to be the most common presentation leading to congestive heart failure, renal failure and severe lower extremities claudication7). Previous studies have reported that mas is commonly diagnosed within the first 3 decades89). Recently, the intensive school healthy screening system substantially aids the earlier detection in developed countries, although the patient has had no specific symptoms . Moreover, the widespread use of radiological modalities such as ct or aortography has led to the better recognition of mas . The etiology of mas is not completely understood, and it can be congenital or acquired . Congenital mas is a developmental defect that is caused by both incomplete fusion and overfusion of the paired embryonic dorsal aorta1011). The most common location of stenosis is the proximal renal artery (up to 80%), and at least 25% of cases may occur in the superior mesenteric artery91112). Acquired mas is caused by takayasu's arteritis, neurofibromatosis, fibromuscular dysplasia, retroperitoneal fibrosis, mucopolysaccharidosis, and williams syndrome13). Severe complications such as congestive heart failure, cerebrovascular disease, and hypertensive encephalopathy present frequently and may be fatal . The response to antihypertensive drugs is known to be poor in hypertension with mas and several previous studies have shown vascular surgery to be the best curative therapy14). As experience with ptra grows, however, the ptra success rate decreased with mas compared with isolated renal artery stenosis because of the extensive stenotic nature of the aorta and renal branches15), even though ptra is successful in reducing hypertension by using different technical approaches in the arteries from one study3). In conclusion, we report a 3-year - old boy presenting with left ventricular hypertrophy who was diagnosed with congenital mas following the identification of severely elevated bp . Clinical suspicion and accurate examination, including bp measurement, would aid early diagnosis of mas and optimal treatment, and are important to prevent the fatal complications from hypertension.
Pseudomonas aeruginosa has become the most common gram - negative bacterial species associated with serious hospital - acquired infections [1, 2].mortality rates outbreaks from p. aeruginosa in burn units can be significantly high (60%).immediate use of effective antimicrobial therapy for p. aeruginosa bacteremia has been shown to significantly reduce mortality . However, the intrinsic resistance of p. aeruginosa to many antimicrobial agents and, in addition, the variety of their increasingly recognized acquired resistance mechanisms make their management in the hospital setting problematic . Of great concern is the growing emergence of multidrug - resistant (mdr) strains of p. aeruginosa [69], and the severity of associated infections . Unfortunately, the development of new antibiotics with activity against gram - negative organisms has not kept pace with the increase in prevalence of multidrug resistant pathogens . To provide clinicians with credible alternative treatments to reduce the increasing human mortality and morbidity associated with the infectious diseases by drug - resistant bacterial pathogens, there is a compelling need to develop new therapeutic agents that are effective against drug - resistant mutants . Structurally, nubiotics are synthetic nucleotides and oligonucleotides with nuclease - resistant backbones, and are fully protonated . Although the exact mechanisms of antibiotic actions for nubiotics are not totally understood, they are thought to be indeed different from that of the traditional antimicrobial drugs and the protonated structures are believed, in part, to make nubiotics to be taken up by bacterial cells more easily . As a family, nubiotics has many members, including nu-2, nu-3, nu-4, and nu-5, which have been shown to have effective therapeutic effect in the burn wound pseudomonas aeruginosa infection model established in mice . Nu-3 [butyl - phosphate-5-thymidine-3-phosphate - butyl] is a potent nubiotics that is a fully protonated deoxynucleotide . The therapeutic efficacy of nu-3 against pulmonary pseudomonas aeruginosa infection was evaluated in vivo animal model, in which liposome - encapsulated nu-3 presented efficacious antibacterial activity . To further assess nu-3 as a potential new antibiotic for topical uses, we performed studies for its acute, multiple - dose dermal toxicity and the irritation to skin and eyes and genetic toxicity . These studies have revealed that nu-3 has hypotoxicity at high dosage, and it has no chronic toxicity and irritation . This study provides solid data to warrant further development of nubiotics as a novel class of antimicrobial agents for treating pseudomonas aeruginosa infection . Balb / c mice and sprague - dawley rats used in this study were purchased from beijing vital river company . The body weight ranged from 1720 g for mice and 250300 g for rats . All the test animals were randomized into groups after being quarantined for 35 days before experiments . Randomly, the body weight variation was within 20% of the mean weight in each sex . All the mice and rats were housed in a room with a temperature of 2025c, a relative humidity of 40%70%, and a 12-hour - light / dark cycle . The use and care of the animals followed the regulations for the administration of affairs concerning experimental animals in china (11 - 14 - 1988). Chinese hamster ovary cells (cho and cho - k1) were purchased from cell culture center, peking union medical university . Cho - k1 cells were grown in dmem / f-12 supplemented with 10% fetal bovine serum (fbs), 2 mm l - glutamine, and penicillin - streptomycin (100 units / ml, 100 g / ml). Mice were intragastrically administered with a total dose of 2500, 1988, 1580, 1256, and 1000 mg / kg / mouse of nu-3 in saline (table 1). Mice were fasted 24 h before given the test materials and were allowed to get food and water adlibitum after intragastric administration . Animals were observed for their death and subjected to a gross necropsy immediately after they died until day 8, after which the survivor were euthanized and subjected to a gross necropsy and histopathology observation for toxicity . Livers were removed and fixed by 4% neutral formalin at room temperature for 48 h. the serial tissue sections at 5-m - thick were obtained after embedded in paraffin . Each slide was stained with hematoxylin and eosin (h&e) then examined under light microscopy (olympus cx31). Throughout the study, the clinical symptoms, neurological behavior and body weight were recorded daily . Three mice of control, male and female group were sacrificed and their lymphocyte suspensions were prepared from the spleen on 24 h after being intragastrically administered with a single dose of nu-3 at 100 mg / kg / mouse . Single - lymphocyte suspensions were incubated in triplicates in 96-well plates at 2 10 cells / well, in rpmi-1640 plus 5% of fetal calf serum (fcs) at 37c in a 5% co2 incubator and stimulated for 48 h with 2 g / ml precoated anti - cd3 and 1 g / ml anti - cd28 (positive control), 2 g / ml of bsa (irrelevant antigen), or no antigen (negative control). T - cell proliferation was evaluated by cell titer 96 aqueous nonradioactive cell proliferation assay was performed according to the manufacturer's instruction (promega, usa). The mixture of mts / pms (20 l each well) was added to each well to develop the color . After 4 h of incubation, the od values of plates were read at 490 nm by a plate reader (magellan, tecan austria gmbh). Data were expressed as stimulation index (si), calculated as the mean reading of triplicate wells of antigen stimulation, and divided by the mean reading of triplicate wells of negative control . There were ten rats in the treatment group (5 males; 5 females) in which rats were treated with 50 l of 100 mg / ml nu-3 in saline . Fur was clipped from dorsal area of the trunk 24 h prior to the test and was reclipped as necessary to allow observation and dosing . The test materials were applied on the shaved area and kept in contact with the help of nonirritating styptic plaster and porous gauze bandage . To ensure that local application of nu-3 remained in the dorsum of animals, the measure of fastening multiply was taken . Briefly, a band - aid was put on the liniment area, and then covered a pad of medical gauze, followed by wrapping up with gauze bandage . Rats were treated with the test materials for 6 h per day . At the end of 6 hours, any residual material was gently removed from the treatment site using cotton bud soaked in distilled water . Cage side observation was made daily, which included evaluation of the effect on skin, fur, eyes, and respiratory and neural behavior . By the 29th day half of the rats the remaining animals were observed for further 14 days, and then they were euthanized, subjected to a gross necropsy and organ weights recorded on day 43 . During the study, body weight, ten rats were used in the test . On the day prior to dosing, an area on the dorsal surface of the skin was clipped free of hair . On the day of dosing, then 10 mg / ml nu-3 in saline was applied to the shaved area of five rats and the skin was covered with nonirritating styptic plaster and porous gauze bandage . After 6 h of exposure period, the coverings were removed and the test area was rinsed with distilled water . The rats were examined for the presence of erythema and oedema according to a skin irritation scoring standard at the intervals of 1, 24, 48, and 72 h . Rat eye irritation test was used to evaluate the degree to the mucous irritation of nu-3 . Six rats were used for this test and each animal served as its own control . 0.03 ml nu-3 of 1 mg / ml concentration with saline was dripped to the left eyes of the rats, and 0.03 ml saline was dripped to the right eyes of the rats as control . All the measures were taken to ensure that the test materials are dripped into the conjunctival sac of the rats . The rats were examined for the presence of changes in eyes according to the eye irritation scoring standard at grading intervals of 1, 4, 24, 48, 72 and 96 h. then sum of scores calculated was classified according to the eye irritation scoring standard . The purpose of this study was to assess the ability of nu-3 to induce mutations at the hgprt locus in cultured mammalian cells . Cultures were purged of preexisting mutants by growth for 2 days in dmem / f-12 medium supplemented with 10% fbs, 5 10 m thymidine, 1 10 m hypoxanthine, and 3.2 10 m aminopterin . The test was performed according to the procedure of o'neill and hsie and o'neill et al . The cells which were purged of preexisting mutants were treated for 4 h with the test and control materials and then allowed an expression time of 79 days; the cells were then replated . After the period of expression of the mutant phenotype, mutant colonies were selected in the following manner . Each culture flask was trypsinized and the cells were seeded in new flasks as follows: (a) 3 flasks each with 200 cells and without the purine analogue 6-thioguanine; (b) 3 flasks each with 2 10 cells and with 6-thioguanine (10 g / ml). After 8 days, selection was determined by the formation of colonies resistant to the purine analogue . Next, the colonies were fixed with methanol for 15 min and stained with giemsa (sigma) for 30 min . Nu-3 was evaluated in the presence and absence of a metabolic activation system derived from rat liver (s9). Dimethylbenzanthracene was used in the positive control for the activated assay and ethyl methanesulfonate was used in the positive control for the nonactivated assay . The nu-3 concentrations for the assay ranged from 500 to 2000 g / ml . The colony counts obtained were used to calculate: (a) cloning efficiency (ce) in absolute values (number of colonies formed divided by the number of cells seeded); (b) the number of mutant colonies observed in each treatment; (c) the absolute ce observed after selection; (d) the mutation frequency (mf) (number of mutant colonies divided by the number of clonable cells which was equal to the number of cells seeded the absolute ce after selection). Nu-3 was evaluated for its ability to induce micronuclei in the bone marrow of balb / c mice according to hayashi et al . . Cyclophosphamide (pc) was used as the positive control, and saline was the negative control . Groups of five male and five female balb / c mice were dosed with nu-3 at 250, 500, and 1000 mg / kg on 2 consecutive days via intragastric administration . All the mice were euthanized 6 h after the last dosing, and the bone marrow was taken to make smear . Bone marrow cells from animals were analyzed for the number of polychromatic erythrocytes (pces) that contained at least one micronucleus . A minimum of 2000 pces was analyzed for the negative control, the positive control and for mice treated with the nu-3 . The pce fraction was determined by counting a minimum of 200 erythrocytes (pces plus normochromatic erythrocytes [nces]). Data were expressed as means sd and subjected to one - way anova with factors of treatment, genotype or wild type . Mice were intragastrically administered with a single dose of nu-3 at 2500 (group 1), 1988 (group 2), 1580 (group 3), 1256 (group 4), and 1000 (group 5) mg / kg / mouse (table 1). Following the single dose, five male and four female mice in group 1, four female in group 2 died in 24 hours . One female in group 2 and one female in group 4 died on day 1 . From day 2 to day 8 none of the mice in all the groups died . According to the mortality of each group, the minimum lethal dose (mld) was calculated to be 1256 mg / kg . Clinical observations of the dying mice attributed to the administration of nu-3 were lethargy, tachypnea, and tremor and disheveled clothing hair . There were no treatment - related effects on mouse behavior in the cage and body weight for males or females (data not shown). In the early dead mice, some pathological changes were found, including livers turning white and brittle with some yellowish brown nidi, the change of the spleen surface into uneven with some hemorrhagic spots, and kidneys showing some gray - white focus on surfaces . It was also observed that there were some damages and hemorrhage in gastric walls and intestinal tracts of mice which died in 5 hours after being administered . There was no obvious pathological change at gross necropsy and histopathological changes in liver for scheduled euthanasia mice in the study (figure 2). To examine if the administration of nu-3 could affect on immune system, the spleen cells were used to test t - cell proliferation . As shown in figure 3, the treatment with nu-3 had no impact on proliferation of spleen t - cells stimulated with either anti - cd28 (positive control) or bsa (irrelevant antigen) compared with the cells from the nontreated mice . Rats were applied with 50 l of 100 mg / ml nu-3 (total dose of 250 mg / kg / day) for 28 days . There were no evident toxic symptoms in both treated and control rats during observation time . Control and test rats showed a similar pattern of weight growth, food and water uptake (figure 4). Although there seemed to be some lagging in water uptake in treated group compared to control (figure 4(b)), the body weight of test group seemed to increase faster (figure 4(a)). There were no treatment - related changes on food uptake in treated rats compared to the controls (figure 4(c)). During the observation time after administrating nu-3 on the back of the body for 28 days, there were no significant differences between the treated and control rats in the body weight gain although the treated rats took less water and food compared to the controls . No obvious pathological changes were found in all organs by gross appearance . Moreover, there was no significant difference (p>.05) in the organ weight between the test groups and the control group (table 2). The results of histopathological analysis of necropsy samples on liver, kidney, spleen and skin of the body back where nu-3 was applied showed no significant pathological changes and toxic effects except that there was a slight increase in skin labrocytes of the treated rats in comparison with the controls' . Hematology and selected serum chemistry analyses also showed no significant differences (p>.05) between control and treated groups (table 3). After applied with nu-3 on the dorsal surface, the rats were examined for the presence of erythema and oedema on the test area according to a skin irritation scoring standard . The results showed that the scores for erythema and oedema were 0 at 1, 24, 48, and 72 h after administration, which was the same to the controls' . The eye irritation scores were recorded according to the eye irritation scoring standard at 1, 4, 24, 48, 72, and 96 h after administration, all of which were shown to be 0 . Therefore, according to the skin and eye irritation scoring standard, all the rats didn't show any toxic symptom after being given nu-3 . The treated skins and eyes were normal compared to the controls . The mutation frequency was assessed based on the number of mutants per 1 10 clonable cells (table 4). In the activated assay, the mean mutation frequency was 3.3 for the saline negative control, 244 for the dimethylbenzanthracene positive control, 5.4 for the 500 g / ml concentration of nu-3; 4.5 for the 1000 g / ml concentration of nu-3 and 6.9 for the 2000 g / ml concentration of nu-3 . In the nonactivated assay, the mean mutation frequency was 3.3 for the saline negative control; 187 for the ethylmethanesulfonate positive control, 4.5 for the 500 g / ml concentration of nu-3, 5.7 for the 1000 g / ml concentration of nu-3 and 8.3 for the 2000 g / ml concentration of nu-3 . These data suggested that nu-3 was not mutagenic in the testing system both in the activated and nonactivated assays in the dose range from 01000 g / ml . However, at a very high concentration (2000 g / ml) there appeared to have an increase in mutation frequency, which suggested a weak positive response . The percentage of pce and nce of the erythrocytic lines in the nu-3-treated groups were not significantly different from that in the control group . There was a significant difference in the numbers of micronucleated pce between the positive control group and all nu-3 treated groups (p <.01) and between the high dosing group and the negative control (p <.03). However, there was no significant difference between the two low - dosing groups and the negative control group (p>.05). In all the test groups, the pce numbers were almost same between the males and females . These results indicated that at low and medium dosages, nu-3 caused no evident cytotoxicity . At a very high dose (1000 mg / kg), an increase in the pces with absolute values outside the normal control levels (i.e., 0.3%0.4%) was observed, suggestive of a weak genotoxic potential for nu-3 . Antibiotic drug development has failed to keep pace with the numbers of emerging antibiotic resistant strains and variants . Consequently, the past decade has seen the emergence of the widely publicized health crisis where the means to combat drug - resistant, plague bacteria and viral pandemics are failing . Nu-3 is a novel small molecule and has been examined in several studies for its antibacterial and therapeutic action, showing that it was as effective as intravenously administered ciprofloxacin, a potent and broadspectrum fluoroquinolone ., we reported that nu-3 is hypotoxical by intragastric administration . In the repeated dermal toxicity test the irritation study on cutis and mucosa revealed that nu-3 did not irritate the skin tissue . Further studies demonstrated that nu-3 had no notable genetic toxicity both in vitro and in vivo . Taken together, our data suggested that nu-3 is a well tolerated and exhibited no toxicity when administrated via intragastric and topical routes . Chemically, nu-3 is a modified nucleotide, which could potentially cause genetic mutations in the target tissues through disrupting dna or rna replication / transcription . To examine if this is the case for nu-3, the golden standard assays in cells and animals were performed and found no meaningful mutational effect on the testing subjects . It is unlikely that nu-3 could compete with dttp in the cellular environment to be incorporated into dna during replication . Second, although the antibacterial activities of nu-3 have been demonstrated both in vitro and in vivo, its mechanism of action is still not clear . One of the possible mechanisms for nu-3 action has recently been proposed, which was supported by the preliminary data showing that nu-3 caused significant changes of the membrane polarity . Lastly, the pharmacological data indicated that the bioavailability and pk profile of nu-3 were similar to those from other nucleoside drugs, which supports the notion of its hypotoxicity for pharmaceutical use (unpublished data). Thus, we suggest that the lack of genotoxicity of nu-3 observed in this study may be due to its unique feature of the mechanisms of action.
According to recent estimates, developing countries in asia and in the middle east will have the largest increase in the prevalence of diabetes by 2030 (1). The prevalence of type 2 diabetes (t2d) is reported to be more than 14% in tehran, iran, with an estimated incidence of new cases in about 1% of population per year (2). There is a strong genetic predisposition but the risk is greatly increased when companied with lifestyle factors such as overweight or obesity, high blood pressure, insufficient physical activity, poor diet and visceral fat . There are some characteristics such as hyper - glycemia, insulin resistance, hyperlipidemia, oxidative stress and inflammation attributed to dm (3). Among these characteristics, hyperglycemia may lead to oxidative stress which can cause cellular damage, pancreatic beta cell dysfunction and insulin resistance (4). Oxidative stress (os) plays a critical role in development and progression of diabetes and complications such as atherosclerosis, coronary heart and renal diseases (3). Vitamin e and c supplementation may improve oxidative stress and glycemic control in t2 dm patients (58). Royal jelly (rj) is a bee product, secreted from the hypopharingeal and mandibular glands of worker bees . Rj produced by honey bees is known to contain three major nutrients including amino acids, vitamins and minerals (3). Additionally, rj has various biological activities such as a hypotensive effect, insulin - like action and antitumor activity (9). Therefore, it is possible that rj may have some effects on insulin resistance, which is considered to be the major cause of dm . In a study, supplementation of rats with different doses of royal jelly (10, 30, and 300 mg / kg) led to a decrease in systolic blood pressure and significantly decreased serum levels of insulin and the homeostasis model assessment ratio, an index of insulin resistance (10). Only one human study reported that 1000 mg royal jelly for 8 weeks reduced malondialdehyde, glucose and hba1c but this dose was not sufficient to influence antioxidant status, on the other hand this study had some limitation especially it was done only in women and insulin resistant as the most important deleterious factor in diabetes, was not assessed (11). Although several animal studies have showed the effects of royal jelly on insulin resistance or oxidative stress, to our knowledge, human studies with respect to insulin resistance as a main cause of diabetes in both sexes is lacking . We hypothesized that rj supplementation can improve insulin resistance and oxidative stress in type 2 diabetic patients . The randomized controlled trial (rct) was conducted in endocrine and metabolism institute of iran university of medical sciences, tehran, iran . A randomized double blind placebo - controlled trial aiming to compare royal jelly effect on insulin resistance (homa - ir), total antioxidant capacity (tac) and malondialdehyde (mda) level in type 2 the inclusion criteria were: (a) ages 2565 years, (b) bmi of 2030 kg / m2, (c) iranian ethnicity, (d) diagnosis of type 2 diabetes for 510 years (e) hba1c of 68%, and (f) receiving oral hypoglycemic drugs . The exclusion criteria were: (a) serum triglyceride level (tg)> 400 mg / dl, serum cholesterol level> 240 mg / dl (b) lactating or pregnancy, (c) diagnosis of heart, liver and renal failure, cancer, acute myocardial infarction, stroke, or serious injuries, (d) receiving vitamin or mineral supplements at least 3 months before the beginning of the study, (e) smoking / alcohol consumption (f) taking oral contraceptive or lipid lowering drugs, (g) insulin infusion, (h) allergy to royal jelly and (i) any other conditions not suitable for trial as evaluated by the physician . The study was approved by the bioethics committee of iran university of medical sciences (irct201103012709n18). Written consent forms were signed and handed back from all participants before participation . Forty - six patients were randomly assigned to receive royal jelly supplements (natural life . Frengrove co. australia) (group a) 1000 mg, 3 times daily or exactly the same placebo (glycerin) (pars minoo inc . Tehran iran) (group b) 1000 mg, 3 times daily for 8 weeks . Participants were instructed to maintain an isocaloric diet, continue their previous eating habits, and not to change their routine physical activities during the study period . Throughout the study period, subjects were directed to continue taking the same dose of any prescribed hypoglycemic agents unless hypoglycemia occurred, in which case they were directed to reduce their dose immediately . Daily food intake was obtained by a 24 hour dietary recall questionnaire and physical activity level by ipaq questionnaire in three days (two regular days and one holiday) at the beginning and end of the study . Participants were randomly assigned to one of the two groups via computer - generated numbers . Homeostasis model assessment for insulin resistance (homa - ir) (fasting glucose (mg / dl) fasting insulin (mol / ml) /405) was used as the major outcome measurement . At baseline and after 8 weeks of treatment the tac, mda and fasting insulin level were measured for both groups . Serum tac was measured by frap method (12), and serum mda was measured by spectroscopy (13). Fasting blood sugar was measured by an enzymatic method (pars azmon co. kit, tehran, iran) using liasys autoanalyzer while insulin was measured by irma method (immunotech co. kit). Height was measured with a wall - mounted stadiometer to the nearest 0.1 cm, weight was measured on a calibrated balance beam scale to the nearest 0.1 kg, and bmi was calculated according to the formula: bmi = weight/ height2 (kg / m2). Demographic data was collected during the initial anthropometric assessment . In designing the study, we considered power of 90% with a two - sided test with = 0.05 (type i error) to detect a 5% difference in serum glucose between the two groups . On the basis of sds, reported in similar studies (14), the number of subjects needed to treat to detect this difference was 20/group . Given an anticipated dropout rate of 25 percent, we set the enrollment target at 25 subjects . Statistical analyses were performed with pc spss 16 (chicago, il, usa). Normal distribution of the variables was checked by kolmogorov smirnov test; student s t test was used to test whether the differences between the mean values of the items studied in both groups were significant . The mean differences in both groups of participants were compared before and 8 weeks after the intervention were evaluated by paired t - test . The randomized controlled trial (rct) was conducted in endocrine and metabolism institute of iran university of medical sciences, tehran, iran . A randomized double blind placebo - controlled trial aiming to compare royal jelly effect on insulin resistance (homa - ir), total antioxidant capacity (tac) and malondialdehyde (mda) level in type 2 the inclusion criteria were: (a) ages 2565 years, (b) bmi of 2030 kg / m2, (c) iranian ethnicity, (d) diagnosis of type 2 diabetes for 510 years (e) hba1c of 68%, and (f) receiving oral hypoglycemic drugs . The exclusion criteria were: (a) serum triglyceride level (tg)> 400 mg / dl, serum cholesterol level> 240 mg / dl (b) lactating or pregnancy, (c) diagnosis of heart, liver and renal failure, cancer, acute myocardial infarction, stroke, or serious injuries, (d) receiving vitamin or mineral supplements at least 3 months before the beginning of the study, (e) smoking / alcohol consumption (f) taking oral contraceptive or lipid lowering drugs, (g) insulin infusion, (h) allergy to royal jelly and (i) any other conditions not suitable for trial as evaluated by the physician . The study was approved by the bioethics committee of iran university of medical sciences (irct201103012709n18). Written consent forms were signed and handed back from all participants before participation . Forty - six patients were randomly assigned to receive royal jelly supplements (natural life . Frengrove co. australia) (group a) 1000 mg, 3 times daily or exactly the same placebo (glycerin) (pars minoo inc . Tehran iran) (group b) 1000 mg, 3 times daily for 8 weeks . Participants were instructed to maintain an isocaloric diet, continue their previous eating habits, and not to change their routine physical activities during the study period . Throughout the study period, subjects were directed to continue taking the same dose of any prescribed hypoglycemic agents unless hypoglycemia occurred, in which case they were directed to reduce their dose immediately . Daily food intake was obtained by a 24 hour dietary recall questionnaire and physical activity level by ipaq questionnaire in three days (two regular days and one holiday) at the beginning and end of the study . Participants were randomly assigned to one of the two groups via computer - generated numbers . Homeostasis model assessment for insulin resistance (homa - ir) (fasting glucose (mg / dl) fasting insulin (mol / ml) /405) was used as the major outcome measurement . At baseline and after 8 weeks of treatment the tac, mda and fasting insulin level were measured for both groups . Serum tac was measured by frap method (12), and serum mda was measured by spectroscopy (13). Fasting blood sugar was measured by an enzymatic method (pars azmon co. kit, tehran, iran) using liasys autoanalyzer while insulin was measured by irma method (immunotech co. kit). Height was measured with a wall - mounted stadiometer to the nearest 0.1 cm, weight was measured on a calibrated balance beam scale to the nearest 0.1 kg, and bmi was calculated according to the formula: bmi = weight/ height2 (kg / m2). In designing the study, we considered power of 90% with a two - sided test with = 0.05 (type i error) to detect a 5% difference in serum glucose between the two groups . On the basis of sds, reported in similar studies (14), the number of subjects needed to treat to detect this difference was 20/group . Given an anticipated dropout rate of 25 percent, we set the enrollment target at 25 subjects . All data were expressed by means sd . The level of significance was determined at p<0.05 . Statistical analyses were performed with pc spss 16 (chicago, il, usa). Normal distribution of the variables was checked by kolmogorov smirnov test; student s t test was used to test whether the differences between the mean values of the items studied in both groups were significant . The mean differences in both groups of participants were compared before and 8 weeks after the intervention were evaluated by paired t - test . Among the 50 type 2 diabetic patients screened at our outpatient clinic, four patients withdrew because they did not want to continue the supplementation program . Then, 46 patients were allocated equally into groups a and b. the mean and standard deviation of demographic and nutrients intake data are shown in table 1 and 2 . There were no significant differences in age, body mass index (bmi), diabetes duration (table 1), energy and nutrient intake (table 2), in each group and between two groups before and after the intervention . The level of physical activity and lipid lowering drugs was not different between two groups at baseline . Differences between groups were evaluated by independent t- test comparison of dietary intake of some nutrients between two groups of study data are expressed as means sd . Differences between groups were evaluated by independent t- test/ sfa: saturated fatty acid, mufa: monounsaturated fatty acid, pufa: polyunsaturated fatty acid as shown in table 3, there was no significant difference in serum glucose, tac, mda and homa - ir between two groups before the intervention . However, after 8 weeks of rj supplementation, serum tac (p=0.016) increased compared with the initial values and serum glucose (p=0.006) and homa - ir (p = 0.015) decreased at the end of study in rj group compared with placebo . There was also significant decrease in mean differences of homa - ir between two groups (p = 0.023) as shown in table 3 . There was no significant difference after rj supplementation compared to before values in serum glucose, insulin levels, tac, mda and homa - ir . Effects of 8 weeks rj supplementation on serum glucose, insulin, tac, mda and homa - ir in type 2 diabetic patients data expressed as a mean standard deviation . Before, after and mean differences and p values are mentioned in the table.-/ (p value ) comparison in each group between before and after values by paired t - test / (p value ) comparison between two groups of before values and also after 8 weeks values of two groups and also comparison of mean differences by independent t - test among the 50 type 2 diabetic patients screened at our outpatient clinic, four patients withdrew because they did not want to continue the supplementation program . Then, 46 patients were allocated equally into groups a and b. the mean and standard deviation of demographic and nutrients intake data are shown in table 1 and 2 . There were no significant differences in age, body mass index (bmi), diabetes duration (table 1), energy and nutrient intake (table 2), in each group and between two groups before and after the intervention . The level of physical activity and lipid lowering drugs was not different between two groups at baseline . Differences between groups were evaluated by independent t- test comparison of dietary intake of some nutrients between two groups of study data are expressed as means sd . Differences between groups were evaluated by independent t- test/ sfa: saturated fatty acid, mufa: monounsaturated fatty acid, pufa: polyunsaturated fatty acid as shown in table 3, there was no significant difference in serum glucose, tac, mda and homa - ir between two groups before the intervention . However, after 8 weeks of rj supplementation, serum tac (p=0.016) increased compared with the initial values and serum glucose (p=0.006) and homa - ir (p = 0.015) decreased at the end of study in rj group compared with placebo . There was also significant decrease in mean differences of homa - ir between two groups (p = 0.023) as shown in table 3 . There was no significant difference after rj supplementation compared to before values in serum glucose, insulin levels, tac, mda and homa - ir . Effects of 8 weeks rj supplementation on serum glucose, insulin, tac, mda and homa - ir in type 2 diabetic patients data expressed as a mean standard deviation . Before, after and mean differences and p values are mentioned in the table.-/ (p value ) comparison in each group between before and after values by paired t - test / (p value * *) comparison between two groups of before values and also after 8 weeks values of two groups and also comparison of mean differences by independent t - test to our knowledge, this is the first rct conducted to assess the effect of royal jelly supplementation on homa - ir, oxidative stress and mda in both sexes of human subjects . Oxidative stress is associated with type 2 diabetes, and there is compelling biochemical evidence suggesting that ros may even play a role, even if only secondary, in the pathogenesis of type 2 diabetes (15). In this study, rj intake reduced serum glucose and homa - ir while it increased serum tac significantly after 8 weeks of intervention in comparison to placebo group but no significant difference was shown before and after rj supplementation in serum glucose, insulin level, tac, mda and homa - ir . Rj reduces the index of insulin resistance (homa - ir) but not blood glucose levels in rats (17). Insulin resistance associates with changes in oxidative stress levels and rj has protective effects against oxidative stress due to its antioxidant peptides . For example, in one study, supplementation with rj for 16 weeks significantly reduced 8-hydroxy-2-deoxyguanosine as a marker of oxidative stress and the average life span was extended by about 25% compared to the control group (18). In another study supplementation with rj was reported to have beneficial effects against genotoxicity and oxidative damages induced by cadmium in albino mice . Rj had a protective effect on the chromosome aberration, micronucleus, and oxidative stress, and this effect was connected with dose . The protective effect of rj on toxicity induced by cadmium may be explained by the therapeutic properties and antioxidant capacity of rj, which is a product of honeybees (20). In another study, yeast cells were cultivated with different concentrations of rj . In the rj treated cells, lower atp pool size did not cause growth decline, which might be due to decreased cell requirements for energy, since decreased intracellular oxidant level was observed in a dose dependent manner indicating lower energy consumption for induction of endogenous antioxidant defending and repair systems . This was also confirmed by 2-d gels (electrophoresis), where down - regulation of enzymatic antioxidant system cu decreased cell requirement for energy in rj treated cells was observed also by glucose consumption rate measured at 6 h, which was for 14.5% lower compared with controls (21). The protecting effects of rj against oxidative status may be related to scavenging abilities of the superoxide radical (20). Study (11), but increase of tac and decrease of insulin resistance which was occurred in our study was not shown in that study . On the contrary, of course, participants of pourmoradian s study were only women and baseline values of mda were higher than of our study, so these might be responsible for mda decreasing . To our knowledge, this is the first randomized, controlled trial on rj supplementation in both sexes of dm patients but it is limited in that it had a small sample size and short duration . Therefore, larger sample size and longer duration are needed before reaching conclusive results . However, administration of rj in diseases accompanied with oxidative stress history is suggested . Moreover, the effect of 10-hydroxydecanoic acid as an unsaturated fatty acid in royal jelly, on homa - ir, oxidative stress and mda needs further investigation . So, administration of rj in diseases accompanied with oxidative stress history may be useful . Ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc .) Have been completely observed by the authors.
The dentigerous cyst is a common non - inflammatory odontogenic cyst of the oral cavity . It's pathogenesis involves the accumulation of fluid between the unerupted tooth crown and surrounding follicle, giving rise to the characteristic clinical and radiographic finding of a cystic lesion surrounding the neck of the tooth (latin: dens, tooth + gerere, to bear). Treatment involves surgical enucleation or marsupialization with or without preservation of the impacted tooth, followed by histopathological evaluation to rule out an odontogenic keratocyst (okc), ameloblastoma, or rarely, malignant transformation . We report an unusual histopathological variant of the dentigerous cyst, the keratinizing dentigerous cyst, which has been reported only once previously in literature . The present case is about a 21-year - old male patient of indian origin reported with a complaint of mild pain and swelling in the lower front region of the jaw for the past 1 year . Intra - oral examination revealed a diffuse swelling in the lower anterior vestibule extending from the region of 43 to 36 . On palpation, the swelling was firm in consistency and egg shell crackling was elicited in some areas . The panoramic radiograph showed a well - defined unilocular radiolucency extending from the mesial aspect of 44 to the mesial root of 36, extending to the mental foramen on the left side . 43 was impacted and the cystic lesion surrounded the crown of the tooth and extended for some distance along the mesial aspect of the root (circumferential variant). Cone - beam computed tomography was also performed and revealed involvement of roots of 42, 41, 31, 32, 33, 34, 35 and mesial root of 46 [figure 1]. Cone - beam computed tomography shows a unilocular lesion surrounding the crown of impacted 43 and extending along the mesial aspect of the root (circumferential type) an excisional biopsy of the lesion was performed under general anesthesia with conventional pre - operative medication cover and endotracheal intubation . A mucoperiosteal flap was raised from 44 to 37 region and the lesion was detached from the soft - tissue using blunt dissection and curetted out from the bony walls . Root canal treatment was carried out for 41, 42, 31, 33, 34 and 35 . The specimen was sent for histopathological examination to rule out an okc or an ameloblastoma . The patient was called for review and a post - operative osteoprotegerin after 1 month . Macroscopically, a cyst in - toto with a tooth, measuring 40 30 40 mm in size [figure 2] and four bits of tissue curetted from the surgical site were sent for histopathological evaluation . The cyst was sectioned in half and processed along with the four bits of curetted tissue . Gross finding of a tooth within a cystic bag filled with keratinaceous material microscopically, a fibrous connective tissue capsule in association with a non - keratinized cystic lining epithelium of varying thickness was observed . The four bits of curetted tissue microscopically showed the presence of hematoxyphilic substance, suggestive of keratin . We report a case of keratinizing dentigerous cyst which, to the best of our knowledge, has been reported only once previously in the literature . Philipsen in 1956 suggested the term okc for all odontogenic cysts, regardless of type, showing keratinization of the epithelium . More recently, an okc is defined by other characteristics of the epithelium such as basal palisading, hyperchromatism of nuclei and cell thickness of the epithelium and not merely the presence of keratinization . The term keratinizing odontogenic cyst has been suggested for any cyst, regardless of the type, that shows keratinization . Characteristically, the epithelial lining of a dentigerous cyst is not keratinized and most of those that have been described as keratinized have been ascribed to adjacent okcs . However, taking into consideration the established criteria, the present case did not show any of the features that currently define okcs . The patient was followed up after 1 year [figure 4] and showed no clinical or radiographic signs of recurrence . (a) intra - oral - labial aspect of the patient 1 year postoperatively (b) intra - oral - lingual aspect of the patient 1 year postoperatively (c) orthopantamograph (opg) of the patient 1 year postoperatively the significance of keratinization in odontogenic cysts is not fully known . However, dentigerous cysts, thought to arise from reduced enamel epithelium, are products of end cells, i.e. Cells that have completed synthesis (enamel formation). Considering the age of the patient, it is possible that the dentigerous cyst is a primordial variant, arising from more primitive cells of the developing enamel organ . Long term follow - up of patients presenting with a keratinizing dentigerous cyst is advised to observe its potential for recurrence or malignant transformation since very little is known about this unusual entity.
Halorhodospira halochloris is an anoxygenic photosynthetic halophile that was isolated from the hypersaline wadi natrun lakes in egypt, residing in the mats near the sediments 8 . The genus halorhodospira was formed by separating species h. halophila, h. halochloris and h. abdelmalekii from genus ectothiorhodospira based on their 16s rrna sequences 4,6 . Its cells are vibroid, motile by bipolar flagella and have internal photosynthetic membranes as lamellar stacks 13 . H. halochloris exhibits growth over an unusually wide range of medium nacl concentrations and is capable of growth down to 5% nacl, which is unusual for extremely halophilic bacteria . Halophilic bacteria employ two differing strategies to protect their cytoplasmic volume against osmotic movement of water to the hypersaline environment 12 ., organic compounds are accumulated in the cytoplasm - these osmoprotectants are known as compatible solutes . The second biochemically, more radical adaptation involves the selective influx of k ions into the cytoplasm . H. halochloris accumulates glycine betaine (n, n, n - trimethylglycine), a compatible solute as its osmoprotectant 5 . In addition to its osmoprotectant activity, glycine betaine also provides protection against mutagenic compounds and radiation - induced damage 9 . Glycine betaine can either be taken up directly from the environment, or be synthesized de novo 11 . We recently used isoelectric focusing of total cell proteins to demonstrate that h. halochloris does not exhibit an acidic proteome, matching its inability to accumulate k 2 . In striking contrast we found that a closely related organism h. halophila accumulates molar concentrations of kcl when grown in high salt medium and has an acidic proteome . Comparative genomics of h. halochloris and h. halophila promises to provide insights into this issue, which has implications both for genome - wide evolutionary processes and the mechanisms of halophilic adaptations . These considerations led us to determine the genome of h.halochloris, which we report on here . Recently we reported the complete genome of h. halophila 1 . In this study, we report the draft genome sequence of h. halochloris, which was obtained through standard roche 454 pyrosequencing using the roche 454-junior instrument . The raw data obtained were trimmed at either end based on the quality score analysis performed using fastqc tool . Poor or bad quality bases, probably originating from sequencing mis - calls, were trimmed off before subjecting it to the assembly software . We performed genome assembly using three different assemblers, namely newbler 16, mira 17 and phrap 18, with the default set of parameters . After comparisons of these assembly attempts based on contig sizes, genome representation and its functional elements, the output of mira 3.4.1 was selected to proceed with further analysis . The final output had some low quality contigs in terms of length and average coverage . All contigs of length less than 1kbp and average coverage <10 were removed as being uninformative, from annotation point of view, and subjected to an individual annotation check using blast 14 . Most of these individual contigs yielded relatively high e - value or no scores with halophiles . Processed contigs were mapped against a distant reference genome (thioalkalivibrio sulphidophilus), as no true known reference genome is currently available, based on 16s analysis, using contiguator 15 . The assembled scaffold comprises 137 contigs, 3,460,134 bases at 20 fold coverage and has a gc content of 63% . For comparison the genome of h. halophila is 2.7 mb in size and has gc content of 67% 1 . The jgi img / er annotation pipeline (http://img.jgi.doe.gov/er) was employed for gene annotation with img submission i d and img project i d, 15725 and 50543 respectively . The numbers of trna and rrna genes were predicted as 46 and 8, respectively . A total of 3,301 putative protein coding genes (cdss) or open reading frames (orfs) were predicted with a total gene count of 3,376 . The genome has been submitted in public databases, ncbi, genbank (gi number: 589289709, genbank accession number: cp007268). The draft genome information reported here provides opportunity for further research into the mechanism involved in halophilic adaptations and allow organisms to thrive in hyper saline environments, how these evolve and how they differ for bacteria and archaea 10.
It is estimated that hypertension affects about one billion people, and the number will increase by 60% until 2025 (7). According to the world health organization and the international society of hypertension, high blood pressure is defined when the systolic blood pressure is 140 mmhg (18.7 kpa) or more, and/or level of diastolic blood pressure is 90 mmhg (12.0 kpa) or more, after repeated measurements . It can occur as isolated systolic or diastolic hypertension but it is usually case of elevated both systolic and diastolic pressure . Due to the changes that occur in the large arteries because of aging, systolic blood pressure after the age of 50 increases with every year of life by about 2 mmhg and diastolic blood pressure by 0.5 to 1 mmhg (4). Most of the patients with hypertension have essential or primary hypertension with unknown cause, and in which the initially high blood pressure is the only manifestation of the disease . In other patients the hypertension is caused by disease of some other organs and therefore is referred to as secondary hypertension . The degree of clinical severity of hypertension is estimated based on the values of blood pressure and current changes in other organ systems (5, 6). For hypertension treatment today at all level of healthcare protection some of them are: * lisinopril: is a potent ace inhibitor and a potent antihypertensives belonging to the third generation of ace inhibitors . It represents a lysine analog of the enalapril, and unlike it is directly active . With such action it interferes with any component of the renin - angiotensin - aldosterone system separately . From this it is used in treatment of hypertension and heart failure, as well as myocardial infarction and diabetic nephropathy prophylaxis (1 - 3). * reduces the reabsorption of electrolytes in distal tubule of the kidney that increases urine output, reducing the volume of blood in the blood vessels and contributes to lowering blood pressure (1 - 3). * our study confirmed high efficacy in the treatment of hypertension with the combination of ace inhibitor lisinopril and the diuretic hydrochlorothiazide, which provides an additional antihypertensives effect . Evaluate patients suffering from hypertension who were treated with a combination of lisinopril / hydrochlorothiazide, in 5 health institutions in bosnia and herzegovina with an emphasis on the effectiveness of it s antihypertensives effect . Determine the mean blood pressure values in the studied group patients by measuring blood pressure in the studied time period: two months (first, second and third measurement). Provide a cross section of the lisinopril / hydrochlorothiazide antihypertensives effect based on three measurements identify the differences between the patients in our study in relation to gender, age and age groups . It is estimated that hypertension affects about one billion people, and the number will increase by 60% until 2025 (7). According to the world health organization and the international society of hypertension, high blood pressure is defined when the systolic blood pressure is 140 mmhg (18.7 kpa) or more, and/or level of diastolic blood pressure is 90 mmhg (12.0 kpa) or more, after repeated measurements . It can occur as isolated systolic or diastolic hypertension but it is usually case of elevated both systolic and diastolic pressure . Due to the changes that occur in the large arteries because of aging, systolic blood pressure after the age of 50 increases with every year of life by about 2 mmhg and diastolic blood pressure by 0.5 to 1 mmhg (4). Most of the patients with hypertension have essential or primary hypertension with unknown cause, and in which the initially high blood pressure is the only manifestation of the disease . In other patients the hypertension is caused by disease of some other organs and therefore is referred to as secondary hypertension . The degree of clinical severity of hypertension is estimated based on the values of blood pressure and current changes in other organ systems (5, 6). For hypertension treatment today at all level of healthcare protection some of them are: * lisinopril: is a potent ace inhibitor and a potent antihypertensives belonging to the third generation of ace inhibitors . It represents a lysine analog of the enalapril, and unlike it is directly active . With such action it interferes with any component of the renin - angiotensin - aldosterone system separately . From this it is used in treatment of hypertension and heart failure, as well as myocardial infarction and diabetic nephropathy prophylaxis (1 - 3). * reduces the reabsorption of electrolytes in distal tubule of the kidney that increases urine output, reducing the volume of blood in the blood vessels and contributes to lowering blood pressure (1 - 3). * our study confirmed high efficacy in the treatment of hypertension with the combination of ace inhibitor lisinopril and the diuretic hydrochlorothiazide, which provides an additional antihypertensives effect . Evaluate patients suffering from hypertension who were treated with a combination of lisinopril / hydrochlorothiazide, in 5 health institutions in bosnia and herzegovina with an emphasis on the effectiveness of it s antihypertensives effect . Determine the mean blood pressure values in the studied group patients by measuring blood pressure in the studied time period: two months (first, second and third measurement). Provide a cross section of the lisinopril / hydrochlorothiazide antihypertensives effect based on three measurements identify the differences between the patients in our study in relation to gender, age and age groups the data used in our study were obtained from the studies and analyzes carried out in 5 health care institutions at the territory of bosnia and herzegovina . The study included 270 patients who underwent three consecutive medical examinations after being on lisinopril / hydrochlorothiazide therapy . Patients evaluated in our study underwent three examinations over a period of two months . The criterion for inclusion was hypertension . Every physician / doctor evaluated a group of patients . After collection, the data are converted into the group study which results we presented in tables and charts . The necessary data for following lisinopril / hydrochlorothiazide performance are collected on the basis of medical examinations and doctor s notes: history and status preasens;measuring blood pressure during the first, second and third examination;maintaining records of adverse events during the three examinations;laboratory tests;diagnostic tests . History and status preasens; measuring blood pressure during the first, second and third examination; maintaining records of adverse events during the three examinations; the research results are presented in tables and charts by the number of cases, percentage, mean with standard deviation and range of values . Analysis of statistical significance of the differences is conducted by pearson and yates chi - square test and one - way analysis of variance (anova). The results of all these tests with p<0.05 or the confidence level of 95% were considered statistically significant . The analysis was performed using the statistical package ibm spss statistics v19.0 (chicago, illinois, usa). Majority of patients required higher dose of lisinopril / hydrochlorothiazide a 20 mg (185 or 68.5%) (table 1) which can be explained by the older age of the patients (5912 years; 34 - 88 years) ((table 2). The numbers in the second column represents n (%) and in case of average values mean + /- comparison of mean age by l / h dose, t=-0.828; p=0.409 there is a decrease in systolic blood pressure from 172.416.9 mmhg in first measurement, to 153.913.8 mmhg during second, and to 140.413.3 mmhg during third measurement . Also there is a decline in diastolic blood pressure from 98.97.7 mmhg during first, to 87.87.4 mmhg during second and to 72.37.9 mmhg during third measurement . Hearth rate also slightly decreased from 79.414 bpm during first, to 75.49.2 bpm during second and finally to 72.37.9 bmp during third measurement . Side effects were recorded in 12 or 4.4% of cases, or in one case the feeling of suffocation and in 11 cases in form of dry cough . Treatment outcome was mostly excellent (200 or 74.1%), then good (60 or 22.2%). In 8 or 3% of cases the treatment outcome was unsatisfactory and 2 or 0.7% of patients had deterioration so the treatment was discontinued . Comparison by lisinopril / hydrochlorothiazide (l / h) dosage comparison of mean age by lisinopril / hydrochlorothiazide dose indicate that patients on l / h 20 was just slightly older with mean age of 59.4 years compared to patients on l / h 10 and average age of 58.1 years, which does not represent a statistically significant difference . Overview of systolic pressure over three measurements mean values of systolic blood pressures was lower in a group of patients receiving lisinopril / hydrochlorothiazide a 10 mg during all three measurements, with statistically significant difference between group during the first and second, but not the third measurement (figure 1). Review of systolic pressure over three measurements review of diastolic pressure through three measurements the mean values of diastolic blood pressure were significantly different only during the first measurement when higher mean value was recorded in the group of patients receiving l / h a 20 mg . During the second measurement the mean values of diastolic bp was identical, and during the third measurement the mean vale of diastolic bp was slightly higher in a group of patients on lisinopril / hydrochlorothiazide a 10 mg but without significant difference (figure 2). Review of diastolic pressure through three measurements review of heart rate through three measurements mean hearth rate was higher during all three measurements in a group of patients on l / h a 20 mg, but with statistically significant difference only during the first measurement (figure 3). Review of heart rate through three measurements overview by gender and lisinopril / hydrochlorothiazide dose women was more represented in both groups of patients without significant difference (table 3). Overview by gender and l / h dose; =0.675; p=0.247 review of side effects in the sample as expected, the higher dose of the medication produced more side effects or 11 (5.9%) in a group of patients on lisinopril / hydrochlorothiazide a 20 mg, compared to one (1.2%) patient in a group of lisinopril / hydrochlorothiazide a 10 mg . Review of side effects in the sample; =3.120; p=0.065 the types of side effects one patient in group on l / h a 10 mg had the dry cough, and majority of patients in the group on l / h a 20 mg and with side effects had the same, with one patient reporting the feeling of suffocation (figure 4). The types of side eff ects (=3.155; p=0.207) treatment efficacy was almost identical with just slightly better efficacy in a group of patients treated with l / h a 10 mg (figure 5). Review of gender structure of the sample indicated that women were slightly more prevalent with 165 or 61.1% cases compared to men with 105 or 38.9% of cases . The analysis of the sample according to the dose of lisinopril / hydrochlorothiazide a 10 mg and 20 mg shows that the majority of respondents was necessary to introduce l / h a 20 mg (68.5%) while l / h a 10 mg was sufficient to regulate hypertension in 31.5% of respondents . Analysis of the average systolic blood pressure during the first examination, the first control, and other controls showed a linear decrease and the difference between groups was statistically significant during the first and second measurement but not during third, which favors rapid action of the l / h . Analysis of the average diastolic blood pressure showed a linear decrease in the difference between the groups where there is a statistically significant difference in the first and second measurements . The combination of ace inhibitor lisinopril and the diuretic hydrochlorothiazide provides an additional antihypertensives effect . Lisinopril is a potent ace inhibitor, a potent antihypertensives belonging to the third generation of ace inhibitors . It is used in treatment of hypertension and heart failure, as well as myocardial infarction and diabetic nephropathy prophylaxis (1 - 3). It reduces the reabsorption of electrolytes in distal kidney tubule which consequently increases urine output, reducing the volume of blood in the blood vessels and contributes to lowering blood pressure (1 - 3). The initial blood pressure during the first measurement averaged at 172.8 mmhg for systolic blood pressure and 98.9 mmhg for diastolic blood pressure . After one month of treatment with l / h a systolic blood pressure was reduced to an average of 153.8 mmhg and dbp to 87.7 mmhg . The reduction was also recorded during the third measurement, when the average systolic blood pressure was 140 mmhg and diastolic blood pressure 81.8 mmhg . Total reduction in blood pressure in average was 32.8 mmhg for systolic and 17.1 mmhg for diastolic, which is consistent with previously published results . During treatment has been reported a total of 12 side effects, or in 12 cases (dry cough) of the total of 270 and these results are similar to those of which are reported in previous studies of lisinopril / hydrochlorothiazide . Lisinopril / hydrochlorothiazide is because of that valuable therapy in the field of internal medicine . Criteria for introduction of l / h in therapy was newly discovered hypertension, hypertension that has not responded to previous treatment or could not be continued because of side effects . During treatment the patient s blood pressure, as the main indicator, was measured at the first examination of the patient, then the first control and the second follow - up examination . The average blood pressure in the first examination amounted to 172/98 mmhg, while in the first follow - up examination was 133/87 mmhg, and the second follow - up examination 140/81 mmhg . Based on these results the l / h is effective drug in the treatment of mild to moderate hypertension . Also was conducted evaluation of documented treatment adverse effects where it was found that there is no deviation from the previous study of treatment of hypertension with similar drugs . Data from this study indicate that both systolic and diastolic blood pressure significantly decreased after 8 weeks of treatment with l / h . Lisinopril / hydrochlorothiazide has proven as effective and safe antihypertensives drug in treatment of mild to moderate hypertension . The total clinical efficacy was assessed as excellent which is interpreted as a reduction in blood pressure to the target value in most patients.
It has become after the introduction of chemotherapy [14] and new radiation regimen [57]. But with increasing cure rates of mostly patients, late toxicities have been reported among survivors . The long - term frequency of cardiopulmonary, gonadal, and neoplastic treatment complications has been alarming after mopp, mopp - like schedules, and irradiation . In addition, subtotal nodal irradiation leads to an increased frequency of second malignancies (solid tumor and leukemia). The relative risk of the late effects is estimated as ranging from 2 to 6 [8, 9]. Modern current therapy uses risk - adapted and risk - responded chemotherapy with restricted doses of alkylating agents, anthracyclines, and bleomycin and low - dose, involved - field (node - field) radiotherapy, with the identification of additional clinical and biologic risk factors in attempt to avoid treatment - associated toxicity while maintaining high cure outcomes . Recent study results demonstrate that patients with favourable disease are excellent candidates for therapy reduction . The optimal therapy program for patients with hl goes on to be discussed, and this study is representative of the original risk - adapted protocol spbhl-05 for therapy of the similar patients with the dual goals of reducing late adverse treatment effects while sustaining efficacy . Between january 2000 and july 2009, previously untreated 60 patients less than 18 years old who had biopsy - proven hl were eligible . All patients were required to have clinical stage according to the modified ann arbor criteria [10, 11]. The clinical staging evaluation included medical history and physical examination, complete blood count, urinalysis, erythrocyte sedimentation rate, blood chemistry, chest x - ray, abdominal ultrasound, thoracic and abdominal computed tomography scan with contrast, bone marrow biopsy, and isotopic bone marrow, spleen, liver, and bone scans . Gallium scan was used to monitor response if need were . In induction chemotherapy, we used vbvp (vinblastine, bleomycin, etoposide, and prednisone) and abvd (adriamycin, bleomycin, vinblastine, and dacarbazine) schedules followed by consolidating radiotherapy . Vbvp was administered as follows: vinblastine 6 mg / m intravenously (iv) on days 1 and 8, bleomycin 10 mg / m iv on day 1, etoposide 100 mg / m iv on days 1 through 5, and prednisone 40 mg / m orally on days 1 through 8 . It gives a possibility to select patients on favourable, intermediate, and unfavourable risk groups . In the calculation of the prognostic index (pi), several unfavorable factors are taken into account: the age of 10 years or more, 4 or more lymphatic zones involved, when any nodal mass more than 5 cm in diameter or / and mediastinal bulky disease when the ratio of the largest transverse diameter of the mass to the transverse diameter of the thorax was at least 0,33, biological b stage that was defined by two or more of the following findings: erythrocyte sedimentation ratio 30 mm / h, fibrinogen 0,4 g / dl, serum albumin 4 g / dl, leukocytes 12 10/mm, and 2-globulin 12% of whole globulin count, systemic b symptoms (fever, night sweats, or a weight loss of> 10% of the normal body weight), and stage iv disease . These factors incorporated into the pi which was defined as the number of adverse factors present at diagnosis . Patients with pi = 02 (favourable risk group) received two vbvp, children with pi = 3 - 4 (intermediate risk group) were treated with two cycles of vbvp alternating with two cycles of abvd, and patients with pi = 5 - 6 (unfavourable risk group) were treated with three cycles of vbvp alternating with three cycles of abvd . Vbvp were repeated every 3 weeks and abvd repeated every 4 weeks as permitted by count recovery . External beam radiation was delivered with megavoltage equipment either a telecobalt or a linear accelerator . Patients who achieved complete response or 75% or greater reductions in all disease manifestation were administered 25 gy . Rt began 2 weeks after the completion of chemotherapy and was given in 1,8 gy fractions, five times per week . Patients who did not reach these status were administered 36 gy in 20 fractions during 4 weeks . In all patients, the fields were limited to the involved sites, as defined by the initial clinical and radiologic examination (involved - field irradiation petrov research institute of oncology . Written informed consent of parents was required before therapy . Patients were evaluated for response after two, four, and six cycles in the chemotherapy arms and after completion of rt . A cr was defined as the disappearance of any clinical and radiological evidence of active disease over a period of 4 weeks . A partial remission (pr) was defined as a 50% or greater reduction in all disease manifestation compared with the initial involvement for at least 4 weeks . Progressive disease was defined as enlargement of measurable tumors by more than 25% or appearance of any new lesions . Stable disease was not satisfying the definition of cr, pr, or disease progression / relapse . Restaging was performed 2 weeks after the end of chemotherapy and 4 weeks after the end of irradiation . Restaging consisted of a control and documentation of all initial disease manifestation by clinical methods including a physical examination, complete blood count, urinalysis, erythrocyte sedimentation rate, blood chemistry, chest x - ray, and abdominal ultrasound, computed tomography of the chest and abdomen . A bone marrow biopsy and isotopic bone marrow, spleen, liver, and follow - up examination including medical history and physical examination, complete blood count and blood chemistry, chest x - ray, and abdominal ultrasound were performed within the first 2 years in 3-month interval, at years 35 in 6-month interval, and from year 5 once a year . Os was defined as the time from beginning of treatment to death, whether disease - related or not, or last follow - up examination . Efs was defined as the time from beginning of treatment to an adverse event (relapse, disease progression, death in remission, and second malignancy) or last follow - up examination . Os, efs and standard errors (se) were estimated by the methods of kaplan and meier . Test of statistical significance in the comparison of survival curves were calculated using the log - rank test . Actual treatment duration was calculated as a time between the first and final cytotoxic drug administration plus 14 days, representing the theoretical duration of last treatment cycle . The total actual dose given represented the sum of all administered doses per square meter of body . Dose intensity was calculated by dividing the total actual dose in milligrams by total actual duration in days and subsequently expressed as the percentage of initially planned theoretical dose intensity . Six of the 60 (10%) were less than 9 years, 54 (90%) were 10 years of age or older . Twenty eight had stage iii and iv disease, while 32 had stage i and ii disease . Histological subtypes were lymphocyte predominance in 2 cases (3.3%), nodular sclerosis in 46 (76.7%), and mixed cellularity in 9 (15%); three cases (5%) were not subclassified . Thus, in the series about 90% had either nodular sclerosis or mixed cellularity, while only 3.3% had lymphocyte predominance . Twenty three patients (38.3%) had one, two, or three sites of lymph node involvement, whereas 37 patients (61.7%) had four or more sites of node involvement . The distribution of prognostic groups, expressed as the pi, is listed in figure 2 . Fourteen patients (23.3%) had none, one, or two adverse factors (favourable risk group). Twenty five patients (41.7%) had three or four adverse factors (intermediate risk group). Twenty one children (35%) had five or six adverse factors (unfavourable risk group). After therapy, 57 (95%) one child had stable disease, and one patient developed progressive disease after five cycles of chemotherapy . A cause of death after five cycles in one case was the accompanying intercurrent viral epiglottitis . With a median followup of 68 months (range 7 to 115 months), the 5-year os is 91.3% (se 3.7%), and the efs is 82.8% (se 5.0%) (figure 3). Of the 57 patients who received the protocol and achieved cr, 9 (15.8%) relapsed at a median time of 50 months from initial diagnosis (range, 6 to 104 months): seven relapsed patients initially were treated according to principles for unfavourable risk group, two others relapsed patients were treated according to principles for favourable and intermediate risk groups . According to the risk group division, 5-year os in the favourable and intermediate risk group were 100%, efs were 92.9% and 90.7%, respectively, and os and efs in unfavourable risk group were 77.1% and 55.6%, respectively, (table 2). The reasons for early termination of chemotherapy were progressive disease and death from viral infection . Decreasing dose - time intensity of schedules 25% was not significantly influences the outcomes (figure 4). Table 3 lists the acute toxicities associated with each chemotherapy regimen during the time the patient was on treatment . The number cycles was administered to 254 (185 vbvp and 69 abvd). Leukopenia and neutropenia were the most common haematologic toxicity with grade 1 - 2 in 13.8% and 12.5%, respectively, grade 3 - 4 in 6.2% and 4.4%, respectively, of the cycles . Hodgkin's lymphoma has one of the best cure rates of all of childhood and adolescent malignancies . Consequently, ongoing studies aim at reducing treatment - related toxicity, including second tumors such as acute myeloid leukemia, non - hodgkin's lymphoma, and solid tumors . Using the modern combination regimen, risk - adapted therapy models might be warrant the excellent cure rates of patients with hl and should help avoid overtreatment and reduce life - threatening toxicity in patients . German - austrian hodgkin's disease study (dal - hd) worked out the well - known risk - adapted treatment for hl . The study was designed to reduce, step - by - step, loads of the treatment in low and intermediate risk group . High - dose and extended - field irradiation were limited to involved - field irradiation at a dose of 2025 gy (dal - hd 90). Combined modality therapy schedule of the dal - hd study included induction chemotherapy (oppa / oepa and copp regimens) followed by radiotherapy . Variables were selected according to the stage of the disease and systemic b symptoms . After stratification, similar strata were randomly assigned to treatment group tg1 (early stages), tg2 (intermediate stages), and tg3 (advanced stages). Patients with stage ia, ib, and iia (tg1) received two cycles; children with stages iib and iiia (tg2) received four cycles; remaining patients with stages iiib and iv (tg3) received six cycles . The total dose was 25 gy in tg1 and tg2 and 20 gy in tg3 . A boost up to 35 gy total dose was given to sites with residual lymphoma . Eighty - three patients were treated using combined modality therapy schedule of the dal - hd study (versions 87 and 90) between june, 1987 and february, 2000 in our clinic . The median follow - up duration was 116 months (range, 8 to 255 months). Seventy - two per cent of all patients received only two or four cycles; 28% received six cycles . Os of 5 years after diagnosis was 93.3%, whereas in dal - hd study, it was 98%; as to efs, it came to 79.9% versus 86%, respectively . According to risk group division, os in in tg2, the contributions were 100% and 96.7%, and in tg3 they were 98% and 87%, respectively . As for efs, the values in tg1 were 94% in dal - hd study and 84.4% in our investigation . In tg2, the contributions were 91% and 81.4%, and in tg3, they were 93% and 72.7%, respectively . There were fewer patients in tg1 (32.5% in the study and 48% in dal - hd study) and more patients in tg2 (39.8% and 22.2%, resp . ). The number of patients in tg3 was approximately identical (27.7% and 29.9%, resp . ). The proportion of the second tumors did not coincide in the studies, being 0.9% and 3.6%, respectively . We do not exclude further detection of second neoplasms because of the higher doses of alkylating agents and anthracyclines in tg2 and tg3 . Overall, it may be conclude that the therapy regimen of the dal - hd study represented a risk - adapted treatment with high efficacy . On the one hand, alkylating agents and anthracyclines causing late adverse sequels included in chemotherapy regimen of the dal - hd study . The cumulative total dose of cyclophosphamide and procarbazine were 2000 mg / m and 6000 mg / m in tg3, respectively, and the total dose of doxorubicin was 80 mg / m that were analogous to three cycles mopp or copp or abvd . On the other hand, in dal - hd study patients were selected according only two parameters: stage of the disease and b symptoms . For these reasons, the new program spbhl-05 has been offered . Our protocol of risk - adapted therapy was extremely effective for disease control in patients from favourable and intermediate risk groups, but treatment results in unfavourable risk group have been less impressive . Treatment of children from unfavourable risk group resulted in inferior event - free and overall survival . Os in the favourable, intermediate and unfavourable risk group were 100%, 100%, and 77.1%, and efs were 92.9%, 90.7%, and 55.6%, respectively; os were 100%, 100% and 77.1%, respectively . Our experience with protocol decreasing cumulative doses of alkylating agent and anthracyclines in combination low - dose involved - field radiotherapy showed excellent outcomes . (2) with a median followup of 68 months (range 7 to 115 months), the 5-year os for all patients was 92.7% (se 3.6%), and efs was 79.6% (se 5.6%). (3) the study showed significantly improved complete remission rate, event - free survival, and overall survival for vbvp (in favourable risk group) or the sequential vbvp - abvd (in intermediate risk group) followed by radiotherapy . Although the followup was short and only 5-year result was available, there were no differences in survival rates between programs (for example, dal - hd). (4) no patients developed second malignancies in our series, while 3 cases of second neoplasms occurred after the dal - hd . While adequate for the purpose of examining the regimen, the median followup of approximately 5 years interrupted a more detailed comparison of the risk of second malignancy between two programs . (6) low - cumulative doses of potentially dangerous medicines were reached by using sequential vbvp - abvd . (7) own prognostic index included 6 adverse prognostic factors (in dal - hd risk groups were selected according only to the stage of the disease and systemic b symptoms). These results suggest that it is feasible to reduction alkylating agent and anthracyclines in children with hodgkin's lymphoma, but this approach results in inferior disease control in those from unfavourable risk group . Stratification of patients on risk group according to own prognostic index is feasible and proven to be effective, but additional stratification is required in unfavourable risk group . Maybe stratification according to the prognostic score by d. hasenclever and v. diehl is needed in this group . Based on our results, it may be reflected that the spbhl-05 represents an effective risk - adapted and response - adapted treatment strategy for hodgkin's lymphoma for children and adolescents.
Creating and maintaining a functional hemodialysis access conduit is challenging . With an increasing number of patients needing hemodialysis per year, the demand for a durable access with minimum complications is also increasing . Among the postoperative complications, dialysis associated steal syndrome (dass) is the most morbid, often resulting in significant neurologic injury or tissue loss . Clinical risk factors previously identified in patients at risk for development of dass include age greater than 60 years, female gender, diabetes, previous limb procedures, and type of fistula constructed [15]. Preservation of the existing access and relief of the ischemia are a priority in the treatment of dass . Bussell and associates described dass in patients with a radial - cephalic arteriovenous fistula (avf) by using pneumatic plethysmography . The diagnosis is clinically suspected when there are new symptoms either immediately after access creation or subsequently on followup after maturation . It is confirmed clinically by physical exam demonstrating a cold distal extremity, pain, pallor, diminished, or absent peripheral pulses in the extremity, muscle wasting, sensory impairment, or even ulceration or gangrene in the late cases [7, 8]. A noninvasive hemodynamic assessment can be performed by measuring the distal and/or forearm doppler pressure and by recording digital pulse wave plethysmography or by comparing digital pressure measurements with and without manual compression of the arteriovenous (av) access [9, 10]. Accompanying volume flows in the feeding artery have been previously measured but have not consistently shown to correlate to the presence or absence of steal . We proposed that there might be a correlation between volume flows and presence of steal in av access and that there may be differences between the types of access . This was a retrospective review of 118 consecutive patients who underwent us exams of their functioning hemodialysis access during a 30-month period . Eighty - two of those patients had evidence by clinical and physical examination of having steal syndrome . Collected data included gender, type of av access, and volume flow measurements in the proximal feeding artery . Academic medical center with 1000 beds in a catchment area of 5 million people . Duplex examinations were performed based on a standardized protocol using a philips iu22 xmatrix color duplex scanners (phillips healthcare, andover, ma) and a l 74 mhz linear transducer with patients in a recumbent position . Transverse and longitudinal b - mode and color flow images were obtained along at least 10 cm or more of the arterial inflow and the arterial anastomosis . Waveforms were recorded from a small sampling volume placed in the central flow stream at attempted angles of 60 relative to vessel walls of the feeding artery . Velocity sampling was done at multiple sites proximal and distal to the anastomosis and the highest volume flow rate selected [11, 12]. A marked or significant reversal of flow especially in the brachial artery distal to the anastomosis was suggestive of severe steal and consequent distal ischemia as described by zamani and colleagues and suggested by van hoek and associates . Plethysmography probe measurements before and after access compression were used to document doubling of maximum wave amplitude upon compression of the fistula outflow [2, 9, 10]. Student's t - test was used to compare continuous variables (volume flows). The chi - squared test was used to compare categorical variables (men and women with and without steal syndrome). One hundred and eighteen consecutive patients of which 82 (69.5%) had clinical evidence of steal were evaluated by duplex ultrasound (us). Table 1 details the number of men and women presenting with and without steal syndrome . Women were more likely to present with steal than men (chi - squared test p <0.04). Mean volume flow in patients with steal was 1542 ml / min compared to 1087 ml / min (p <0.002) in patients without evidence of steal . However, a significant difference in flow volumes between those with steal and without steal syndrome was only seen in patients with brachial - cephalic upper arm avf (p <0.002). When comparing between types of access in patients with steal syndrome, brachial - cephalic upper arm avfs had significantly higher volume flows than the upper extremity av graft (avg) group (p = 0.04). Table 2 shows the mean volume flow by access type in patients with and without steal . Complications of vascular access, including thrombosis, bleeding, infection, pseudoaneurysm, and distal ischemia [14, 15] are a large cause of morbidity in the hemodialysis population in the united states . Though uncommon among these, the most morbid is hand ischemia or steal syndrome . It can often result in significant neurologic injury, motor deficit, or tissue loss . Management has proven to be a challenge partly because of the desire to maintain access while alleviating the ischemia in this difficult population with often advanced peripheral vascular disease . Steal phenomenon is particularly frequent in patients with forearm and upper arm avfs and in patients with prosthetic straight or loop grafts . Because of low resistance in the venous outflow, the av access takes not only the antegrade flow into the feeding artery but also steals retrograde flow from the hand via the palmar arch and jeopardizes its adequate perfusion . Interestingly, reversed blood flow in the artery distal to the anastomosis has been observed in radial - cephalic avfs but has not been documented in brachial artery based av access . Some minimal element of steal may occur in 75%90% of patients after creation of the vascular access [15, 16]. The steal phenomenon is converted into a steal syndrome when compensatory mechanisms to maintain peripheral arterial perfusion fail . The steal syndrome is characterized by pain at rest, pain during hemodialysis sessions, ulcerations, mostly acral necrosis, and even tissue loss . Preoperative risk factors for a steal syndrome are female gender, age> 60 years, and diabetes mellitus construction of an autogenous fistula, multiple previous operations on the limb, and use of the brachial artery as the donor vessel [15]. Our series corroborates these findings with more steal phenomenon seen in women and in patients with brachial - cephalic upper arm avfs . Although angiography has been considered as the gold standard for imaging of vascular access abnormalities, duplex ultrasound may be helpful in some aspects since it provides information both on the morphology and on the function of the vascular access . In addition, us offers the advantage of a noninvasive procedure with lower cost and avoidance of contrast media . Volume flow measurements traditionally have been used to diagnose fistula dysfunction with lower flow rates corresponding to higher failure rates [17, 18]. For instance, bay and associates described a series of over 2700 patients in whom serial volume flow measurements were done to predict failure rates over followup . Some authors have argued that dialysis access volume flow and the presence of steal are related . Overall we found a significantly higher flow rate in access with steal syndrome than in those without steal . In particular, a significant difference in flow volumes in patients with and without steal was only seen with a brachial - cephalic upper arm avfs . This finding may be clinically relevant and may have potential implications for its surgical management . Owing that our data suggest that brachial - cephalic avfs develop steal in part due to high volume flow rates compared to those without steal, treatment may be potentially influenced by the decision to decrease or reduce the flow through the avf . Thus, flow reduction techniques such as a simple plication of the inflow may be considered in this instance . This is based on the premise that increasing fistula resistance or decreasing the flow through it will indirectly increase perfusion to the distal extremity . Tordoir and associates have suggested that blood shunting through the avf may cause stealing of blood and hypoperfusion of distal tissues . In addition, they have shown that high - flow avfs have a greater risk of ischemia than avfs with normal flow volumes, with the caveat that when combined with peripheral arteriosclerotic disease the latter may also lead to ischemia . They suggest that augmentation of arterial inflow by interventional techniques and/or avf blood flow - reducing surgical procedures may eliminate pain and heal ulcers in this particular case . We did not find an overall significant difference between flow volumes when comparing upper arm av grafts, brachial - basilic upper arm transposition, or radial - cephalic forearm avfs with and without steal . In previous work by van hoek and colleagues, the intensity of steal was not related to the magnitude of access flow . However, similar to our report, individuals with brachial - cephalic upper arm avfs were at higher risk of developing complaints associated with reduced hand circulation compared to those with a radial - cephalic forearm avf or an upper arm avg . Fistulas, unlike grafts, have an intact endothelial lining that allows them to actively dilate and remodel over extended periods . In addition, fistulas have side branches that reduce resistance to flow and ligation of accessories or spontaneous occlusion of side branches within a fistula increases resistance and results in an access that hemodynamically mimics the profile of a graft . In our series, none of the patients with avfs had branch ligations . These factors may partially explain our finding of higher volume flows observed in patients with steal having a brachial - cephalic upper arm avf compared to the avg group . First, the scope of this retrospective study focused on us evaluation of volume flows in av dialysis access . We did not capture data on the status of the forearm and digital arteries nor measured digital pressures or indices . Thus, we do not have a picture of the underlying arterial pathology that clearly contributes to the multifactorial nature of steal . Similarly, by not correlating with arteriographic images, we do not have the ability to evaluate the formation of arterial collaterals as a potential compensatory mechanism in physiologic steal . Thus, in patients with congestive heart failure or those with decrease heart function, volume flow measurements will be affected negatively . Similarly, increases in peripheral vascular resistance (pvr) as often seen in diabetics will also affect access flow . Work by wijnen and colleagues describes this relationship and found that access flow was significantly and positively related to the cardiac output and cardiac index and inversely related to pvr . We limited the investigation to us derived volume flow measurements and its correlation with the presence of ischemic steal syndrome . This has important implications precisely if we want to quantitate and compare the volume flow after intervention and assess its correlation with the persistence or absence of symptoms of steal and av access function . Finally, we believe that our observations may contribute to the understanding of dass, and we intend that the us data generated in this work be validated by prospective studies in the future . Accurate history taking, physical exam, and noninvasive us studies are important in confirming the diagnosis . In patients with dass, significantly higher volume flows were seen with a brachial - cephalic upper arm avf in patients with steal compared to those without . This may have potential implications in the management of this complication . A physiologic basis of this us finding may be present, which warrants further investigation into the dynamics of flow and resistance in different av access conduits and their interplay with the underlying arterial pathology in the development of ischemic steal syndrome.
Diagnosis, treatment planning, and treatment monitoring in endodontics, depend to a large scale on finding the true etiologic factor(s) of the current disease . The goal of endodontic treatment is to prevent and when required, to cure apical periodontitis (ap). Like other diseases, ap can be resolved only if the etiological factor is eliminated or at least disabled . As a basic rational, egress of microorganisms or their byproducts from the necrotic root canal system or the extension of pulp inflammation, are the main etiologies of ap . In cases of a periapical lesion of a nonvital tooth, nonsurgical orthograde endodontic treatment is suggested . However, in some cases, despite root canal treatment, the ap persists, and the endodontic treatment is considered a failure which necessities apical surgery . While root canal therapy is an attempt to exclude the irritants from the root canal system, in root - end surgery the theory is to attempt to confine them within the canal boundaries . A serious consequence of traumatic injuries of teeth with immature root formation is the contusion of the apical part of the pulp and severance of pulpal blood supply, which can result in pulp necrosis, especially if the possibility of pulp revascularization is unlikely . Anachoresis through the apical foramen and bacterial contamination of the periodontal ligament (pdl) appear to be the source of infection of the compromised pulp . In cases of asymptomatic untreated pulp infection, occurrence of ap is inevitable which is frequently symptom - free and discovered primarily by the radiographic appearance . On the other hand, the number of patients demanding for orthodontic treatment with history of traumatic injuries cannot be underestimated . It is strongly emphasized that orthodontic treatment of previously traumatized vital teeth, especially in maxillary incisors, can lead to pulp necrosis and in case of already necrotic pulp associated with ap, it can worsen the situation . In cases of immature nonvital teeth, one - visit apexification has been defined as the nonsurgical placement of an endodontic biomaterial into the apical part of the root canal . As a result many biomaterials with good sealing ability have been proposed for this treatment, with mineral trioxide aggregate (mta) being the most frequent one . Calcium - enriched mixture (cem) cement has been introduced as a hydrophilic tooth - colored biomaterial with favorable sealing ability . It is shown that pdl regeneration, cementogenesis, and dentinogenesis occur in contact with this biomaterial, like mta . This article represents a report of a treatment case in an orthodontic patient with a large periapical lesion in anterior segment of maxilla, which was diagnosed to be related to a necrotized immature tooth that had been traumatized before . A 16-year - old male patient was referred by his orthodontist with main chief complaint being a dull pain sensation in the upper maxillary region . According to his parents, his upper left central incisor (i.e., tooth no . 21) had been slightly discolored after a trauma during bicycle riding at the age of nine . His orthodontic treatment had started 2 months before referral and he had been experiencing this dull pain for the last 2 weeks . On clinical evaluation, the periradicular region of the upper left central incisor was tender on palpation . An orthoradial periapical radiograph revealed a periapical lesion with its epicenter being the apical foramen of the tooth no . The tooth was nonresponsive to vitality tests using cold as well as electric pulp testing (vitality scanner, model 2006, analytic technology, redmond, wa). According to patients medical and dental history a presumptive diagnosis of pulp necrosis of traumatic origin associated with extensive ap considering the history of trauma and the apical size of the canal in tooth no . 21, which was assessed to be ~1 mm and taking into account the fact that for a tooth to be labeled as mature, the size of apical foramen needs to be less than 1 mm wide in direct periapical radiographs, it was assumed that the process of apical closure had ceased prior to tooth maturation, due to pulp necrosis . In other words, this phenomenon was independent of orthodontic treatment and could be cured without having to unload the tooth . The treatment plan consisted of single - visit orthograde endodontic intervention and placement of an apical plug . (a) pretreatment radiograph; note the extensive lesion around the apex of tooth no . 21, also consider the 1 mm wide apical foramen and the otherwise healthy crown . (b) the posttreatment radiograph showing the ~5 mm calcium - enriched mixture (cem) plug and the restoration of the tooth . (c) 4-month radiography indicating the bone healing process starting from the periphery of the lesion toward the center . (d) seven - month radiograph still indicates the progression of bone healing and a slight increase in root length (white arrowhead). (e) two - year follow - up; the lesion has perfectly healed and the new hard tissue in the most apical part of the root, beyond the cem plug is surrounded by periodontal ligament (white arrowhead) on a subsequent visit, after local anesthesia with 2% lidocaine containing epinephrine 1:80000 (daroupakhsh, teharn, iran), the root canal therapy was done using the race rotary files (race, fkg dentaire sa, switzerland) to #50/0.04 with simultaneous 5.25% naocl irrigation . Then the canal was dried using paper points (ariadent, tehran, iran). Cem cement powder and liquid (bioniquedent, tehran, iran) were mixed according to the manufacturer's instructions and a ~5 mm plug was placed in the apical area of the canal . The opacity and length of the plug was confirmed with a periapical radiograph, a prefabricated metal post was cemented in the canal and the tooth was restored using composite resin (herculite ultra flow and optibond solo, kerr, orange, ca) [figure 1b]. The patient was put on a regular follow - up plan . By the end of the 1 week all the symptoms faded away; the 4 month postoperative radiograph revealed the incomplete bone replacement process starting from the periphery of the lesion, as the sign of healing [figure 1c] and progressed to the 7 month [figure 1d]. [figure 1e], shows the 2-year follow - up radiograph which shows the complete bone healing . An outstanding finding beyond the apical end of the cem plug was the increase in root length, which was radiographically visible from the 3 month; and was also surrounded by pdl in the final clich [figure 1e]. This case report represented the treatment challenges of an exacerbated large periapical lesion in a maxillary left incisor due to orthodontic load, the etiology of which was assumed to be a previous trauma . The treatment was based on a single - visit root canal therapy with emphasis on reduction of bacterial load as well as establishment of a three - dimensional seal using cem apical plug . The 24-month follow - up revealed not only the perfect bone healing, but also a slight increase in root length, beyond the cem plug . Typically, from a histological point of view, the radiolucent periapical lesions of endodontic origin can be granuloma or to a lesser incidence, cysts . Traditionally, lesions larger than 10 mm were considered cysts; and many clinicians held the view that all cysts do not heal by nonsurgical root canal therapy . It should be pointed out with emphasis that based on radiographs, apical lesions cannot be differentially diagnosed into cystic and noncystic lesions . In 1998, nair made it clear that there are cysts and cysts . He pointed out two distinct categories of radicular cysts, namely those containing cavities completely enclosed in epithelial lining (true cysts) and those containing epithelium - lined cavities that are open to the root canals (bay / pocket cysts). It is also stated that bay cysts tend to response to nonsurgical endodontic intervention, while those few cysts categorized as true, have to be surgically removed . Strong evidences suggest that subsequent to removal of etiologic factors, immunological system contributes to the breakdown of epithelial cyst linings . The low prevalence of true cysts (<10%) and the favorable potential outcome of root canal treatment, with ap prevented or resolved in ~80% of treated teeth are significant considerations in clinical management of apical lesions . In other words, the majority of apical lesions whether cysts or granuloma heal without surgery; nonsurgical approach in cases of ap is the first treatment option, based on the current concept of treatment decision making; and in case of persistent symptoms the surgical removal of the lesion is considered as the next step . The treatment outcome for this patient, confirms this statement that although the lesion responded favorably to nonsurgical treatment, the possibility for surgical intervention was kept in mind in case it did not . The treatment plan was scheduled to be single - visit with effective canal disinfection, without ceasing the orthodontic load . Current evidence indicates the nonsuperiority of single- or multiple - visit root canal treatment in cases of artificial apical stop (i.e., apical plug). The success of treatment for this case was based on appropriate asepsis and filling of the root canal, without increasing the chair - side treatment sessions and thus the chances of canal re - infection . Moreover, although orthodontic treatment for vital teeth with history of trauma can lead to pulp necrosis, in this case the size of apical foramen revealed the occurrence of tooth necrosis to be before root maturation and thus there was no need to unload the tooth . During the posttreatment follow - up, in the absence of symptoms, if radiographs still suggest the same disease, this should not be considered a satisfactory outcome . In other words, even after a 1-year posttreatment follow - up the lesion may decrease in size, probably due to initial decrease in the irritants within the canal, but it still might be radiographically detectable which is interpreted as treatment outcome being unfavorable . Nonendodontic disease, true apical cysts, and healing in progress, should all be carefully considered as a differential diagnosis, the latter putting an emphasis on importance of pulp vitality testing prior to conducting root canal therapy . In this regard, the case history is reviewed, noting previous radiographs and the time elapsed since previous treatment (to recognize healing in progress and thus avoid premature diagnosis). For the presented case the 2-year follow - up revealed the complete healing of the lesion . Last but not least, is the increased root length in this case, which started 4 months posttreatment . In an animal study, cementum formation was reported in contact with mta and cem cement and over the dentinal surface of the resected root ends in all samples which showed entrapped cementoblasts and insertion of pdl fibers . This phenomenon can be due to its sealing ability, biocompatibility, high alkalinity, and antibacterial effect . In the current case, it can be assumed that the increase in root length beyond the cem plug, which was surrounded by pdl [figure 1e], may be due to new cementum formation . When used as an apical barrier, cem cement shows desirable properties in terms of induction of hard tissue formation . Whatever the nature of the apical lesion, in the current case, nonsurgical root canal therapy was favorably able to heal the large lesion . If the orthodontic treatment is not the cause of ap, its continuing has no interference with the endodontic treatment.
In the 19th century, robert koch postulated a causal relationship between a pathogenic microbe and a disease . This was later extended to the role of autoantibodies in the pathogenesis of human disease by witebsky et al . . In 1957, they proposed the fulfillment of several criteria to proof the pathogenic effects of autoantibodies, namely, the direct demonstration of free, circulating, or cell - bound antibodies by indirect means, the recognition of specific antigen against which the antibody is directed, the production of antibodies against the same antigen in experimental animals and finally the appearance of pathological changes in the corresponding tissues of an actively sensitized experimental model that is similar to that in the human disease . Molecular mimicry was proposed as a mechanism by which infectious agents trigger an immune response against autoantigens, resulting in the development of autoimmune diseases . Similar criteria must be satisfied to conclude that a disease is triggered by molecular mimicry . They are as follows: (i) the establishment of an epidemiological association between the infectious agent and the immune - mediated disease; (ii) the identification of t cells or antibodies directed against the patient's target antigens; (iii) the identification of microbial mimics of the target antigen; (iv) reproduction of the disease in an animal model . Although there have been a number of diseases proposed to exhibit the mechanism of molecular mimicry, none has been proven in examples of human diseases based on fulfilment of all four criteria . Guillain - barr syndrome (gbs), characterized by limb weakness and areflexia, has become the most frequent cause of acute flaccid paralysis since the near elimination of poliomyelitis in the world . Most gbs patients have had either gastrointestinal or upper respiratory symptoms one to three weeks prior to the onset of their neurological symptoms, making gbs the prototype of postinfectious autoimmune diseases . Gbs can be classified into two major subtypes, acute inflammatory demyelinating polyneuropathy (aidp) and acute motor axonal neuropathy (aman) depending on whether the myelin or the axonal components of the peripheral nerves are affected . Ean can be transferred to animals by t cells sensitized to peripheral nerve proteins such as p2 protein . However, no investigators have shown conclusive evidence that such autoreactive t - cell response is seen in patients with gbs, indicating that ean is not a true model of aidp . In this paper, we describe the development of a true model of aman, which fulfills all the four criteria of molecular mimicry as well as witebsky's postulate as stated above . This verifies gbs as the first paradigm of an autoimmune disease triggered by molecular mimicry . We also discuss how this disease model has helped uncover the precise mechanism of muscle weakness in gbs, which will potentially lead to the development of better treatments . Gram - negative bacterium campylobacter jejuni, a leading cause of acute gastroenteritis, is the most common antecedent microorganism in gbs . A prospective case - control study detected evidence of recent c. jejuni infection in 26% of patients with gbs in comparison to only 2% of the household controls (a member of the patient's household) and 1% of the age - matched hospital controls . A study showed that c. jejuni infection was associated with aman, but not aidp, although this finding has yet to be verified by other investigators . Autoantibodies are considered to be the pathogenic components which trigger gbs because plasma exchange is proven to be an effective treatment in gbs . Gangliosides constitute a large family of predominantly cell - surface glycosphingolipids bearing a ceramide moiety anchored in the external leaflet of the lipid bilayer and a sialylated oligosaccharide core exposed in the extracellular space . Autopsy studies show that, in aman, igg is deposited on the axolemma of the spinal anterior root, indicating that igg, which binds effectively with complement, is an important factor in the development of aman . Patients who develop aman subsequent to c. jejuni enteritis have igg antibodies against gm1 . In contrast, patients who have c. jejuni enteritis with no neurological sequelae did not have similar autoantibodies . These findings suggest that gm1 is an autoantigen for igg antibodies in patients with aman subsequent to c. jejuni enteritis . Igg anti - gd1a antibodies are also associated with aman subsequent to c. jejuni infection . Lipo - oligosaccharides (loss) are a family of phosphorylated glycolipids anchored on the outer surface membrane of c. jejuni . A c. jejuni strain isolated from an aman patient carrying igg anti - gm1 antibody expressed an oligosaccharide structure [gal 13 galnac 14 (neuac 2 - 3) gal] which protruded from the los core (figure 1). This terminal structure was identical to that of the terminal tetrasaccharide of the gm1 ganglioside . This was the first definitive evidence of molecular mimicry between human peripheral nerves and antecedent agents in gbs . Further evidence regarding the role of antiganglioside antibodies in gbs came in the form of adverse reactions to therapeutic gangliosides extracted from bovine brain tissue . These were previously used in the treatment of various neurological disorders . In a study investigating seven patients who developed aman following intravenous bovine brain ganglioside administration, anti - gm1 antibodies were discovered in six patients and anti - gd1a and -gt1b antibodies in one patient . These findings set the scene for the work done in the development of a suitable experimental animal model for gbs . An aman model was established by the sensitization of rabbits with a bovine brain ganglioside mixture that included gm1 or with an isolated gm1 . The rabbits developed high titres of igg anti - gm1 antibodies, followed by an acute onset of flaccid limb weakness with a monophasic course . Pathological findings in their peripheral nerves showed predominant wallerian - like degeneration with neither lymphocytic infiltration nor demyelination features . Igg was deposited on the axons of the anterior roots, internodal axolemmas, and nodes of ranvier . Cauda equina and spinal nerve root specimens from the paralyzed rabbits showed macrophage infiltration in the periaxonal space . These findings correspond well with pathological findings for human aman [10, 18]. The most straightforward way of verifying that molecular mimicry between microbes and autoantigens cause gbs is to establish a gbs model by the immunization of animals with components of antecedent infectious agents . An aman model was established by the immunization of rabbits with c. jejuni los bearing a gm1-like structure . On sensitization with this gm1-like los, rabbits developed igg anti - gm1 antibodies and subsequent flaccid limb weakness (figure 2). Their nerve roots had occasional macrophages in the periaxonal spaces surrounded by an almost intact myelin sheath . These findings, which are compatible with the features of human aman, provided the evidence that rabbits inoculated with c. jejuni los constitute a valid aman model . This represents the first replica of human autoimmune disease in an animal model immunized by a microbial mimic of a self - antigen . In contrast to the active immunization models above, a passive transfer model was developed to further proof the molecular mimicry theory . Ex vivo nerve - muscle preparations from gd1a - overexpressing mice were exposed to mouse igg anti - gd1a monoclonal antibodies in the presence of a complement source . This allowed the morphological and neurophysiological features of such an exposure to be investigated further . Dense antibody and complement deposits were shown to only be present on the presynaptic motor axons . This was accompanied by severe ultrastructural damage and electrophysiological blockade of motor nerve terminal function . A similar paralyzing effect was found when human sera with igg anti - gd1a reactivity were used instead . These findings indicate that anti - gd1a antibodies arise from molecular mimicry and are the likely trigger of peripheral nerve injury . In summary, gbs is the first true model of molecular mimicry having fulfilled all four criteria as follows: (i) establishment of an epidemiological association between gbs and c. jejuni infection by a prospective case - control study, (ii) identification of autoantibodies against gm1 and gd1a gangliosides in patients with aman subsequent to c. jejuni enteritis [11, 12], (iii) identification of molecular mimicry between gm1 or gd1a and los of c. jejuni isolated from aman [13, 14], and (iv) reproduction of the aman models by active immunization of rabbits with gm1 or the c. jejuni los [16, 19] as well as by passive transfer of anti - gd1a antibodies in mice . This makes gbs the first disease in humans to verify that an autoimmune disease is triggered by molecular mimicry . In both human and rabbit aman, the earliest pathological changes consist of lengthening of the node of ranvier with distortion of the paranodal myelin [17, 21]. Voltage - gated sodium channel dysfunction is postulated to occur at the nodes of ranvier . There has been some controversy as to whether anti - gm1 antibodies truly affect sodium channels at the nodes of ranvier [23, 24]. In the spinal anterior roots of aman rabbits, this binding of autoantibodies triggers complement activation with deposition of c3 components followed by membrane attack complex formation at the nodal axolemma . The sodium channel clusters are then altered by the destruction of their stabilizing components which include the axonal cytoskeleton at nodes, schwann cell microvilli, and paranodal axo - glial junctions . This apparent disappearance of sodium channel clusters significantly lowers the safety factor for impulse transmission, thus causing muscle weakness in aman . This is a novel mechanism by which autoantibodies can modulate sodium channel properties to cause the development of certain neurological disorders . Plasma exchange and intravenous immunoglobulins are equally effective treatments in gbs . Despite the administration of either immunotherapeutic agent, there are still mortality and significant morbidity in patients with gbs; 310% death and 20% immobile after 6 months . Research into new treatment options to improve the final outcome of gbs is urgently required . As previously described, complement activation products are deposited on the outer membrane of schwann cells in aidp patients, and on the axolemma of motor fibres in aman . These postmortem studies show that complement activation followed by membrane attack complex formation is an important mechanism for the glial and neuronal injury seen in gbs . This suggests that complement inhibitors have the potential of being a new, more rational treatment for gbs . Nafamostat mesilate is a synthetic serine protease inhibitor that has been used clinically in japan for over 20 years with no serious adverse effects to patients . As the complement system contains several serine proteases such as c1r, c1s, and c3/c5 convertases, nafamostat mesilate can efficiently inhibit these serine proteases, thus blocking the formation of the membrane attack complex . This inhibition of complement deposition and sodium channel cluster disappearance has been demonstrated in aman rabbits . These experimental findings along with the autopsy findings would justify establishing clinical trials to investigate the use of complement inhibitors as a potential treatment for gbs . In this paper, we have demonstrated how the animal model of aman has contributed to the proof of the molecular mimicry theory . Only the first criterion has been achieved with the association of aidp with cytomegalovirus infection . Until the target antigen in aidp is ascertained, identification of its microbial mimics and reproduction of the disease in an animal model remain unresolved.
The need for new antibiotics has arisen due to the widespread resistance to current drugs . Despite this need, the antibiotic pipeline in the past few decades has been relatively dry in terms of new antibacterial classes when compared with progress against other diseases . One strategy to fight bacterial resistance is to inhibit enzymes that are not the targets of current antibiotics but, instead, act in the same pathways as existing drugs since this might enable the restoration of drug sensitivity via combination therapy . The undecaprenyl diphosphate product (upp) is essential for bacterial cell growth because of its role in the formation of bacterial cell wall peptidoglycan, scheme 1, and it is not produced by humans . Smithkline beecham screened their compound collection against upps but reported no chemically tractable low micromolar hits . Novartis pursued tetramic and tetronic acids and dihydropyridin-2-ones, but noted issues associated with human serum albumin binding and a lack of in vivo activity . Previously, we reported several potent upps inhibitors together with x - ray crystallographic (or modeled) binding modes for a variety of chemical classes including lipophilic bisphosphonates, phthalic acids, diketo acids, anthranilic acids, benzoic acids, aryl phosphonates, bis - amines, and bis - amidines . The most promising of these compounds, a bis - amidine, was shown to have potent activity in biochemical assays, in cellular assays, and in a murine model of mrsa infection . Since upps must bind multiple substrates (ipp, fpp, or more elongated prenyl - pp intermediates) and many inhibitors are to some degree substrate mimics, it is common to observe numerous inhibitors simultaneously bound to upps, with up to 4 binding sites being occupied . However, it is unclear whether inhibitory activity is due to binding to one specific site or to multiple sites . It has been shown that some inhibitors occupy only site 4, an allosteric site distant from the catalytic center, while others bind to site 1, the substrate binding site, complicating docking studies and, regardless of the inhibitor - binding mode, the flexibility of upps creates challenges for virtual screening . Here, to help reduce these problems we employed the 12 crystallographic structures described in previous work to select those that provided maximal enrichment in retrospective virtual screening studies . We then made prospective predictions using these structures, leading to novel upps inhibitors, some with promising antibacterial activity . Following the methods described in previous work, we docked 112 known upps inhibitors having ic50 values <100 m, together with 1000 decoys from the schrdinger decoy collection (having an average molecular weight of 400 da), to escherichia coli upps (hereafter, ecupps). Docking was performed using the glide program, and compounds were ranked by their glide xp score . The proteins were prepared by stripping water and ligand molecules, capping, and neutralizing any unsolved loops, followed by preparation with the schrdinger protein preparation wizard using standard parameters . After docking, compounds were ranked by their docking score, and then area under the curve (auc) analyses were performed . Retrospective enrichment was quite good for 2/12 structures (pdb codes 2e98 and 4h3a), so we docked into these structures for the prospective studies (figure 1). 2e98 is an ecupps x - ray structure containing four lipophilic bisphosphonates (bph-629; ic50 300 nm), which bind to sites 14, one inhibitor to each site.4h3a is an ecupps structure containing a diketo acid inhibitor (bph-1330) which has a 2 m ic50, and the inhibitor binds (in the solid state) only to site 4 . These structures thus have significant differences: only site 4 is occupied in 4h3a, while in 2e98, all four sites are occupied and the protein is in a wide - open conformation (figure 2). Stereo presentation of the x - ray structures chosen for further virtual screening from docking and roc analysis . (b) 4h3a showing one inhibitor bound to site 4 . To find new inhibitors, we began with a library of 450,000 commercially available compounds, the chembridge experimental library . The library was filtered to exclude compounds that had undesired, toxic or reactive functional groups; known promiscuous binders; mw> 460 da or mw <250 da; more than 4 chiral centers; polar surface area (psa)> 150 or psa <50; number of rotatable bonds> 10; or clogp> 5 or clogp 2 . Next, the selected compounds (100,000) were loaded into schrdinger s virtual screening workflow, where they were prepared with ligprep and then docked using the filtering procedure for efficiency and only retaining the top 20% of compounds in the two rapid, initial docking modules htvs (top 20% retained) and sp (top 20% retained). Finally, glide xp was used to assign a final docking score to each molecule . Auc analyses on active and decoy data sets were previously performed using the glide xp module, however, this was impractical for the large filtered chembridge library . Therefore, we relied on htvs and sp modules to provide early filtering before employing the more time intensive xp protocol . We then extracted the 400 top scoring compounds (docking score less than 7 kcal / mol). Binary molprint2d fingerprints were generated using canvas, and 40 clusters were generated using k - means clustering . Of these 40 clusters, the top scoring compounds from each cluster were visually inspected and a representative was chosen from each cluster, resulting in a final list of 100 compounds . These were purchased from chembridge (chembridge corporation, san diego, ca) and then assayed for upps inhibition activity . Similarity searches based on these active compounds were then performed using pubchem and scifinder, and additional compounds were obtained and tested . Molecular weights and purities were verified by mass spectrometry and sds page, respectively . Briefly, the condensation of fpp with ipp catalyzed by upps was monitored by using a continuous spectrophotometric assay in 96-well plates with 200 l reaction mixtures containing 400 m 2-amino-6-mercapto-7-methylpurine ribonucleoside (mesg), 350 m ipp, 35 m fpp, 20 mm trishcl buffer (ph 7.5), 0.01% v / v the ic50 values were obtained by fitting the inhibition data to a rectangular hyperbolic dose response function using graphpad prism 4.0 software (graphpad software, san diego, ca). The ic50 values for the most active hits were verified using a radiometric assay with 2.5 m fpp, 25 m [h]ipp, and 0.01% v / v triton x-100 . Subtilis (atcc 6051), escherichia coli (atcc 29425), and saccharomyces cerevisiae (atcc 208352) were purchased from the american type culture collection . Bacillus subtilis strain 168, bacillus anthracis strain sterne, listeria monocytogenes strain 4b f2365, staphylococcus aureus usa300 (methicillin - resistant), e. coli mc400, pseudomonas putida, and enterococcus faecalis u503 (vancomycin - resistant) were from our laboratory strain collection . Ic50 values for e. coli growth inhibition were determined by using a microbroth dilution method . The culture was then diluted 500-fold into fresh lb medium, and 100 l was inoculated into a 96-well flat bottom culture plate (corning inc ., the starting concentration of each compound was 0.3 mm, and this was 2-fold serially diluted . Plates were incubated for 3 h at 37 c to midexponential phase . A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) cell proliferation assay (atcc) ten microliters of mtt reagent was added into each well, followed by incubation for 24 h until a purple precipitate was visible . Then, 100 l of detergent reagent was added and plates were further incubated in the dark at 23 c for 2 h. the absorbance was recorded at 570 nm . For each inhibitor, two independent experiments were performed and the ic50 values found were averaged . A 16 h culture of b. subtilis was diluted 50-fold into fresh luria the culture was then diluted 500-fold into fresh lb medium, and 100 l was inoculated into a 96-well flat bottom culture plate (corning inc ., the starting concentration of each compound was 0.5 mm and was then serially diluted . A nonlinear regression analysis was carried out on the data obtained using origin 6.1 . For each inhibitor, two independent experiments were performed and the ic50 values found were averaged . The protocol was the same as with the b. subtilis assay protocol except that ypd medium was used and the 96-well plate was incubated for 36 h instead of 1216 h. b. subtilis strain 168, b. anthracis strain sterne, e. coli mc4100, and p. putida were grown to stationary phase in 10 ml of lb broth at 37 c . S. aureus usa300 (methicillin - resistant), e. faecalis u503 (vancomycin - resistant), and l. monocytogenes strain 4b f2365 were grown to stationary phase in 10 ml of brain heart infusion (bhi) medium at 37 c . The cultures were adjusted to an od600 of 0.016 in the designated medium before being added to 96-well microplates . Successive 2-fold dilutions of compounds 1 and 4 were added to the cultures (0.2564 g ml). As a control, kanamycin (132 g ml) was added to samples of e. coli, b. subtilis, b. anthracis, p. putida, and l. monocytogenes . Gentamycin was used as a control for s. aureus and e. faecalis with dilutions from 1 to 64 g ml . As a negative control, an equal volume of dmso the minimum inhibitory concentration (mic) that suppressed at least 99% of bacterial growth was established based on culture turbidity in the microbroth dilution assay . The assay was repeated in three replicates, and values were averaged, or a range was reported . The purities of the key compounds investigated, obtained from chembridge (1 and 4), were determined by high - performance liquid chromatography and structures verified by nmr spectroscopy and high resolution mass spectrometry (supporting information figures s2s9) and were consistent with the structures provided by the vendor . In previous work, we obtained moderate correlations between enzyme inhibition activity and docking scores within a congeneric series of upps inhibitors (lipophilic bisphosphonates) using docking methods, so we first examined whether we could obtain similar correlations between docking scores and experimentally determined ic50 values for the 112 known actives . There was no significant correlation between docking scores and pic50 values (pic50 = logic50), figure s1 in the supporting information . The wide variety of potential binding modes (sites 1, 2, 3, and 4) and protein conformations would be expected to make it difficult to achieve a good correlation between a scoring function and the experimentally determined pic50 values, in addition to the assumptions made in scoring functions that cause inaccuracy when compared to experimental affinities . Nevertheless, docking studies can provide enrichment of active compounds from large libraries, even though docking scores rarely correlate well with activity when structurally diverse compounds are involved . We thus next employed an area - under - the - curve (auc) analysis, also known as the receiver - operating - characteristic (roc), a method that has been shown to be useful in validating structure - based virtual screening protocols and is a standard method for evaluating such protocols . We therefore tested 12 ecupps x - ray structures for their ability to separate actives (ic50 <100 m) from decoys (presumed inactive compounds in the decoy library). Several ecupps x - ray structures showed a good separation of active from decoy compounds, with auc values of 0.8 . These structures also demonstrated early enrichment, as evidenced by the steep initial slope of the curve . This means that the best scores were given primarily to active compounds and suggests that, in screening a large compound library, the best scoring compounds would be enriched in upps inhibitors . We thus picked the two x - ray structures (pdb codes 2e98 and 4h3a) that provided significant early enrichment and a high auc in the validation studies, for predictive studies . Using these two x - ray structures, we screened the chembridge express - pick compound library (after filtering) and determined 400 hits with glidexp scores less than 7 kcal / mol (lower energy is better). Since many highly ranked compounds were chemically very similar, we clustered the top scoring compounds and selected representatives from each cluster to ensure chemical diversity among the compounds to be tested . The screening of the chembridge express - pick compound library using the validated docking protocol resulted in the discovery of three new upps inhibitor classes: the 4-oxo-2-thioxo-1,3-thiazolidines, also known as rhodanines (e.g., compound 1), dihydroxyphenyls (the resorcinol, compound 2), and pyrimidinetrione (a barbiturate analogue, compound 3). All three compounds are predicted to bind in either site 1 or 3 of the 2e98 crystal structure (figure 3), although x - ray crystallographic studies will be required to confirm this binding mode (and our attempts to obtain crystal structures of these systems have not been successful). In any case, the three new inhibitors discovered represent upps inhibitors with drug - like physicochemical properties, passing the common drug - like filters as described in the methods and materials section . The most potent of the 3 compounds was the 4-oxo-2-thioxo-1,3-thiazolidine 1 (ic50 2.6 m against s. aureus upps), which in an initial screen for bioactivity was also found to be active against b. subtilis, mic (minimal inhibitory concentration) 3 g / ml (table 1). For this reason, we chose to next investigate analogues based on the 4-oxo-2-thioxo-1,3-thiazolidine core . Three new classes of upps inhibitors discovered via virtual screening: (a) chemical structure and computed docking mode of compound 1, a rhodanine derivative; (b) chemical structure and docking mode of compound 2, a resorcinol derivative; (c) same for compound 3, a barbiturate . We next obtained 16 additional compounds from chembridge, from sigma - aldrich, and from the drug synthesis and chemistry branch, developmental therapeutics program, division of cancer treatment and diagnosis, national cancer institute (419, table 1), containing the 4-oxo-2-thioxo-1,3-thiazolidine core and tested them for activity against saupps and ecupps, as well as a preliminary activity screen against b. subtilis, e. coli, and s. cerevisiae (the latter as a general cytotoxicity control, since it lacks upps). The alkyl carboxylic acid containing compounds with the 4-oxo-2-thioxo-1,3-thiazolidine core were active in assays against b. subtilis, and the most potent compound was 4 (an analogue of 1). 4 was roughly equipotent against saupps and ecupps with an ic50 of 2 m . Additionally, 4 was active against b. subtilis with an mic 0.43 g / ml and was very weakly active (200 g / ml) against s. cerevisiae, indicating that 4 was not generally cytotoxic . Since 1 and 4 showed significant activity in enzymatic and bacterial growth assays, we subsequently tested them against several pathogens . Both 1 and 4 gave mic values in the high ng / ml to low g / ml range against b. anthracis sterne, mrsa, vre, and l. monocytogenes, table 2 . This promising antibacterial activity suggested the potential utility of these upps inhibitors in synergizing with other cell wall agents but where significant resistance has emerged, such as with methicillin (mrsa) and vancomycin (vre). The compounds were tested against a panel of both gram - positive (top five) and gram - negative (lower two) bacteria . To investigate the possibility of synergistic interactions with known cell wall biosynthesis inhibitors, we determined the fractional inhibitory concentration index (fici) values for three systems: mrsa, using 1 + methicillin; vre, using 1 + vancomycin; and b. anthracis, using 1 + ampicillin . The fici is defined aswhere fic(a) and fic(b) are the fractional inhibitory concentrations of drugs a and b, mic(a) and mic(b) are the mic values of drugs a and b acting alone, and mic(ab) and mic(ba) are the mic values of the most effective combination of drug a or b in the presence of drug b or a. using this method, fici values of <0.5 represent synergism,> 0.5 and <1.0 represent additivity,> 1 and <2 represent an indifferent effect, and> 2 represents drug antagonism ., the fici for 1 + methicillin in mrsa is 0.11, which indicates strong synergism during late stage growth . However, with both vre (1 + vancomycin) and b. anthracis (1 + ampicillin) the fici values are in the 12 range, which indicates an indifferent effect . In vitro synergy assays . Isobolograms for growth inhibition of vre, mrsa, and b. anthracis strain sterne . (a) 1 and vancomycin inhibition of e. faecalis u503 (vancomycin - resistant, vre). Fici = 1.78 . (b) 1 and methicillin inhibition of s. aureus (usa300) what is particularly interesting about the most active species investigated here (1) is that it has a structure that is very similar to that found in the drug epalrestat, an aldolase reductase inhibitor that is used to treat diabetic neuropathy, and is approved for clinical use in japan, china, and india . This is encouraging because rhodanines as a class are known to often have activity in widely different assays, and indeed computer programs such as pains categorize, e.g., 14 (as well as epalrestat) as possible this can mean that the compounds cause false positives in assays, or that they may be multitarget inhibitors . In some cases multitargeting may be undesirable; however, in the context of anti - infective development, multitargeting is expected to increase efficacy as well as decrease the possibility of resistance development, both very desirable features . First, we carried out an in silico screen of 100 known upps inhibitors and 1000 decoys using 12 reported upps x - ray structures . The two x - ray structures providing the best enrichment in an auc - roc analysis were then used to screen a subset of 100,000 compounds selected for drug - like activity from an initial chembridge library of 450,000 compounds . We then tested the 100 in silico hits in vitro against saupps and ecupps, leading to several m upps inhibitors (as deduced from both ppi release and radioactive assays). The most potent lead was 1, which is structurally quite similar to epalrestat, in clinical use to treat diabetic neuropathy . 1 (and its analogue 4) inhibited the growth of gram positives; they did not inhibit the growth of gram negatives (important with e. coli in the context of maintaining commensal microflora), and they had no activity against s. cerevisiae . Activity against b. anthracis, a vancomycin - resistant enterococcus spp ., and listeria monocytogenes was good, in the 0.1254 g / ml range, and there was very strong synergy (fici = 0.11) with methicillin and 1 in a mrsa strain of s. aureus, suggesting that 1 could be a promising lead (in combination therapies) for treating staph infections.
Due to the lower level and impaired binding activity of cell - surface receptors on monocytes, a poorly controlled diabetic state increases susceptibility to infections such as endotoxemia . In the diabetic patients, infection is more serious and difficult to eradicate . However, the severity of cardiopulmonary dysfunction in type 1 diabetes during systemic endotoxemia remains unclear . As a systemic infection, endotoxemia is mainly caused by the endotoxin (lipopolysaccharides (lps) from gram - negative bacteria . Lipopolysaccharides binds to cd14 receptor with lps - binding protein when entering the mammalian bloodstream . As a result, nuclear factor-b is activated to induce monocytes, macrophages, and endothelial cells to release cytokines such as tumor necrosis factor - alpha (tnf-) and causes tissue damage . Also, arterial pressure is decreased due to the dilation of peripheral vessels in rats during endotoxemia . Tnf- plays an important role in this endotoxin - induced shock because serum tnf- level increases during lethal endotoxemia [9, 10]. In addition, acute respiratory distress syndrome, which is caused by acute lung injury, is another complication of endotoxemia . A great number of monocytes and macrophages are present in alveoli and release tnf-, which damages pulmonary vessels and increases lung vascular / epithelial permeability . Hence, septic patients may suffer from critical shock and acute respiratory distress syndrome as a group experience high mortality . The mortality from cardiovascular disorders in type 1 diabetes is 4-to-37 times higher than that in the general population . Severely uncontrolled diabetic state may initiate pathologic events leading to the capillary leak of acute respiratory distress syndrome as well . Therefore, we hypothesized that the cardiopulmonary dysfunction induced by systemic endotoxemia will be more marked in type 1 diabetes . In the present study, we used type 1-like diabetic rats to investigate hemodynamic dysfunction and lung injury after intravenous lps infusion . Also, the changes in tnf- in serum and lung lavage fluid were determined . Ninety - six adult male wistar rats (320 20 g) were purchased from the animal center of national cheng kung university medical college (tainan city, taiwan). They were housed in groups of four at an ambient temperature of 24 1c and maintained under a normal light - dark cycle (14:10 h; lights on at 6:00 am). All protocols were approved by the institutional animal care and use committee of national cheng kung university, tainan, taiwan . All experimental procedures were conducted in compliance with the national institutes of health's guide for the care and use of laboratory animals . Adequate anesthetic level was maintained to abolish the corneal reflex and pain reflexes induced by tail pinching throughout the course of all experiments (approximately 8 h each) after a single intraperitoneal dose (1.4 g / kg) of urethane . Animals were randomly assigned to four groups (24 rats for each group): (a) normal rats receiving 0.9% normal saline administration (ns), (b) normal rats receiving lps administration (nl), (c) diabetic rats receiving normal saline administration (ss), and (d) diabetic rats receiving lps administration (sl). An animal model of type 1 diabetes, streptozotocin-(stz-) treated rats, was prepared by intravenously injecting rats with stz (sigma, st . Louis, mo) (60 mg / kg) via the femoral vein to irreversibly destroy pancreatic cells after the animals had fasted for 72 h . Rats with hyperglycemia (blood sugar> 300 mg / dl) and polyuria were considered diabetic . At the 5th week after stz administration, survival time, and fresh solutions of lps (from escherichia coli 0111:b4, sigma chemical co.) were prepared in phosphate buffered saline (ph 7.40) at a concentration of 10 mg / ml for infusion (15 mg / kg) into the femoral vein to induce endotoxemia . Different subgroups of animals (8 rats for each subgroup) were used for each of the four experiments: (i) determination of survival rate in normal and diabetic rats after receiving an injection of lps or saline; (ii) determination of the change of hemodynamic parameters during endotoxemia and lung edema at 180 min after lps or saline injection; (iii) determination of arterial blood gas, lung protein leakage, tnf- level in serum and bronchoalveolar lavage, and lung histopathology at 180 min after lps or saline injection . Animals were anesthetized with an intraperitoneal injection of urethane (1.4 g / kg) and the right femoral artery was cannulated using polyethylene catheters (pe-50). Mean arterial pressure (map) and heart rate (hr) were recorded using a polygraph (mp35, biopac systems inc ., goleta, ca, usa) every 20 minutes from the arterial tube in the femoral artery throughout endotoxemia . In order to determine arterial ph, arterial partial pressure of o2 (pao2), co2 (paco2), and o2 saturation (so2) of rats, 0.4 ml of arterial blood was sampled from the femoral artery 3 h after administration of lps using a syringe rinsed with heparin . Venous blood samples were taken from rats at 3 h after administration of lps or saline . Blood samples were allowed to clot for 30 minutes at room temperature and then were centrifuged for 20 minutes (2000 g, 4c). The supernatants were harvested and stored at 70c until measurements were taken . At the same time, bronchoalveolar lavage samples were collected by perfusing saline from the endotracheal tube . The concentrations of tnf- were determined using a double - antibody sandwich elisa (r&d systems, minneapolis, mn, usa) according to the manufacturer's instructions . The optical density of each well was determined by microplate photometers (multiskan ex, 110120 v, thermo fisher scientific inc ., albumin content of the bronchoalveolar lavage fluid was determined to assess the damage to endothelial cells of the lung capillaries . The lavage samples were analyzed using a bio - rad protein assay system (bio - rad hercules, ca) with bovine serum albumin (bsa) as the standard . The albumin content in the bronchoalveolar lavage fluid was calculated by dividing the albumin by dried weight of lung to evaluate pulmonary capillary / endothelial cell permeability . Three hours after administration of lps (or saline), the entire lungs were dissected, weighed (wet weight), and dried (at 63c for 48 hours). The wet / dry weight ratio of lung tissue was calculated to evaluate lung edema . Rats were perfused with saline followed by 4% paraformaldehyde buffer at 180 min after lps administration . Serial sections (4 m) of right upper lobe were stained with hematoxylin and eosin for microscopic evaluation . The characteristics of lung damage include vascular congestion, hemorrhage, polymorphonuclear leukocytes (pmn) infiltration, and edematous changes of alveolar wall . Each characteristic was scored (0: normal; 1: mild; 2: moderate; 3: severe) by a pathologist and overall lung injury was further calculated according to the sum of the score . Moreover, the degree of leukocyte infiltration, which was assessed as the pmn / alveoli ratio, can stand for lung damage . We selected 10 randomly areas at high - power field (hpf, 400x) of each blind sample . Then alveoli and pmns in each area were counted and pmn / alveolus ratio was expressed by dividing the sum of pmns in 10 hpf fields by that of alveoli . Statistical analysis was conducted using analysis of variance (anova) for factorial experiments, and repeated - measures anova (followed by scheffe after hoc test) for comparing hemodynamic parameters . The wilcoxon rank - sum test was used for analysis of survival time the kaplan - meier test for survival rates . Significant differences between groups were assumed to be present at values of p <0.05 . Blood glucose in diabetic rats was substantially (approximately 5-fold) and significantly (p <0.001) higher than that in normal rats (361 9 mg / dl versus 73 3 mg / dl). Body weights in stz - treated rats were significantly lower than those in controls (262 10 gm versus 385 9 gm, p <0.05) at the 5th week after injection of stz . After administration of lps, the survival time of stz - treated rats was markedly shorter than that of control rats (180.0 14.6 min versus 224.4 17.9 min, p = 0.027). Also, the survival rate of diabetic rats was significantly lower than the rate for control rats (figure 1). Figure 2 shows that tnf- levels in serum and bronchoalveolar lavage fluid were significantly raised by administration of lps in both normal and diabetic rats . Actually, the tnf- levels for normal or diabetic rats were undetectable with saline treatment . Moreover, the lps - induced increases of tnf- levels in serum and bronchoalveolar lavage fluid were statistically higher in diabetic rats as compared with control normal rats . The time course for changes in cardiovascular parameters in normal and stz - treated rats after the administration of saline or endotoxin is shown in figure 3 . Mean arterial pressure was higher in diabetic rats than that in normal rats with saline administration . Moreover, heart rate and mean arterial pressure were significantly decreased in diabetic rats than those in control ones . The ratio of wet lung weight to dry lung weight and the albumin content in lung increased significantly after lps administration, and these parameters were indistinguishable between nl and sl groups (figure 4). Histopathologic examination of the lungs demonstrated several changes after 3 hours of lps administration (figure 5). The interstitial spaces of aveoli became thickness with polymorphonuclear leukocytes infiltration as well as edematous changes of alveolar walls were showed in both normal and diabetic groups . Quantitative analyses of histology were shown in figure 6 . The lung injury score and pmns / alveoli ratio were significantly increased in nl and sl groups compared with that of ns and ss groups . However, the values of lung injury score and pmns / alveoli ratio are indistinguishable between sl and nl group . Baseline values for pao2 and sao2 for diabetic rats were significantly lower than those for control rats (table 1). During endotoxemia, paco2 diminished significantly, whereas pao2 and sao2 were significantly greater in control and diabetic rats . In the present study, we demonstrated alterations in cardiopulmonary function in both control and type 1 diabetic rats during systemic endotoxemia . We found higher tnf- levels in serum and bronchoalveolar lavage fluid in diabetic rats during systemic endotoxemia . Shorter survival times and the hemodynamic abnormalities observed in diabetic rats were worse after lps challenge . However, the difference is minor from normal rats . A previous report showed a lower survival rate with a higher tnf- level during endotoxemia . Multiple organ injury and irreversible cardiovascular collapse are induced in animals receiving recombinant human tnf . Conversely, monoclonal antibodies against tnf- and agents which antagonize lps - induced production of tnf- are effective in preventing lps - induced lethality in vivo and in vitro [9, 17, 18]. Thus, tnf- level seems to be related to survival rate . In the present study, the finding of a high level of tnf- in the serum and bronchoalveolar lavage fluid of diabetic rats appears to be responsible for the higher mortality rate associated with lps treatment . Regarding hemodynamic parameters, our data show that the value of mean arterial pressure is higher in diabetic rats than control rats during saline administration . These changes are consistent with previous studies that used the stz - induced diabetic model for chronic and continuous hemodynamic measurement [19, 20]. Those studies showed that the onset of hyperglycemia in the earliest stage of diabetes increases mean arterial pressure . Moreover, studies in patients with early uncomplicated type 1 diabetes also show an increase in arterial pressure during poor glycemic control [21, 22]. This increase is correlated with angiotensin ii, due possibly to overstimulation of the renin - angiotensin system in response to hyperglycemia . In addition, endothelial dysfunction, which can be seen in type 1 diabetic patients, was found in the rat's aorta at the 4th week after stz injection . Heart rate was reported to be increased in order to compensate for the endotoxin - induced low blood pressure, which is consistent with our results during the early stages of endotoxemia . However, later on, the animals were unable to compensate, and heart rate dropped dramatically . Our data demonstrated that blood pressure drops in diabetic rats significantly than that in normal rats after lps challenge whereas the difference among lps - treated normal and diabetic groups was minor . The present results are in good agreement with those of previous findings showing that endotoxin administration to diabetic animals resulted in more severe cardiovascular dysfunction than in nondiabetic animals . High levels of tnf- were found in serum and lung in rats with endotoxemia, and more albumin was present in endotoxemic lungs than in control ones . Yoshinari et al . Found numerous polymorphonuclear cells in thickened alveolar interstitial tissue during endotoxemia . The tnf- released by polymorphonuclear cells damages the pulmonary vessels and increases alveolar - vessel permeability which causes lung protein leakage . Some evidence has shown that albumin content in the bronchoalveolar lavage fluid increases significantly, and lung compliance is reduced after endotoxin treatment . In this study, we showed that diabetic rats have significantly higher tnf- levels in lavage fluid from the lungs, but not greater albumin content and lung edema during endotoxemia . Rojas et al . Have shown that tnf- and lung edema have different time courses during endotoxemia . The tnf- content was significantly higher at 24 h than at 6 h after lps injection . However, the degree of lung edema was lower at 24 h than at 6 h. there seemed to be a tnf- threshold for causing lung injury and another, much higher, threshold for inducing more serious damage . In contrast, previous studies showed that diabetes may be associated with a lower - than - average risk of acute lung injury after intratracheal instillation of lps, and the concentrations of tnf- in the bronchoalveolar lavage fluid of intratracheal lps - treated diabetic rats were markedly reduced . It is possible that intratracheal instillation of lps and intravenous lps administration cause different pulmonary responses in diabetic rats . In addition, the response to lps may be different between stz - induced and alloxan - induced diabetic rats . Increases in vasodilators and vasoconstrictors that are induced by endotoxemia cause vessel dysfunction and redistribute blood flow . The insufficient blood flow for several organs slows down metabolic rate and decreases oxygen consumption . As a result, the generation of co2 decreases [33, 34]. . The higher mortality rate of stz - treated rats may correlate with higher levels of tnf- after lps administration, but the mechanisms underlying the differences in survival rate between diabetic rats and nondiabetic rats are still unclear . To explore whether tnf- plays a critical role during endotoxemia in diabetic rats, it will be necessary to study in vivo blockade of tnf-. Moreover, it is important to determine whether glycemic normalization in diabetic rats can reduce tnf- levels and improve survival after lps injection . Also, further studies will be necessary to determine the responses of other inflammatory mediators (e.g., nitric oxide, macrophage inflammatory protein-1, etc .) During endotoxemia . In conclusion, the difference of cardiopulmonary dysfunction between normal and stz - induced diabetic rats was minor whereas the tnf- levels in serum and bronchoalveolar lavage fluid were increased more markedly in diabetic rats receiving an lps challenge.
Histological features of nonalcoholic fatty liver disease (nafld) include steatosis, hepatocellular ballooning, the formation of mallory bodies, apoptosis / necrosis, and inflammation . Around 1020% of patients with nafld have nonalcoholic steatohepatitis (nash), which can develop into cirrhosis and hepatocellular carcinoma [25]. Because excess nutrition intake is one of the main causes, nafld is often accompanied by obesity, insulin resistance, hypertension, and/or dyslipidemia, which are manifestations of the metabolic syndrome . The two - hit theory is increasingly being adopted to explain the pathogenesis of nafld and nash . In this theory, the first hit consists of the accumulation of fatty acids / triglycerides in the liver, while the second hit involves oxidative stress, mitochondrial dysfunction, and inflammation, which ultimately cause liver damage . It is also clear that inflammatory cytokines and insulin resistance are closely associated with fatty liver during the progression of nafld . In previous studies that examined lipid metabolism in the context of nafld, dysregulation of cholesterol metabolism, we focus on the role of cholesterol and its metabolites on the pathogenesis of nafld, and also the validity of cholesterol management as a method of treating this disease . Hepatic lipid homeostasis represents a balance between lipid uptake, synthesis, catabolism, and secretion . Therefore, steatosis, a typical characteristic of nafld, is expected to be caused by disordered lipid metabolism, particularly inhibition of fatty acid oxidation and enhanced lipogenesis . Many factors involved in hepatic lipid metabolism pathways have been identified, even though the precise cellular networks are not fully elucidated . Amp - activated protein kinase (ampk) works as a metabolic master switch, and its activity is regulated by adiponectin and tumor necrosis factor- (tnf). Inhibition of ampk results in the activation of sterol regulatory element - binding protein-1c (srebp-1c), which upregulates fatty acid synthesis - associated enzymes, such as acetyl - coa carboxylase (acc) and fatty acid synthase (fas). This leads to enhanced fatty acid synthesis and overproduction of triglycerides, ultimately resulting in liver steatosis . Fatty acids are used for -oxidation in mitochondria and peroxisomes under the regulation of peroxisome proliferator - activated receptor- (ppar). Fatty acids are ligands for ppar, which transactivates the expression of genes involved in the transport, oxidation, and export of free fatty acids, including carnitine palmitoyltransferase-1 (cpt-1), the rate - limiting enzyme in fatty acid -oxidation . The relationship between nafld and lipid metabolism has been extensively investigated in studies that analyzed the hepatic gene expression profile in animals fed a high - fat diet and in liver biopsy samples from nafld patients [1014], and their expression profiles have been compared with those in normal individuals . Figure 1 summarizes the pathological changes in the nafld liver, which may lead to the accumulation of triglycerides, free fatty acids, and cholesterol . To our knowledge first, hepatic steatosis develops because of upregulated fatty acid synthesis, but it is questionable whether downregulation of fatty acid oxidation is also involved [1518]. Third, insulin resistance, which is common in nafld, causes fatty liver, while increases in hepatocyte fatty acids levels cause hepatic insulin resistance . Fifth, disturbed insulin signaling in hepatocytes leads to steatosis associated with the activation of srebp-1c and the induction of fatty acid synthesis . Recent findings suggest that the cannabinoid system is also involved in the development of fatty liver [2224]. In animal studies, cannabinoid 1 (cb1) receptors were activated by a high - fat diet via induction of the synthesis of endocannabinoids, such as 2-arachidonoylglycerol and anandamide . Cb1 receptor activation enhanced the expression of several lipogenic factors, including srebp-1c, acc and fas, and downregulated cpt-1, resulting in increased de novo fatty acid synthesis and suppression of fatty acid oxidation . However, in the context of lipid metabolism, the signaling pathway downstream of the cannabinoid receptor has not been identified . In humans, cholesterol is absorbed from the diet and synthesized by cells in various tissues . A healthy man weighing 60 kg contains approximately 140 g of cholesterol, but only 1% of the total cholesterol is involved in a dynamic metabolic cycle . In one study, the mean intake of dietary cholesterol was estimated to be 300500 mg / day . They also reported that the dietary cholesterol aggregates into micelles with biliary cholesterol (8001300 mg / day) in the duodenum . Physiologically, approximately 50% of the cholesterol is absorbed in the jejunum via a cholesterol transporter niemann - pick c1-like 1 (npc1l1) expressed on the brush border membrane . The cholesterol is then transported to the liver in the form of chylomicrons and chylomicron remnants . Npc1l1, which may facilitate the hepatic accumulation of cholesterol, is expressed on the canalicular membrane of hepatocytes in humans . Another transporter pump system involving atp - binding cassette (abc) g5/g8 excretes cholesterol into bile . The main metabolic pathways of cholesterol in hepatocytes include (1) cholesterol de novo synthesis (acetyl - coa - mevalonate - cholesterol pathway); (2) cholesterol uptake in the form of ldl and chylomicron remnants; (3) cholesterol excretion into the blood in the form of vldl; (4) cholesterol excretion and uptake through bile via abcg5/g8 and npc1l1, respectively; (5) synthesis of bile acids and their excretion . Under normal conditions, these pathways interact with each other to maintain cholesterol levels within a specific range . However, in nafld patients, these systems are highly disorganized . Srebps act as regulators of hepatic cholesterol levels and activate genes involved in the synthesis of cholesterol and free fatty acids . Srebp cleavage - activating protein (scap) has a cholesterol - sensing domain that senses intracellular cholesterol levels and directs the activity of srebps . Physiologically, when intracellular cholesterol levels are low, srebps are first translocated to the golgi apparatus by scap and undergo proteolytic cleavage . Next, the cleaved activated form of srebp is released to the nucleus . When intracellular cholesterol levels are high, scap activity and srebp activation are suppressed . However, in the context of nafld, the regulatory loop of srebp is disturbed, even if the intracellular levels of cholesterol and/or fatty acids are high . In our study using liver biopsy samples from nafld patients, despite excess cholesterol accumulation in hepatocytes, de novo cholesterol synthesis remained greatly enhanced even though srebp-2 expression was downregulated . In the liver of these patients, as evidence of excess cholesterol accumulation, cholesterol uptake was reported to be suppressed because of markedly downregulated expression of ldl receptor (ldlr). Cholesterol excretion was enhanced via overexpression of abcg5/g8, apolipoprotein b, and microsomal triglyceride transfer protein (mtp), but it was considered that the secretion of cholesterol reaches a plateau in nafld patients . Even in this situation, cholesterol synthesis continued with upregulated expression of hmg - coa reductase and synthase, farnesyl p - p synthase and squalene synthase [3032]. This is because excess levels of cholesterol and its oxysterol metabolites, which are agonists for liver x receptor- (lxr), cause excess fatty acid synthesis and steatosis by activating the lxr-srebp-1c pathway . Lxr expression was also upregulated in the liver of nafld patients [31, 32]. As shown in figure 1, cholesterol uptake in the form of ldl is limited by the intracellular accumulation of fatty acid and cholesterol, while fatty acid and cholesterol synthesis are upregulated in the nafld liver . These findings suggest that the feedback system regulating intracellular lipids levels is disrupted in nafld . In some nutritional investigations, it has been suggested that high - fat, high - fat plus low - protein, high - carbohydrate, and/or high - cholesterol diets are the main causes of nafld [3336]. Although many nafld patients show excess nutrition intake, obese, and/or insulin resistance, not all patients exhibit these features . In our nutritional analysis on japanese population, naturally, the dietary intake levels of total energy, fat, and carbohydrate were markedly higher in obese nafld patients with insulin resistance than those in nonobese nafld patients and healthy volunteers . The most interesting finding was that cholesterol intake was significantly higher in nonobese nafld patients than in obese nafld patients although cholesterol intake in obese patients was also significantly higher than that in healthy volunteers . In our hepatic expression analysis of lipid metabolism - associated genes, we found that lxr expression levels were significantly higher in nonobese nafld patients than in obese nafld patients . Of note, cholesterol overload can upregulate lxr expression and activate fatty acid synthesis by increasing oxysterol levels, metabolites of cholesterol that act as agonists for lxr and activate the lxr-srebp-1c pathway . These nutrition and gene expression profiles indicate that excess cholesterol intake (i.e., cholesterol supply) itself can be a strong stimulant for the development of steatosis, even though the total calorie intake may be within the normal range . Recent reports using model animals support our findings in nonobese nafld patients . Fatty liver without obesity can be established in animal models by feeding them with a hypercholesterolemic diet containing normal calorie level [3840]. Although this animal model showed marked hypercholesterolemia, which was not observed in our patients, this may be because the diet for animals contains a very high cholesterol content (0.21.25%). Moreover, serum cholesterol levels may be preserved in nafld patients because dietary cholesterol is promptly taken up into the hepatocyte cholesterol pool . The progression from simple steatosis to steatohepatitis (nash) usually involves the second hit, such as oxidative stress and inflammation . In some studies of nutritional animal models, the accumulation of cholesterol rather than fatty acids / triglycerides plays a critical role in this progression, possibly because of increased susceptibility to oxidative cell death . It has also been suggested that the regulation of cholesterol can control c - reactive protein levels and insulin sensitivity . Conversely, in some reports, the progression of triglyceride accumulation and suppression of fatty acid oxidation were not hepatotoxic and actually protected against worsening liver damage . Ezetimibe, a blood cholesterol lowering agent, is a npc1l1-specific inhibitor and selectively blocks 54% of cholesterol absorption from the intestine in humans and in animals [43, 44]. Ezetimibe is quickly absorbed, enters the enterohepatic circulation, and has a half - life of 24 hours . From a nutritional point of view, it is important that ezetimibe does not inhibit the absorption of fat - soluble vitamins . In our clinical study, nonobese nafld patients showing excess intake of dietary cholesterol were treated with ezetimibe . After starting the therapy, although significant changes were not seen in their body weight, their serum alt levels decreased by 49.3 16.1% and 45.3 24.2% at 6 and 12 months, respectively . Npc1l1 knockout mice with excess nutrition intake were resistant to fatty liver, while ezetimibe elicits therapeutically significant effects in animal models of nafld [46, 47]. These findings demonstrate that over intake and hepatic accumulation of cholesterol, leading to the activation of the lxr-srebp-1c pathway, are closely related to the development of nafld . Accordingly, inhibiting cholesterol absorption or reducing dietary cholesterol intake may offer a reliable therapeutic strategy for nafld . It has also been reported that hmg - coa reductase inhibitors (statins) improve serum alt levels in nafld patient [4850]. Considering these findings, reducing hepatocytic accumulation of cholesterol may represent a fundamental treatment strategy for nafld . To establish treatments focusing on cholesterol management, for example, cholesterol - restricted diet or lipid modulators (ezetimibe and statins) may be less effective in obese nafld patients with insulin resistance than in nonobese patients . This is because other factors associated with obesity and insulin resistance are involved in the development of fatty liver, and these factors may mask the effect of ezetimibe . For example, does the therapeutic effect of statin in combination with ezetimibe surpass that of monotherapy? Can long - term cholesterol management with ezetimibe and/or statins really improve steatosis as well as alt levels in nafld? It is also important to assess whether the clinical effects of cholesterol management are observed in patients with steatohepatitis (i.e., nash) as well as patients with simple steatosis, and whether there is a synergistic / additive effect of cholesterol management in combination with antioxidant therapy or liver protection therapy . Lifestyle modifications offer simple therapeutic targets for nafld . Cognitive nutritional support, which is aimed at reducing calorie - intake and avoiding overeating, should be developed alongside pharmaceutical treatments to prevent the disease progression to cirrhosis and hcc . Excess cholesterol intake, in particular, the accumulation of cholesterol rather than triglycerides may play a critical role in the progression from simple steatosis to steatohepatitis . Accordingly, strategies targeting cholesterol accumulation offer basic therapeutic approaches for nafld patients, and cholesterol management therapy seems to represent a promising treatment for nafld . The potential clinical benefit of cholesterol management for treating nafld with respect to hepatic steatosis and injury should be estimated in appropriately designed trials.
The methodology has been described in detail in a previously published article.9 in summary, this study involved the prospective collection of data on all trauma patients attended to by the scottish ambulance service (sas) and emergency medical retrieval service (emrs) in scotland, for a complete year . Patients were notionally triaged to appropriate levels of care, on the basis of clinical need . A modeling of all mathematically possible trauma system configurations was then undertaken, using a combination of network analysis and multiobjective optimization, identifying optimal network configurations . The study was considered by the north of scotland research ethics service and deemed a service evaluation and therefore did not require ethical approval . It was approved by the sas caldicott guardian (body responsible for electronic patient data access). Scotland has a population of 5.3 million, concentrated in four major conurbations (fig . Prehospital care is provided by the sas and the emrs . Between july 2013 and june 2014, every patient 15 years and older, for whom a final diagnostic code relating to physical injury was recorded by the ambulance crew, was notionally triaged, using the field triage decision scheme.12 we did not include children because the organization of a pediatric trauma system differs . To enable the notional triage, patients who met the criteria of step 1 of the triage algorithm (physiologic derangement) or step 2 (critical injuries) were triaged to the highest level of care (mtc, equivalent to a north american level i trauma center). Patients who met the criteria of step 3 (mechanistic criteria) or step 4 (special considerations) were triaged to trauma unit (tu) care . (a tu is broadly equivalent to a north american level ii / iii trauma center .) All other patients were triaged to local emergency hospital (leh) care (equivalent to a level iv / v trauma center or nondesignated hospital). We used incident location data to calculate drive times and flight times from every incident location to every hospital in scotland, which could potentially become an mtc or tu . In total 1), of which 4 (in the major cities of glasgow, edinburgh, aberdeen, and dundee) could also become mtcs, yielding more than 2 million mathematically possible configurations . Calculated drive times were adjusted to account for traffic conditions, considering time of the day, day of the week, road type, and population density . Ambulances conveying patients triaged to mtc care were assumed to travel at blue - light speeds . Flight times considered stand - to times, flight time from base to incident location, average loading times, and flight time to destination and assumed night - flying capability . We then analyzed which hospital each patient would have gone to, given any particular configuration of trauma system, using a set of decision rules (fig . 3).9 we used an access time threshold of 45 minutes, as used by many trauma systems . Patients who could not have reached the desired level of care within this time frame were modeled as having been diverted to a lower - level center and flagged as we assumed a maximum possible number of seven flights per helicopter per day based on average mission duration . For each analyzed configuration, we calculated the predicted total and median system travel times and the number of patient exceptions . We used a multiobjective optimization algorithm, which searches for optimal solutions to problems with two or more conflicting objectives, to determine geospatially optimized trauma system configurations . Specifically, we used the non - dominated sorting genetic algorithm (nsga-2),13 with the objectives of minimizing total system travel time and minimizing the number of exceptions, reflecting the time to definitive care paradigm.4 the output is composed of a set of mathematically equal solutions, which cannot be mathematically or numerically ranked, as they are all deemed optimal (or pareto optimized). We selected the predicted annual volume of severely injured patients in the mtcs and the number of helicopters required as constraints . The choice of the former was based on the recognized relationship between trauma center case volume and mortality.4,14,15 we modeled minimum thresholds of at least 650 (high volume), 400 (moderate volume), and 240 (low volume) severely injured patients per year, as defined by injury severity score (iss). The number of severely injured patients per hospital was calculated from the number of patients triaged to each category, using coefficients based on scottish trauma audit group registry data, and the published sensitivity of the field triage decision scheme.16 the annual volume of severely injured patients in mtcs was calculated from the number of patients who were primarily admitted as well as the number of severely injured patients transferred secondarily from tus, assuming a 90% transfer rate, to allow for patients deemed not to require specialist care or receiving palliative care . In essence, we attempted to maximize mtc case volume, using the smallest number of helicopters . If no feasible or acceptable solutions were obtained for a given case volume threshold, we increased the number of helicopters, to a maximum of double the current configuration . The performance characteristics of each trauma system configuration, which we examined, included the estimated number of primarily admitted patients (directly from the scene) with severe injury (per center), the number of interfacility transfers of patients with severe injury, the total number of severely injured patients (including interfacility transfers) per center, the median access time for all patients, the median access time for patients triaged to mtc care and taken to an mtc, and the number of helicopter flights required . Data were collated using microsoft excel (microsoft, redmond, wa). The modeling and multiobjective optimization were performed using matlab (mathworks, natick, ma). The methodology has been described in detail in a previously published article.9 in summary, this study involved the prospective collection of data on all trauma patients attended to by the scottish ambulance service (sas) and emergency medical retrieval service (emrs) in scotland, for a complete year . Patients were notionally triaged to appropriate levels of care, on the basis of clinical need . A modeling of all mathematically possible trauma system configurations was then undertaken, using a combination of network analysis and multiobjective optimization, identifying optimal network configurations . The study was considered by the north of scotland research ethics service and deemed a service evaluation and therefore did not require ethical approval . It was approved by the sas caldicott guardian (body responsible for electronic patient data access). Scotland has a population of 5.3 million, concentrated in four major conurbations (fig . Prehospital care is provided by the sas and the emrs . Between july 2013 and june 2014, every patient 15 years and older, for whom a final diagnostic code relating to physical injury was recorded by the ambulance crew, was notionally triaged, using the field triage decision scheme.12 we did not include children because the organization of a pediatric trauma system differs . To enable the notional triage, bespoke data collection screens were added to the ambulances electronic patient record system . Patients who met the criteria of step 1 of the triage algorithm (physiologic derangement) or step 2 (critical injuries) were triaged to the highest level of care (mtc, equivalent to a north american level i trauma center). Patients who met the criteria of step 3 (mechanistic criteria) or step 4 (special considerations) were triaged to trauma unit (tu) care . (a tu is broadly equivalent to a north american level ii / iii trauma center .) All other patients were triaged to local emergency hospital (leh) care (equivalent to a level iv / v trauma center or nondesignated hospital). We used incident location data to calculate drive times and flight times from every incident location to every hospital in scotland, which could potentially become an mtc or tu . In total 1), of which 4 (in the major cities of glasgow, edinburgh, aberdeen, and dundee) could also become mtcs, yielding more than 2 million mathematically possible configurations . Calculated drive times were adjusted to account for traffic conditions, considering time of the day, day of the week, road type, and population density . Ambulances conveying patients triaged to mtc care were assumed to travel at blue - light speeds . Flight times considered stand - to times, flight time from base to incident location, average loading times, and flight time to destination and assumed night - flying capability . We then analyzed which hospital each patient would have gone to, given any particular configuration of trauma system, using a set of decision rules (fig . 3).9 we used an access time threshold of 45 minutes, as used by many trauma systems . Patients who could not have reached the desired level of care within this time frame were modeled as having been diverted to a lower - level center and flagged as we assumed a maximum possible number of seven flights per helicopter per day based on average mission duration . For each analyzed configuration, we calculated the predicted total and median system travel times and the number of patient exceptions . We used a multiobjective optimization algorithm, which searches for optimal solutions to problems with two or more conflicting objectives, to determine geospatially optimized trauma system configurations . Specifically, we used the non - dominated sorting genetic algorithm (nsga-2),13 with the objectives of minimizing total system travel time and minimizing the number of exceptions, reflecting the time to definitive care paradigm.4 the output is composed of a set of mathematically equal solutions, which cannot be mathematically or numerically ranked, as they are all deemed optimal (or pareto optimized). We selected the predicted annual volume of severely injured patients in the mtcs and the number of helicopters required as constraints . The choice of the former was based on the recognized relationship between trauma center case volume and mortality.4,14,15 we modeled minimum thresholds of at least 650 (high volume), 400 (moderate volume), and 240 (low volume) severely injured patients per year, as defined by injury severity score (iss). The number of severely injured patients per hospital was calculated from the number of patients triaged to each category, using coefficients based on scottish trauma audit group registry data, and the published sensitivity of the field triage decision scheme.16 the annual volume of severely injured patients in mtcs was calculated from the number of patients who were primarily admitted as well as the number of severely injured patients transferred secondarily from tus, assuming a 90% transfer rate, to allow for patients deemed not to require specialist care or receiving palliative care . In essence, we attempted to maximize mtc case volume, using the smallest number of helicopters . If no feasible or acceptable solutions were obtained for a given case volume threshold, we increased the number of helicopters, to a maximum of double the current configuration . The performance characteristics of each trauma system configuration, which we examined, included the estimated number of primarily admitted patients (directly from the scene) with severe injury (per center), the number of interfacility transfers of patients with severe injury, the total number of severely injured patients (including interfacility transfers) per center, the median access time for all patients, the median access time for patients triaged to mtc care and taken to an mtc, and the number of helicopter flights required . The modeling and multiobjective optimization were performed using matlab (mathworks, natick, ma). In total, 80,391 casualties were attended to by the sas and emrs over the year, underwent notional triage, and were included in our analysis . A total of 1,599 (1.9%) of incidents occurred on islands . Of the patients, 80,202 (99.8%) were retrieved by road ambulance and 192 (0.2%) by helicopter . A total of 7,095 (8.8%) were notionally triaged to mtc care, 33,567 (41.8%) to tu care, and 39,728 (49.4%) to local emergency hospital care . The groups, as expected, differed in terms of demographic and physiologic characteristics (table 1). The spatial distribution of the incidents has been described previously.17 characteristics of study population there were 21 optimized configurations for a network with high- or moderate - volume mtcs . (modelling and optimization with both volume thresholds produced the same set of results .) Nine of these configurations would require five helicopters, and the remainder, six . There were no feasible high- or moderate - volume configurations based on the current fleet size of three helicopters or four helicopters . The nine optimized configurations with the smallest number of helicopters all had one mtc, in glasgow, and between 10 and 17 tus (table 2, configurations a to i). Configurations with a smaller number of tus would be predicted to result in an increase in median travel time, a decrease in the proportion of patients reaching their destination within 45 minutes, an increase in the number of helicopter flights required, and a small decrease in the number of exceptions (as a result of more patients being taken to mtcs by helicopter). The reconfiguration of services in scotland is currently focused on mtcs, and it is therefore likely that all hospitals that could become tus would be designated as such . This configuration (configuration a in tables 2 and 3) would result in an estimated 864 severely injured patients per year being taken to the mtc in glasgow . Of these, an estimated 328 would be primary admissions, and 536 would be secondary transfers . The latter figure includes both patients who could not primarily reach an mtc within the desired time, for geographic reasons, despite being triaged to mtc care, and those who were incorrectly triaged and mistakenly taken to a tu, as a result of the limited sensitivity of the triage protocol . The median access time for patients triaged to mtc care, who would be taken to an mtc, was estimated to be 21.9 minutes (table 3). Optimized network configurations predicted performance of optimized network configurations if mtcs with lower annual case volumes were accepted, there would be 11 optimized configurations . Two of these could be realized with four helicopters, whereas the remainder would require at least five aircraft . The two configurations requiring only four helicopters are also shown in table 2 (configurations j and k). Both had two mtcs, in glasgow and edinburgh, and either 15 or 16 tus . If all 16 hospitals that could become tus were designated as such (configuration j in tables 2 and 3), the glasgow mtc would be estimated to have moderate volume (494 severely injured patients per year, including secondary transfers), and the edinburgh mtc would have low volume (381 severely injured patients per year, including secondary transfers). As before, this number includes both undertriaged patients and those who were correctly triaged but could not reach an mtc within 45 minutes . The estimated median access time for patients triaged to mtc care, who would be taken to an mtc, would be 18.2 minutes, which is slightly shorter than for a single - center configuration . If the number of helicopters was increased to five, an additional nine configurations would become feasible, all with two mtcs, in glasgow and edinburgh, and 10 to 16 tus . The implementation of a trauma system with one or two mtcs and with the tasking criteria described is estimated to require an increase in primary helicopter retrievals . The configurations described would necessitate between 2,832 and 4,065 missions to be flown per year, depending on the choice of configuration, equating to approximately nine retrievals per day . However, only 941 to 1,117 of these flights would be for patients triaged to mtc care, the remainder being for patients triaged to tu care, injured in remote locations (table 3). A sensitivity analysis with a 60-minute (as opposed to 45 minutes) access time threshold revealed a small number of additional configurations . Networks with high- or moderate - volume mtcs alone could be optimized with a single mtc in glasgow, as before, or a single mtc in edinburgh . Networks with low - volume mtcs had two mtcs, in glasgow and edinburgh (as before) or in glasgow and dundee . However, the latter combination would have been discounted if two fewer patients had been admitted to dundee . Furthermore, this configuration would also be associated with markedly longer access times . In summary, lengthening the access time threshold to 60 minutes does not alter the conclusions . There were 21 optimized configurations for a network with high- or moderate - volume mtcs . (modelling and optimization with both volume thresholds produced the same set of results .) Nine of these configurations would require five helicopters, and the remainder, six . There were no feasible high- or moderate - volume configurations based on the current fleet size of three helicopters or four helicopters . The nine optimized configurations with the smallest number of helicopters all had one mtc, in glasgow, and between 10 and 17 tus (table 2, configurations a to i). Configurations with a smaller number of tus would be predicted to result in an increase in median travel time, a decrease in the proportion of patients reaching their destination within 45 minutes, an increase in the number of helicopter flights required, and a small decrease in the number of exceptions (as a result of more patients being taken to mtcs by helicopter). The reconfiguration of services in scotland is currently focused on mtcs, and it is therefore likely that all hospitals that could become tus would be designated as such . This configuration (configuration a in tables 2 and 3) would result in an estimated 864 severely injured patients per year being taken to the mtc in glasgow . Of these, an estimated 328 would be primary admissions, and 536 would be secondary transfers . The latter figure includes both patients who could not primarily reach an mtc within the desired time, for geographic reasons, despite being triaged to mtc care, and those who were incorrectly triaged and mistakenly taken to a tu, as a result of the limited sensitivity of the triage protocol . The median access time for patients triaged to mtc care, who would be taken to an mtc, was estimated to be 21.9 minutes (table 3). If mtcs with lower annual case volumes were accepted, there would be 11 optimized configurations . Two of these could be realized with four helicopters, whereas the remainder would require at least five aircraft . The two configurations requiring only four helicopters are also shown in table 2 (configurations j and k). Both had two mtcs, in glasgow and edinburgh, and either 15 or 16 tus . If all 16 hospitals that could become tus were designated as such (configuration j in tables 2 and 3), the glasgow mtc would be estimated to have moderate volume (494 severely injured patients per year, including secondary transfers), and the edinburgh mtc would have low volume (381 severely injured patients per year, including secondary transfers). As before, this number includes both undertriaged patients and those who were correctly triaged but could not reach an mtc within 45 minutes . The estimated median access time for patients triaged to mtc care, who would be taken to an mtc, would be 18.2 minutes, which is slightly shorter than for a single - center configuration . If the number of helicopters was increased to five, an additional nine configurations would become feasible, all with two mtcs, in glasgow and edinburgh, and 10 to 16 tus . The implementation of a trauma system with one or two mtcs and with the tasking criteria described is estimated to require an increase in primary helicopter retrievals . The configurations described would necessitate between 2,832 and 4,065 missions to be flown per year, depending on the choice of configuration, equating to approximately nine retrievals per day . However, only 941 to 1,117 of these flights would be for patients triaged to mtc care, the remainder being for patients triaged to tu care, injured in remote locations (table 3). A sensitivity analysis with a 60-minute (as opposed to 45 minutes) access time threshold revealed a small number of additional configurations . Networks with high- or moderate - volume mtcs alone could be optimized with a single mtc in glasgow, as before, or a single mtc in edinburgh . Networks with low - volume mtcs had two mtcs, in glasgow and edinburgh (as before) or in glasgow and dundee . However, the latter combination would have been discounted if two fewer patients had been admitted to dundee . Furthermore, this configuration would also be associated with markedly longer access times . In summary, lengthening the access time threshold to 60 minutes does not alter the conclusions . This study has shown that a novel notional triage and mathematical optimization methodology can be used to inform the planning of a major national care system . That the analysis is based on a complete and large national cohort of prospectively collected data adds to the robustness of the findings . Our analysis indicates that a trauma system configuration with one mtc, in glasgow, or two mtcs, in glasgow and edinburgh, would be optimal, based on observed data . These findings are at variance with the widely held belief that the geographic distribution and associated long access times would preclude a configuration with a single center.10 while such a configuration would result in a high proportion of patients who could not reach definitive care primarily and a high number of secondary transfers, these results should be viewed in the context of an inclusive trauma system, which would facilitate best possible care, even for those injured in remote areas . The findings are also at variance with the intuitive assumption that, if two mtcs were required, these would be best placed as far apart as possible, in glasgow and aberdeen . These results are explained by the spatial distribution of the incidents and the clustering of case volume in the glasgow / edinburgh area in particular, as reported in our previous article,17 which exerts a gravitational pull . Trauma system design is influenced by a number of forces, which vary with setting . The principal reasons include the economic benefits and the prestige of trauma center status . In the united kingdom and in scotland in particular, there are no economic benefits to a hospital being designated as an mtc, but as the national health service is more susceptible to political influences, local opinion can strongly influence decision making . Robust data, as generated by the geos study, can help to make the process more objective and transparent . Following the decision to regionalize trauma care in scotland, the scottish government s national planning forum had originally recommended a trauma system with four mtcs, in glasgow, edinburgh, dundee, and aberdeen.10 this study shows that such a configuration might not be optimal . For the two solutions identified as optimal in our analysis, for a given minimum mtc case volume, the decision as to which solution is best therefore relates, partly, to what is deemed an adequate center volume . The relationship between case volume and outcome is well recognized, but the improvements in mortality, which are seen with higher case volumes, are probably not the consequence of higher volume per se, but rather the ability to justify a different service delivery framework . A dedicated trauma service to coordinate and deliver care for the severely injured is key to improving outcomes but is only justifiable when there is a sufficient case volume . In addition, mortality is not the only measure of a high - quality service . The precise position of the inflection point on the volume / outcome curve is therefore not known, and it is probable that both configurations described would result in mtcs large enough to sustain a specialist service . Furthermore, choosing between the two optimal configurations identified by the modeling should also consider other factors, such as hospitals capacity, and issues that are less quantifiable, such as organizational commitment and resilience . Capacity is difficult to model because it is influenced by the number of admissions and the length of stay . Data on the latter were not available in our prehospital data set . In terms of the number of emergency department attendances alone, however, it seems probable that a configuration with two mtcs, in glasgow and edinburgh, would be better able to deal with the predicted increase in volume than a single - mtc configuration . Both the single- and two - mtc system configurations (but, in fact, also the four - mtc configuration proposed by the scottish government) would require an increase in aeromedical retrievals, although a proportion of the primary helicopter retrievals predicted by this study were for patients triaged to tu care, with a low probability of major trauma who were injured in remote locations . Some of these patients might not always require helicopter transport or could be taken to a local emergency hospital, if a degree of provider judgment was applied . The anticipated need for increased aeromedical retrieval reflects scotland s geography and a probable underprovision of lift capacity, given the population characteristics . The cost of operating additional helicopters may seem substantial but should be viewed in the context of setting up and running additional mtcs, which is also considerable . The combined set - up cost for four mtcs is estimated to be in the region of 12 to 17 million ($19$27 million). The strengths of the study lie in its prospective, systemwide, population - based design; its use of actual incident location data; and its application of multiobjective optimization to network analysis, which enables multiple, conflicting objectives to be considered . The use of prehospital triage decisions implicitly considers both undertriage and overtriage and thus provides a realistic model of patient flow . Several previous studies1820 have attempted to quantify access to trauma center care using isochrone analysis of census data, relying on the assumption that the distribution of the injured population mirrors that of the population in general, which does not always hold true.21 these issues are overcome by network analysis, a well - established technique for solving siting problems in operations research, which was used by branas et al.22,23 in their seminal trauma resource allocation model for ambulances and hospitals (tramah) study . However, the tramah study was limited by the use of retrospectively calculated severity scores, obtained from trauma registries, to stratify injury severity . In reality, patient flow is determined by prehospital triage decisions, as used by the geos modeling . The positive predictive value of triage for determining severe injury is limited, and the resulting overtriage has implications for transport services and hospitals capacity . No provision was made for provider judgment in triage, and the model is dependent on the sensitivity of steps 1 and 2 of the field triage decision scheme for detecting severe injury . We used a conservative estimate of 45.5%, derived from a large multicenter study from the united states.16 it is possible that the performance of the field triage decision scheme in scotland differs because of variations in case mix and application . Calculated drive times are estimates and did not consider the effects of weather, both of which may impact on the accuracy of the data . We considered making allowances for no - fly weather conditions, but the available data on which to base such modeling are limited, particularly as the entire flight path rather than just the incident location would have to be considered . Similarly, we did not make allowances for weather - related decreases in driving speeds . However, the model was built on a large data set, collected over a full year, to account for any seasonal variation in the geographic distribution of the incidents . It is possible that the injuries observed over this time may not be representative of what would happen every year, but the profile of the data is similar to that seen in previous years,2426 which provides reassurance that the results are generalizable . This study adds to the literature on trauma system design, and while the data in this study pertains to the configuration of a trauma system for scotland, the methodology could easily be adopted and adapted to other settings . In particular, the technique could also be used to compare mathematically optimized configurations with existing ones, to provide a form of quality assurance of the configuration of existing trauma systems . This latter application could help to address the issue of trauma center proliferation, which is an increasingly recognized problem in north america . Furthermore, trauma is not the only time - critical condition that requires complex care delivered by a hierarchical clinical network . The effect of hospital volume on outcome following percutaneous coronary intervention for myocardial ischemia, thrombolytic therapy for stroke, and the repair of ruptured abdominal aortic aneurysms is well recognized,2729 and these treatments might also benefit from geospatially optimized systems of care . Trauma system design is influenced by a number of forces, which vary with setting . The principal reasons include the economic benefits and the prestige of trauma center status . In the united kingdom and in scotland in particular, there are no economic benefits to a hospital being designated as an mtc, but as the national health service is more susceptible to political influences, local opinion can strongly influence decision making . Robust data, as generated by the geos study, can help to make the process more objective and transparent . Following the decision to regionalize trauma care in scotland, the scottish government s national planning forum had originally recommended a trauma system with four mtcs, in glasgow, edinburgh, dundee, and aberdeen.10 this study shows that such a configuration might not be optimal . For the two solutions identified as optimal in our analysis, for a given minimum mtc case volume, the decision as to which solution is best therefore relates, partly, to what is deemed an adequate center volume . The relationship between case volume and outcome is well recognized, but the improvements in mortality, which are seen with higher case volumes, are probably not the consequence of higher volume per se, but rather the ability to justify a different service delivery framework . A dedicated trauma service to coordinate and deliver care for the severely injured is key to improving outcomes but is only justifiable when there is a sufficient case volume . In addition, mortality is not the only measure of a high - quality service . The precise position of the inflection point on the volume / outcome curve is therefore not known, and it is probable that both configurations described would result in mtcs large enough to sustain a specialist service . Furthermore, choosing between the two optimal configurations identified by the modeling should also consider other factors, such as hospitals capacity, and issues that are less quantifiable, such as organizational commitment and resilience . Capacity is difficult to model because it is influenced by the number of admissions and the length of stay . Data on the latter were not available in our prehospital data set . In terms of the number of emergency department attendances alone, however, it seems probable that a configuration with two mtcs, in glasgow and edinburgh, would be better able to deal with the predicted increase in volume than a single - mtc configuration . Both the single- and two - mtc system configurations (but, in fact, also the four - mtc configuration proposed by the scottish government) would require an increase in aeromedical retrievals, although a proportion of the primary helicopter retrievals predicted by this study were for patients triaged to tu care, with a low probability of major trauma who were injured in remote locations . Some of these patients might not always require helicopter transport or could be taken to a local emergency hospital, if a degree of provider judgment was applied . The anticipated need for increased aeromedical retrieval reflects scotland s geography and a probable underprovision of lift capacity, given the population characteristics . The cost of operating additional helicopters may seem substantial but should be viewed in the context of setting up and running additional mtcs, which is also considerable . The combined set - up cost for four mtcs is estimated to be in the region of 12 to 17 million ($19$27 million). The strengths of the study lie in its prospective, systemwide, population - based design; its use of actual incident location data; and its application of multiobjective optimization to network analysis, which enables multiple, conflicting objectives to be considered . The use of prehospital triage decisions implicitly considers both undertriage and overtriage and thus provides a realistic model of patient flow . Several previous studies1820 have attempted to quantify access to trauma center care using isochrone analysis of census data, relying on the assumption that the distribution of the injured population mirrors that of the population in general, which does not always hold true.21 these issues are overcome by network analysis, a well - established technique for solving siting problems in operations research, which was used by branas et al.22,23 in their seminal trauma resource allocation model for ambulances and hospitals (tramah) study . However, the tramah study was limited by the use of retrospectively calculated severity scores, obtained from trauma registries, to stratify injury severity . In reality, patient flow is determined by prehospital triage decisions, as used by the geos modeling . The positive predictive value of triage for determining severe injury is limited, and the resulting overtriage has implications for transport services and hospitals capacity . No provision was made for provider judgment in triage, and the model is dependent on the sensitivity of steps 1 and 2 of the field triage decision scheme for detecting severe injury . We used a conservative estimate of 45.5%, derived from a large multicenter study from the united states.16 it is possible that the performance of the field triage decision scheme in scotland differs because of variations in case mix and application . Calculated drive times are estimates and did not consider the effects of weather, both of which may impact on the accuracy of the data . We considered making allowances for no - fly weather conditions, but the available data on which to base such modeling are limited, particularly as the entire flight path rather than just the incident location would have to be considered . Similarly, we did not make allowances for weather - related decreases in driving speeds . However, the model was built on a large data set, collected over a full year, to account for any seasonal variation in the geographic distribution of the incidents . It is possible that the injuries observed over this time may not be representative of what would happen every year, but the profile of the data is similar to that seen in previous years,2426 which provides reassurance that the results are generalizable . This study adds to the literature on trauma system design, and while the data in this study pertains to the configuration of a trauma system for scotland, the methodology could easily be adopted and adapted to other settings . In particular, the technique could also be used to compare mathematically optimized configurations with existing ones, to provide a form of quality assurance of the configuration of existing trauma systems . This latter application could help to address the issue of trauma center proliferation, which is an increasingly recognized problem in north america . Furthermore, trauma is not the only time - critical condition that requires complex care delivered by a hierarchical clinical network . The effect of hospital volume on outcome following percutaneous coronary intervention for myocardial ischemia, thrombolytic therapy for stroke, and the repair of ruptured abdominal aortic aneurysms is well recognized,2729 and these treatments might also benefit from geospatially optimized systems of care . This study has shown that a novel combination of notional triage, network analysis, and mathematical optimization methodology can be used to inform the planning of a major national care system . Scotland s nascent trauma network would be optimally configured with one or two mtcs, and the latter configuration, in particular, seems feasible with regard to the capacity of the proposed centers and the additional need for aeromedical retrieval resources . Whether explicitly considered or not, there is a geographic dimension to the design of any clinical network . The need to balance conflicting objectives such as accessibility, center case volumes, and need for aeromedical transport is a particular feature of networks caring for patients with highly acute conditions.
Injections of botulinum toxin type a (btx - a, botox cosmetic, allergan inc, ca, usa) have become one of the most popular cosmetic procedures worldwide (pitman 2005). In the us, more than 2.2 million cosmetic injections were reported in 2003, only a year after it received food and drug administration (fda) approval for treatment of glabellar lines (batra et al 2005). Today it is the most common cosmetic procedure performed in the us (wise and greco 2006). In contrast to other products used for cosmetic procedures, btx - a is minimally invasive, relatively easy and quick to administer, has a relatively immediate and noticeable effect and a quick recovery period, and is often more affordable than other cosmetic treatments (klein 2004). Alternative procedures involve the use of injectable fillers (eg, silicone, collagen), resurfacing techniques (eg, dermabrasion, chemical peels, laser resurfacing), and surgery, which may lead to serious and permanent adverse effects, eg, photosensitivity, scarring, surgical morbidity, and involve a long recovery period (lemperle et al 2001; cather et al 2002). Btx - a is a neuromuscular blocking agent that works by producing transient, dose - dependent, focal weakening of the facial muscles involved in expressions that lead to a tired, disapproving or aged look, such as frowning or wrinkling the forehead (klein 2004; carruthers et al 2004). The ideal candidates for btx - a injections are men and women between 40 and 60 years of age whose facial wrinkles are primarily caused by repeated, habitual muscle contraction over time (allergan pi 2005). Despite the manufacturer s labeling for the use of btx - a in adults younger than 65 years of age, there have been anecdotal reports of btx - a use in older patients . However, there is little published information on the efficacy and safety of btx - a for cosmetic reasons in this population . Prior to its use for cosmetic reasons, local injections of btx - a were successful in treating other disorders characterized by inappropriate and excessive muscle contraction, such as dystonia, blepharospasm, and spasticity (cote et al 2005). Btx - a is one of eight potent botulinum toxin serotypes produced by the bacteria clostridia botulinum, a gram - positive spore - forming anaerobe . Commercially available btx - a is a purified and diluted form of the neurotoxin . Following injection into a muscle, btx - a binds to the presynaptic nerve terminal and selectively blocks the release of acetylcholine at the neuromuscular junction, thereby preventing muscle contraction . The effect is temporary and reversible following the sprouting of new axons and development of extrajunctional acetylcholine receptors over time . Acetylcholine synthesis and storage are not affected (frampton and easthope 2003; klein 2004; allergan pi 2005). Btx - a is most effective for wrinkles that form because of muscle contraction, where weakening of the muscle smoothes and flattens the overlying skin . After a btx - a injection, an improvement in appearance occurs within 1 to 14 days, peaks at approximately 4 weeks, and begins wearing off after 1012 weeks; therefore, a repeat injection approximately every 34 months is necessary to maintain the cosmetic effect of btx - a (klein 2004; allergan pi 2005). Btx - a is not effective for facial wrinkles caused by mechanisms other than muscle contraction, such as sun damage, environmental pollutants, or subcutaneous soft tissue atrophy . Because btx preparations vary in potency, commercially available botulinum toxins, including botox cosmetic, dysport (ispen ltd, slough, uk), and myobloc (elan pharmaceuticals, san francisco, ca, usa), are not interchangeable . Cosmetic uses for botulinum toxin include treating the vertical lines between the eyebrows (glabellar lines) and on the bridge of the nose, squint lines or crows feet at the corners of the eyes, forehead horizontal lines, periorbital lines and nasolabial folds around the mouth, and the thick platysmal bands around the neck, also known as turkey neck (blitzer and binder 2002; carruthers et al 2004; klein 2005). The recommended dose for glabellar lines is 20 units distributed among 5 injection sites; however, the dosing is often individualized according to the location and size of the muscle as well as the depth of the wrinkle . The btx - a doses used for cosmetic purposes are much lower than those used in therapeutic cases (allergan pi 2005). It is difficult to tell for sure whether patients over the age of 65 respond differently to btx - a than younger patients; there have been no studies investigating cosmetic uses of btx - a specifically in the elderly, and there have not been enough elderly patients enrolled in clinical studies to make any meaningful comparisons (allergan pi 2005). However, since the elderly are more likely to have thinner and less elastic skin, weaker facial muscles, and wrinkles that over time are caused by gravity - induced tissue sagging rather than muscle contraction, the elderly are not expected to respond as well to btx - a treatment (norman 2003; rhodes et al 2003). Btx - a may help soften wrinkles that are noticeable even without muscle contraction, but additional resurfacing procedures are often needed to lead to visible differences in the appearance of the wrinkle (patel et al 2004). Treatment of forehead lines, for example, would require injections to the frontalis muscle, which many older people use to raise their eyebrows and eyelids to see . Older patients may also have extra skin under the brow (pseudoptosis) which could be worsened by btx - a treatment . Older patients who receive btx - a for glabellar lines may be more at risk for complications such as eyelid ptosis if they have a reduced or absent orbital septum (fagien 2003; klein 2004; carruthers et al 2004). Because of their delicate skin, older patients are also more susceptible to bruising from btx - a injections . The risk for bruising is even greater among patients taking medications that inhibit clotting, such as vitamin e, aspirin, nonsteroidal antiinflammatory drugs, and herbal products such as ginseng, ginko biloba, and garlic . Many physicians advise avoiding these medications and products 1014 days prior to treatment . A full medication and supplement history this is particularly important for elderly patients, who are more likely to be taking multiple medications or supplements (rhodes et al 2003; klein 2004; mclean and le couteur 2004). Conservative dosing, injection of low volumes, and proper placement of the injection can reduce the possibility of spread of the toxin to unintended muscles . Electromyographic guidance may be helpful in selecting the appropriate muscles for injection (klein 2004; carruthers et al 2004; vartanian and dayan 2005). The manufacturer recommends starting at the lowest possible effective dose for elderly patients (allergan pi 2005). The most common side effects are related to the injection technique and include local redness, bruising, swelling, and mild pain (allergan pi 2005). Patients who have severe wrinkles (which require a higher dose of btx - a) or who have altered facial anatomy, and those with pre - existing neuromuscular disease are more at risk for complications . Other side effects include eyelid ptosis, focal facial weakness, and headache . Serious adverse events included dysphagia, flu - like symptoms, and hypersensitivity reactions . Complications that result from weakened muscles adjacent to the injection site are due to the diffusion of toxin to unintended muscles; this may be minimized through proper injection technique (klein 2004; naumann and jankovic 2004; batra et al 2005; vartanian and dayan 2005). Topical anesthetics applied prior to injection, use of smaller gauge needles, slow injection, small doses, and application of ice immediately after injection can help minimize pain and discomfort following btx - a injections . Many physicians encourage patients to remain upright for 34 hours after the procedure, avoid massaging the treatment area, and actively contract the injected muscle to encourage the uptake of toxin by the intended muscles (carruthers and carruthers 1998; carruthers et al 2004; klein 2004). Proper dilution, storage, and handling of btx - a are also essential for minimizing adverse outcomes (cote et al 2005). Btx - a should not be used in patients who are pregnant, nursing, or have neuromuscular disease . Patients taking medications that could potentiate the effects of btx - a should also not receive btx - a . Btx - a is also contraindicated in the presence of infection or inflammation at the injection site and in patients with known hypersensitivity to btx - a or any ingredient in the formulation (allergan pi 2005). Reports of serious cardiovascular events, including arrhythmias, have been reported in post - marketing surveillance of adverse events related to the cosmetic uses of btx - a . The incidence was 2.8% (1/36) for serious arrhythmia and 5.6% (2/36) for any serious cardiovascular event . In many of these cases the patients had preexisting cardiovascular risk factors that may have been related to the adverse event . Nevertheless, btx - a should be used with caution in patients with cardiovascular comorbidities, which are more common among the elderly (wenge 2000; cote et al 2005). Physicians should consider the suitability of btx - a for elderly patients, taking into account the etiology of their wrinkles, skin fragility, facial anatomy, concomitant medications and medical conditions, risk of adverse effects and the likelihood of treatment benefit . Although btx - a has a low perceived risk of side effects, older patients may be more susceptible to these effects . The benefit from btx - a treatment is also questionable since wrinkles in older people are more likely to be caused by factors other than repeated muscle contraction . Precautions such as a obtaining a full medication and medical history, beginning with low doses, and proper injection technique are especially important for optimal outcomes in elderly patients who are deemed to be good candidates for btx - a cosmetic injections.
Odorants are troublesome in water samples, because they dramatically influence the esthetic quality and consumers' acceptability of drinking water [1, 2]. These odorants are often associated with the metabolites that are produced in the degradation of cyanobacteria, actinomyces, fungi, and blue - green algae [35]. The odorants geosmin (gsm) and 2-methylisoborneol (2-mib) are commonly found in lakes and reservoirs [6, 7], which people can smell their odors in water samples even at the concentration of 10 ng l or less, but it would be difficult to identify and quantify these two trace volatile organic compounds (vocs) [812]. As a result, the low threshold of detection can result in consumer complaints about the odors in recreational waters, aquatic products, and tap water, especially during the outbreak period of algal blooms, even if some other quality indicators of water, such as turbidity, number of algal cells, and suspended matter, are acceptable . In recent years, according to the investigation by chinese academy of sciences [14, 15], the results indicated that freshwater lakes were suffering the odor problems in china, especially in lake taihu, lake chaohu, and lake dianchi . These unpleasant odors occurring in these lakes or therefore, we had conducted this study in 2012, aiming to obtain the basic data of common odorants (gsm and 2-mib) in environmental waters in jiangsu province . To be more specific, the headspace solid - phase microextraction coupled to gas chromatography mass spectrometry (hs - spme / gc - ms) was applied to determine the levels of gsm and 2-mib in water samples, and this method can be obtained in our early reports [16, 17]. In addition, the water samples were collected in detail as follows: firstly, for water sources of tap water, four kinds of water samples were collected respectively from four cities in jiangsu, including city a (little lakes as the source), city b (the changjiang river as source), city c (lake taihu as source), and city d (underground water as the source); secondly, for conventional treatment process of tap water, including coagulation and sedimentation, filtration, and disinfection, the water samples were collected respectively from above processes; thirdly, for different seasons of water samples, we collected the samples in three periods in lake taihu, including the period when a river is at its normal level (november to december), drought period (february to march), and wet season (july to august) when the algae bloom . Two common odorants in water, gsm and 2-mib, and internal standard 2-isobutyl-3-methoxypyrazine (ibmp) were obtained from sigma - aldrich (usa) at a concentration of 100 mg l in methanol and 1 mg l mixed standard solutions of two target compounds in methanol, and all of them were stored in the dark at 4c . Deionized water was prepared on a water purification system (gradient a10) supplied by millipore (billerica, ma, usa). Sodium chloride (analytical grade, china), which was added to the samples before extraction, was conditioned by heating at 450c for 4 h before use . Spme apparatus was purchased from supelco (usa), including fiber 30/50 m dvb / car / pdms (number 57348-u), fiber holder, 60 ml specialized vials for spme, sampling stand, magnetic stirrer, and injection catheter (number 57356-u). After putting nacl and a stir bar in a 60 ml vial, 40 ml of mixed standard solutions for standard curve or 40 ml environmental water samples were added, and 2 l ibmp (1 mg the vial was sealed with polytetrafluoroethylene (ptfe) septum cap and placed in a water bath . Several minutes after the temperature was achieved in the vial, the outer needle of fiber was used to penetrate the septum, and the fiber was exposed to the headspace for extraction . After 30 min exposure, the fiber was immediately inserted into gc injection port for desorption . According to our early study [16, 17], the hs - spme / gc - ms was applied to determine the levels of gsm and 2-mib in water samples, and the optimum conditions for hs - spme were as follows: temperature of extraction and desorption, 65c and 260c, respectively; time of extraction and desorption, 30 min and 5 min, respectively; ionic strength, 25% (w / v); rotation speed, 600 rpm; solution ph, 5.0 . A varian 300 gc / ms / ms (varian inc ., ca, usa) with ion trap and mass spectrometer was obtained with a varian vf-5 ms capillarity column (30 m 0.25 mm 0.5 m). The temperature of the oven started at 40c and was held for 5 min . Then the temperature was 8c min to achieve 160c (total time 20 min) followed by 20c min to achieve 260c (total time 25 min). The electron impact (ei)-ms conditions were as follows: ion - source temperature, 230c; ms transfer line temperature, 250c; solvent delay time, 5 min; ionizing voltage, 70 ev . The mass spectrogram in full scan mode was obtained at the m / z range of 80200 u. based on the ms scan function (sim mode), the process was divided into three main segments . The method of internal standard was applied to construct calibration curve and determine concentrations of 2-mib and gsm in water . Based on different sources for tap water, four representative cities were chosen to collect water samples, including city a (little lakes as the source), city b (the changjiang river as source), city c (lake taihu as source), and city d (underground water as the source). Five water works were selected from each city, and raw water, output water, and end water (for consumers) were obtained respectively from each water work in drought period february to march by manual sampling, meaning 15 samples in total in one city . Water samples from the water works were analyzed by the proposed method, which were kept in 350 ml sample vials with ptfe - faced silicone septum and stored at 4c before analysis . The conventional treatment of tap water includes four processes, as raw water, coagulation and sedimentation, filtration, and disinfection, and end water was provided for consumers by pipe network as shown in figure 1 . The five water works were selected from city b (shorter distance to laboratory than other three), and one sample was obtained from each process, that is to say, 4 samples in total in one water work . For different seasons of water samples, we collected the samples in three periods in lake taihu, including the period when a river is at its normal level (november to december), drought period (february to march), and wet season (july to august) when the algae bloom . In addition, lake taihu was used as sampling point, because the algal overgrowth appears every summer in this lake, and the consumers complain that the uncomfortable odors exist in their drinking water during the outbreak period of algal blooms . The five water works were selected from city c, and raw water, output water, and the end water were obtained respectively from each water work, 15 samples in total for one period . The analysis of variance (anova) was applied to the present work, aiming to obtain the significant differences when comparing average values in the two or more groups . More specifically, two - way anova was applied to compare the gsm and 2-mib levels in four cities (tables 1 and 2), while one - way anova was applied in water samples from different processes of tap water treatment and different seasons (tables 3 and 4). According to tables 1 and 2, the significant differences of concentration cannot be observed for both gsm and 2-mib among the four cities (p> 0.05), excepting the 2-mib levels of raw water (p <0.01). More specifically, the 2-mib level of raw water in city d was lower than that in three other cities (p <0.01), and the most potential reason could be that the soil or rocks work as natural barrier or filter, keeping the larger particles permeating into raw water . As a result, the level of 2-mib may be lower in city d. however, there are no significant differences for the levels of gsm among four cities; in other words, the lower 2-mib levels of raw water in city d never necessarily equal the lower levels of gsm in city d similarly . It can be explained that the levels of two odorants were associated with multiple factors, including the species of algae, acidic condition of environment, rainfalls, and light density . For example, growth and production of 2-mib was characterized in the cyanobacterium phormidium sp . Another example is that the actinomycetes can cause the odorants, but the total colony counts did not necessarily indicate the direct role of actinomycetes in odor problems in environmental water, because their activities would be restricted by other environmental factors . Consequently, the lower 2-mib occurred in city d, but gsm did not, even though they shared the same water source . In addition, the decreased levels of gsm or 2-mib were observed among the raw water, output water, and end water (p <0.05), as implied in table 4 . Because both of gsm and 2-mib were volatile organic chemicals (vocs), the concentrations would decrease in the water transferred from water works to destination, and similar results are obtained by early reports in china . Meanwhile, the concentration of total residual chlorine in pipe network would necessarily reduce the levels of gsm and 2-mib [22, 23]. But, however, an experimental error (1070%) may be observed due to the presence of free residual chlorine, and this error can decline after dechlorination (less than 10%) [22, 24]. Consequently, the average values of two odorants in table 3 may increase from filtration to chlorination . Based on table 3, it is indicated that the concentration of gsm and 2-mib decreased significantly as treatment process moved forward (p <0.0001). The reasons can be as follows: firstly, after the processes of coagulation, sedimentation, and filtration, larger particles had been removed, such as algae, silt, and other plankton, which can produce the musty and earthy odorant in tap water, and the same result was implied in early study . The last process, chlorination, can kill or remove main cyanobacteria and actinomyces by oxidation [26, 27]. And also some reports indicated that the presence of chlorine would substantially reduce the observed gsm and 2-mib concentrations, and its impact was larger for lower organic compound concentrations, under higher residual chlorine conditions . The water samples of different seasons were collected from city c, the lake taihu of which was used as source water and blue - green algae blooming in summer in this lake . According to table 4, the significant differences were observed for the levels of gsm and 2-mib among the three periods (p <0.01); in other words, the gsm and 2-mib levels of raw water were higher in period of wet water than corresponding levels in other two periods . The most important reason was the algae blooming and reservoir's eutrophication in summer, resulting in higher concentrations of odorants, while the algae or cyanobacteria were dead or inactive in other periods . Moreover, the concentrations were found to correlate with corresponding air and water temperatures, and the average water temperature was 12, 16, and 27c for normal level, drought, and wet period, respectively . The concentrations of odorants followed a trend of higher levels during the warm seasons [2830]. Consequently, the levels in wet season (in summer) were higher than those in other two periods . Also, the results implied that levels of odorants were approximately equal between the periods of normal level and drought . The levels of gsm and 2-mib in water samples were less than 10 ng l, the odor threshold concentration (otc) [12, 31]. And the conventional treatment process of tap water plays a significant role in removing common odorants in running water for these source waters . In particular the largest decrease in concentrations occurs after sedimentation suggesting that removal of gsm and mib in intact algae cells is important.
Corvids collected as part of the wnv dead bird surveillance programs in manitoba and ontario, canada, in 2001 and 2002 were used . Laboratories in each province received dead birds, collected oropharyngeal or cloacal swabs or tissues, performed the antigen - capture assay, and shipped tissues or swabs to the national microbiology laboratory for confirmatory testing . In both laboratories, only birds lacking signs of obvious decay or decomposition were included for testing . If specimens could not be shipped on the date of collection, most were held at 4c . On rare occasions, birds were frozen at 20c by submitters before shipment or by the diagnostic laboratories before testing . However, most specimens were held at approximately 4c for 1 to 4 days before processing . Thirty - one american crows (corvus brachyrhynchos) and 28 blue jays (cyanocitta cristata), collected from june to december in 2001 and held at 20c were tested in march and april 2002 . Swabs were taken from the oropharyngeal cavity and cloaca of each bird . In 2002, testing was restricted to crows (n = 222) from which only oropharyngeal swabs were collected and tested by vectest . Polyester swabs (vwr canlab, mississauga, ontario) were applied to the oropharyngeal cavity and the cloaca . Swabs were placed in microcentrifuge tubes containing 1 ml of ba-1 diluent (similar to that described by nasci et al . ). The antigen - capture assay was carried out according to the manufacturer s instructions except that the aliquot of swab eluate was 125 l, not 250 l . To confirm the infection status of birds, approximately 25-mg pieces of brain and kidney from each bird were pooled in microcentrifuge tubes and stored at 80c before shipment to the national microbiology laboratory . At that laboratory, 1 ml of ba-1 diluent and a tungsten bead (qiagen, inc . [canada], mississauga, ontario) were added to each tube, and tissues were homogenized on a retsech mixer mill (qiagen). Beginning in mid - august, 2002, a total of 109 american crows, 31 blue jays, 6 common ravens (corvus corax), and 4 black - billed magpies (pica pica) were examined . Two cloacal swabs (one each from a common raven and an american crow) and one oropharyngeal swab from a blue jay were not taken; hence swabs from both sites were available for a total of 147 birds . Specimens were collected by the procedures described above . However, after the 125-l aliquots of swab eluate were removed, the remainder of the sample was frozen at 80c and shipped to the national microbiology laboratory for confirmatory rt - pcr testing . Tissues were removed and tested for wnv by using rt - pcr on 42 (29 crows, 9 blue jays, 3 common ravens, and 1 magpie) of the 150 birds . In both laboratories, swabs were incubated at 37c for 30 min and shaken intermittently; if debris was present, tubes were centrifuged for 5 min at 4,000 rpm . Aliquots of 125 l of the manufacturer s grinding solution and of each swab sample were mixed in a microcentrifuge tube . This mixture was centrifuged by using a vortex mixer (vwr international, mississauga, ontario), and test strips were inserted into the supernatant fluid for 15 min, then removed and examined . During this study, two and five different persons from the manitoba and ontario laboratories, respectively, interpreted or read the test strips . As illustrated by nasci et al . (4), presence of wnv was confirmed by the formation of a reddish purple line on the test strip, corresponding to the wnv - positive location and the development of a test control line . Real - time taqman rt - pcr assays to detect wn viral rna, as described by lanciotti et al . (1), were conducted on all swabs and representative tissue samples from manitoba and all tissue samples from ontario . Briefly, rna was extracted from 150 l to 200 l of swab eluates or tissue homogenates by using qiaamp viral rna kits (qiagen). A final volume of 70 l of eluted rna was stored at 80c until used . Five microliters of rna was combined with the appropriate primers and probes in buffer with the taqman rt - pcr ready - mix kit (pe applied biosystems, foster city, ca). Samples were subjected to 40 amplification cycles in an abi prism 7700 sequence detection system instrument (pe applied biosystems), according to the manufacturer s protocol for taqman rt - pcr cycling conditions . All samples were screened with primers and probe specific to the 3 ntr, and positive extracts were confirmed by reamplification with the env primers and probe (1). Swabs or tissues were considered positive only if positive results were obtained with both primer and probe sets . Sensitivity and specificity of the antigen - capture assay applied to oropharyngeal and cloacal swabs from corvids were calculated by using the results of taqman rt - pcr assays on swab or tissue samples as the true outcome (6). To establish how storage conditions and duration of storage affected the sensitivity of the antigen - capture assay, cultures of egypt 101 strain of wnv (approximately 4 x10 pfu / ml) polyester swabs were then dipped into each virus solution, placed into 1 ml of manufacturer s grinding solution, and held at 20c, 4c, or room temperature (approximately 18c). The test strips were applied to aliquots of each solution 0, 3, 5, 7, 10, and 14 days later, as described previously . To assess virus viability in grinding solution, 50 l of each tested grinding solution and virus dilution was placed into 450 l of ba-1 diluent, and aliquots of 200 l of this mixture were added to vero cells reared on standard nutrient medium (i.e., dulbecco s modified eagle medium and 10% fetal bovine serum [fbs]) at 37c and 5% co2 . After a 1-h adsorption, 2 ml of nutrient medium (same as above but 5% fbs) was added to tissue culture wells, which were incubated as above for up to 7 days . Positive (wnv in ba-1 as above) and negative (media only) controls were maintained under the same conditions, and cells in culture were observed daily for cytopathic effect (cpe). Cells from positive controls and the highest concentration of virus used (i.e., 4 x 10 pfu / ml) in grinding solution for each temperature and sampling date were acetone fixed, exposed to wnv - specific antibodies (produced in rabbits and supplied by h. weingartl, canadian food inspection agency, winnipeg, manitoba) and conjugated goat anti - rabbit immunoglobulin (ig) g (kirkegaard & perry, gaithersburg, md) and assessed for fluorescence under a uv microscope . Thirty - one american crows (corvus brachyrhynchos) and 28 blue jays (cyanocitta cristata), collected from june to december in 2001 and held at 20c were tested in march and april 2002 . Swabs were taken from the oropharyngeal cavity and cloaca of each bird . In 2002, testing was restricted to crows (n = 222) from which only oropharyngeal swabs were collected and tested by vectest . Polyester swabs (vwr canlab, mississauga, ontario) were applied to the oropharyngeal cavity and the cloaca . Swabs were placed in microcentrifuge tubes containing 1 ml of ba-1 diluent (similar to that described by nasci et al . ). The antigen - capture assay was carried out according to the manufacturer s instructions except that the aliquot of swab eluate was 125 l, not 250 l . To confirm the infection status of birds, approximately 25-mg pieces of brain and kidney from each bird were pooled in microcentrifuge tubes and stored at 80c before shipment to the national microbiology laboratory . At that laboratory, 1 ml of ba-1 diluent and a tungsten bead (qiagen, inc . [canada], mississauga, ontario) were added to each tube, and tissues were homogenized on a retsech mixer mill (qiagen). Beginning in mid - august, 2002, a total of 109 american crows, 31 blue jays, 6 common ravens (corvus corax), and 4 black - billed magpies (pica pica) were examined . Two cloacal swabs (one each from a common raven and an american crow) and one oropharyngeal swab from a blue jay were not taken; hence swabs from both sites were available for a total of 147 birds . Specimens were collected by the procedures described above . However, after the 125-l aliquots of swab eluate were removed, the remainder of the sample was frozen at 80c and shipped to the national microbiology laboratory for confirmatory rt - pcr testing . Tissues were removed and tested for wnv by using rt - pcr on 42 (29 crows, 9 blue jays, 3 common ravens, and 1 magpie) of the 150 birds . In both laboratories, swabs were incubated at 37c for 30 min and shaken intermittently; if debris was present, tubes were centrifuged for 5 min at 4,000 rpm . Aliquots of 125 l of the manufacturer s grinding solution and of each swab sample were mixed in a microcentrifuge tube . This mixture was centrifuged by using a vortex mixer (vwr international, mississauga, ontario), and test strips were inserted into the supernatant fluid for 15 min, then removed and examined . During this study, two and five different persons from the manitoba and ontario laboratories, respectively, interpreted or read the test strips . As illustrated by nasci et al . (4), presence of wnv was confirmed by the formation of a reddish purple line on the test strip, corresponding to the wnv - positive location and the development of a test control line . Real - time taqman rt - pcr assays to detect wn viral rna, as described by lanciotti et al . (1), were conducted on all swabs and representative tissue samples from manitoba and all tissue samples from ontario . Briefly, rna was extracted from 150 l to 200 l of swab eluates or tissue homogenates by using qiaamp viral rna kits (qiagen). A final volume of 70 l of eluted rna was stored at 80c until used . Five microliters of rna was combined with the appropriate primers and probes in buffer with the taqman rt - pcr ready - mix kit (pe applied biosystems, foster city, ca). Samples were subjected to 40 amplification cycles in an abi prism 7700 sequence detection system instrument (pe applied biosystems), according to the manufacturer s protocol for taqman rt - pcr cycling conditions . All samples were screened with primers and probe specific to the 3 ntr, and positive extracts were confirmed by reamplification with the env primers and probe (1). Swabs or tissues were considered positive only if positive results were obtained with both primer and probe sets . Sensitivity and specificity of the antigen - capture assay applied to oropharyngeal and cloacal swabs from corvids were calculated by using the results of taqman rt - pcr assays on swab or tissue samples as the true outcome (6). To establish how storage conditions and duration of storage affected the sensitivity of the antigen - capture assay, cultures of egypt 101 strain of wnv (approximately 4 x10 pfu / ml) polyester swabs were then dipped into each virus solution, placed into 1 ml of manufacturer s grinding solution, and held at 20c, 4c, or room temperature (approximately 18c). The test strips were applied to aliquots of each solution 0, 3, 5, 7, 10, and 14 days later, as described previously . To assess virus viability in grinding solution, 50 l of each tested grinding solution and virus dilution was placed into 450 l of ba-1 diluent, and aliquots of 200 l of this mixture were added to vero cells reared on standard nutrient medium (i.e., dulbecco s modified eagle medium and 10% fetal bovine serum [fbs]) at 37c and 5% co2 . After a 1-h adsorption, 2 ml of nutrient medium (same as above but 5% fbs) was added to tissue culture wells, which were incubated as above for up to 7 days . Positive (wnv in ba-1 as above) and negative (media only) controls were maintained under the same conditions, and cells in culture were observed daily for cytopathic effect (cpe). Cells from positive controls and the highest concentration of virus used (i.e., 4 x 10 pfu / ml) in grinding solution for each temperature and sampling date were acetone fixed, exposed to wnv - specific antibodies (produced in rabbits and supplied by h. weingartl, canadian food inspection agency, winnipeg, manitoba) and conjugated goat anti - rabbit immunoglobulin (ig) g (kirkegaard & perry, gaithersburg, md) and assessed for fluorescence under a uv microscope . The sensitivity and specificity of the vectest assay for detecting wnv in oropharyngeal and cloacal swabs are presented in the table . Data from ontario reflect the sensitivity and specificity of this antigen - capture assay by using swab eluates in relation to the infection status (taqman rt - pcr on kidney and brain) of corvids . In contrast, with the exception of 42 birds in which tissue samples were used to establish wnv infection status, the manitoba results evaluate the sensitivity and specificity of the antigen - capture assay by using swab eluates in relation to the detection of virus in the same sample by taqman rt - pcr . Data from the ontario birds from 2001 need to be interpreted with caution since sample size was small and the birds were frozen for up to 10 months . However, cloacal swabs appeared to be less sensitive than oropharyngeal swabs in detecting an infected bird . The sensitivity and specificity of the antigen - capture assay, when oropharyngeal swabs from crows collected in 2002 were used, were 83.3% and 95.8%, respectively, indicating that this assay is acceptable for use as a field detection test in a wnv surveillance program . The advantages of this assay compensate for lower sensitivity only in areas where infected crows are relatively common . The manitoba data also indicate that this antigen - capture assay is sufficiently sensitive and specific to detect wnv in oropharyngeal and cloacal swabs from crows when compared to rt - pcr results . Sixty - two of 109 crows were positive by rt - pcr on either oropharyngeal or cloacal swab, although the number of birds that would have been positive using tissues was not determined . All but three birds positive on oropharyngeal swabs were also positive on cloacal swabs . Small sample sizes and the relatively small number of positive samples on rt - pcr make evaluation of the utility of this antigen - capture assay on blue jays, common ravens, and magpies difficult . In general, the test s specificity seems superior to its sensitivity (table). Further evaluation is necessary before there can be confidence in this assay as a means of detecting wnv in these species . Cloacal swabs were not available from one bird in each of these groups of birds . An oropharyngeal swab was not taken from one of the blue jays . In manitoba, the vectest assay was not evaluated against rt - pcr on tissue for all birds, and thus the sensitivity and specificity cannot be related to the true infection status of each bird . However, indirect evidence suggests that the manitoba antigen - capture results approximate those that would have be obtained had rt - pcr on tissues been used in the comparison . On the 29 manitoba crows in which tissues were used to establish infection status by rt - pcr, the sensitivity and specificity (i.e., 83.3% and 94.1%, respectively) of the antigen - capture assay on oropharyngeal swabs were comparable to those from ontario . Likewise, the data from manitoba are similar to the data on sensitivity and specificity of the antigen - capture assay on swab eluates from crows in ontario and compatible with the high prevalence or titers of virus in oropharyngeal or cloacal samples described by komar et al . Vectest samples had higher mean cycle threshold (ct) values (i.e., ct values are a measure of the overall virus titer in samples; higher ct values indicate lower viral loads) for both primer and probe sets (i.e., 26.1 4.5, generic; 26.0 4.9, envelope) compared to bona fide positives (i.e., 21.0 3.4, generic; 19.7 3.7, envelope). This observation suggests that the viral titers in the false - negative samples were below the threshold for consistent detection when the antigen - capture format was used . Similar but lower levels of sensitivity have been reported for this antigen - capture assay when it has been used to detect wnv in field - collected mosquitoes (4). Regardless of the storage conditions of swab samples, vectest strips always detected wnv at viral concentrations> 4 x 10 pfu / ml but never at titers of #4 x 10 pfu / ml . The vectest assay detected wnv at 4 x 10 pfu / ml when the grinding solutions were held at any of the three temperatures for up to 7 days; thereafter wnv was only consistently detected at 4 x 10 pfu / ml when solutions were held at 20c . None of the tissue cultures inoculated with grinding solution containing wnv had evidence of cpe or positive indirect fluorescent - antibody assay results, whereas all of the positive controls did . Based on these results, swabs can be placed in grinding solution for up to 7 days before the test strips are applied and held from 20c to 18c, without loss of sensitivity . As noted by nasci et al . (4), once in the grinding solution, the titer of wnv is substantially reduced, as would be any potential biological hazard associated with handling or shipping the swab samples . In noncorvid species the vectest assay also has low sensitivity when compared with immunohistochemistry to detect viral antigen in fixed tissue and rt - pcr on frozen tissue . The sensitivity of this antigen - capture assay was 46.7% for oropharyngeal swabs taken from 27 different raptors (of various species) in ontario during 2002 (g.d . This obviates the use of this assay as a reliable screening test for wnv infection in rehabilitation centers, veterinary clinics, zoos, animal disease diagnostic laboratories, and other settings where potential exposure to a biocontainment level 3 agent is of concern . The advantages of this antigen - capture assay over conventional molecular - based diagnostic procedures such as real - time taqman rt - pcr include simplicity of procedures, no requirement for expensive and technically demanding instruments, and much shorter turn - around times for testing (i.e., results are available in 15 min). In addition, testing swabs rather than tissues eliminates the need to dissect submitted birds, thus decreasing the processing time and cost of testing and the risk for lacerations to laboratory workers (7). The greatest limitation to the use of this system in wnv surveillance programs is the potential loss of information on early season wnv activity . This loss could result from the lower sensitivity of the antigen - capture assay compared to the real - time rt - pcr taqman assay . However, the loss of temporal sensitivity likely would be offset somewhat by the rapid turn - around times possible with the antigen - capture format . In addition, in our experience, the vectest assay does not appear to work as effectively in noncorvid (or all corvid) species . Thus, its usefulness would be greatly diminished in jurisdictions that test all avian species as part of their wnv surveillance programs . The poorer performance of the vectest assay in noncorvids may be related to species - specific differences (and variability) in virus titers in excretions and secretions at the time of death (3). The vectest assay will likely replace taqman rt - pcr assays as the front - line diagnostic test for future wnv dead bird surveillance programs in canada . When this antigen - capture assay is used, only index cases of wnv infection in dead birds detected in a given jurisdiction such as a health unit or municipality need to be confirmed with rt - pcr assays before public notification of virus activity . This procedure would compensate for the slightly lower specificity of the antigen - capture assay and should result in markedly decreased workloads for laboratories using molecular diagnostic procedures . Vectest positives obtained thereafter need not be confirmed by an alternative assay since the impact of false positives in a jurisdiction with previously confirmed virus activity should be negligible.
Bladder cancer is the 7th most common cancer in men and the 17th most common cancer in women in the world . Occupational risks, environmental risks, dietary habits, and cigarette smoking are lifestyle factors influencing the development of bladder cancer . It is a common malignancy requiring a high degree of surveillance because of the frequent recurrences and the poor clinical outcome of invasive disease . Hematuria is often the onset symptom of bladder cancer; therefore, cytologic analysis of urine becomes the initial evaluation method, followed by cystoscopy with or without biopsy . However, cytologic analysis has a limited value because it is operator - dependent and has low sensitivity . Cystoscopy still has some limitations [3, 4]; for example, it is invasive, time - consuming, and expensive, requires sedation or anesthesia, and sometimes leads to iatrogenic injury . Also, evaluation of lesions located in the base or neck of the bladder or in the diverticulum is difficult because of the limited perspective of the cystoscope [5, 6]. To date, serum biomarkers for bladder cancer have some practical value, but they lack optimal sensitivity and specificity in diagnosis and disease categorization . Recently, many radiologic imaging techniques have been used to detect and evaluate bladder tumors, but none is reliable in detection of bladder cancer . Therefore, it is important to establish early detection methods with high sensitivity and specificity for bladder cancer . In this study, hsp74 as a potential tumor antigen was identified by 2-de and western blot using bladder cancer cell line blz211 . Moreover, keratin 1 as an associated protein with hsp74 was found by coimmunoprecipitation . These two molecules, in conjunction, might play a certain role in the progression of bladder cancer and might be seen as potential therapeutic target, which is more inspiring, though further investigations are needed . In addition, these data represent the first report, to our knowledge, of a functional link between hsp74 and keratin 1 in bladder cancer cells . Cells were maintained in rpmi-1640 supplemented with 10% fetal calf serum, 100 u / ml penicillin, and 100 g / ml streptomycin, at 37c in a humidified atmosphere of 5% co2 . Blz211 cells were harvested from monolayer cultures by trypsinization and the cell pellet was washed with sterile pbs and suspended in the same medium . Approximately 6 10 cells were injected i.p . Into 6-week - old female balb / c mice . Spleen cells from these mice were fused with sp2/0 myeloma cells (spleen cells versus myeloma cell = 5: 1). 2 - 3 weeks after fusion, culture supernatants were analyzed using elisa method for antibody production . Positive clones were selected and subcloned twice by semisolid cloning . 1 10 blz211 cells were suspended in 200 l of lysis buffer (7 m urea, 2 m thiourea, 4% chaps, 0.5% triton x-100, 0.5% ipg buffer, 2 mm tbp, and 50 mm dtt) supplemented with protease inhibitor cocktail and vortexed at 4c for 1 h, and then the cell lysate was clarified by centrifugation at 40,000 g for 1 h. the supernatant was collected and protein concentration was determined by bradford assay using bsa as a standard . An ipgphor apparatus (amersham - pharmacia biotech, uppsala sweden) was used for ief with 13 cm ph 310 no - linear or ph 47 immobilized ph gradient (ipg) strips (ge healthcare bio - science ab) at 20c.the strips were rehydrated overnight with 250 l of rehydration buffer (7 m urea, 2 m thiourea, 4% chaps, 0.5% triton x-100, 0.5% ipg buffer, 2 mm tbp, and 30 mm dtt) containing 50 g proteins . Isoelectric focusing protocol was followed as (1) 60 v, 12 h, step and hold mode; (2) 200 v, 2 h; step and hold mode; (3) 500 v, 1 h, gradient mode; (4) 1000 v, 1 h, gradient mode; (5) 8000 v, 1 h, gradient mode; (6) 8000 v, step and hold mode until 30 kvht was reached . Strips were then equilibrated in 10 ml equilibration buffer (6 m urea, 30% w / v glycerin, 4% w / v sds, 50 mm tris - hcl ph 8.8) with 1% w / v dtt for 15 min at room temperature . Strips were removed and incubated in equilibration buffer with 2.5% w / v iodoacetamide for another 15 min . After equilibration, the strips were embedded onto an 11%, 1 mm sds / page gel and were fixed in place with a 0.5% w / v agarose overlay . Gels were run in a se600 (amersham - pharmacia biotech, uppsala sweden) at 10 ma / gel for 20 min then 20 ma / gel until the bromophenol blue dye reached the bottom of the gel . One of 2-de gels with separated proteins were immunoblotted onto nitrocellulose membrane (0.45 m pore size; bio - rad) and blocked overnight at 4c in superblock blocking buffer in tris - bufferedsaline (pierce) to block the nonspecific bindingsite . As first antibodies, the mab diluted 1: 10 in blocking buffer and the membranes were incubated for 1 h at room temperature . After one hour incubation at room temperaturewith the second antibodies, the immunoproducts were visualizedwith ecl western blotting detection reagents (amershambiosciences) and exposure to x - ray films . Each wash was performedwith tris - buffered saline - tween (10 mmol / l tris - hcl (ph 7.6), 100 mmol / l nacl, and 1 ml / l tween 20). One of 2-de gels was fixed in 30% alcohol containing 1% acetic acid for 2 h and was sensitized in 30% alcohol containing 0.2% na2s2o3 and 6.8% sodium acetate for 30 min . The grids were removed with plastic forceps and washed by 3 immersions of 5 min each in double - distilled water . The grids were washed by 3 immersions of 1 min each in double - distilled water . 2-de gel was developed in 0.74% formaldehyde containing 2.5% na2co3 for 48 min . At clear spot times, the grids were washed by 3 immersions of 5 min each in double - distilled water . The images were saved in tiff format and then exported to the imagemaster 2d platinum software 5.0 (amersham - pharmacia, sweden). Spot detection parameters were best adjusted using, first, the smooth parameter which was set to a value of 1.5 allowing the detection of all real spots and split as many as possible overlapping spots . Then, the minimum area was set to eliminate spots that have an area smaller than 15 pixels . Total protein of blz211 cells was used to immunoprecipitate and coimmunoprecipitate interacting proteins (prey proteins) according to the protocol of profound co - immunoprecipitation (co - ip) kit (pierce). The purified antibody was put into the spin cup containing the gel for antibody immobilization . The blz211 cells were washed once with pbs (product number 28372; 0.1 m phosphate, 0.15 m nacl, and ph 7.2). Lysate was collected and transferred to a microcentrifuge tube for centrifugation of samples at ~13,000 g for 510 minutes to pellet the cell debris . The prey complex and controls were put to the appropriate gel in the spin cups, and 0.4 ml of co - ip buffer was added . 100 l elution buffer was put to the gel in the spin cup and the tube was centrifuged . 5 l of the 5x sample buffer was added to the sample and applied to the gel for electrophoresis . The mobile phase buffer b contained acetonitrile and 0.1% methanoic acid, 120 min linear gradient elution, and flowed at 1 l / min . This consisted of a full mass scan (m / z 4002000), zoom scan on the most abundant ion to determine charge state, and a tandem mass spectrometry (ms / ms) scan to collect collision - induced dissociation (cid) spectra on peptides . Automated analysis of cid spectra to determine the amino acid sequence of peptides was performed on computer (sequest software; thermofinnigan) as described by yates iii et al . . Blz211 cells were cultured in 6-well plate with cover slips for 24 hr . The cells on cover slip were fixed for 10 min by immersion in 20c precooling methanol and washed in phosphate - buffered saline (pbs) three times . Cells were incubated in 0.5% tritonx-100/pbs for 5 min at room temperature and washed in pbs three times and incubated with goat serum for 1 h at room temperature . Cells were incubated for 4 h at 4c on a rocker platform in 1:10 dilutions of experimental mcab in pbs . . Then cells were washed in pbs three times, incubated with secondary antibody for 40 min at 4c in 1: 40 dilutions of fluorescein - conjugated goat anti - mouse igg in a pbs media, followed by incubation with dapi for 5 min at room temperature, and washed in pbs three times . The cover slips were subjected to another washing cycle before being monitored for specific fluorescence under an immunofluorescence microscope . 35 bladder cancer tissues and adjacent normal tissues were used to analyze the expression of hsp74 with mcab . Endogenous peroxidase activity was quenched and antigen retrieval was done by 5 min heating in citric acid . After blocking, sections were incubated with hsp74 mcab at 4c overnight . Comparisons between groups for bladder cancer tissues and adjacent normal tissues were performed by applying chi - square test as indicated . Fusion cells were inoculated in a 96-well plate, in which clone growth appeared in 62 wells . Using immunohistochemistry method, the subcloning with limited dilution assay did not stop until the preliminary screening positive hybridoma cells were 100% . To analyze the proteome of blz211 cells, soluble proteins from the blz211 cells western blot showed 3 protein spots were more intense in blz211 cells (figure 1(b)). One of the protein spots was detected by lc - ms / ms with an apparent mass of 94299.96 da, score 70.4, accession 6226869, peptides (hits)7(70000), and pi of 5.18, which was identified as hsp74 (figure 2). Keratin 1 (figure 4) was identified associated with hsp74 by coimmunoprecipitation (figure 3), lc - ms / ms . Compared with ncbinr database showed score 198.2, accession 11935049.0, peptides (hits)20(191000), theory pi 8.16, and mw 66066.74 da . The results showed that they are all distributed in cellular membrane, cytoplasm and nucleus (figure 5). The cellular membrane and cytoplasm show all red, and the cellular nucleus shows some red and some blue . Among the 35 bladder cancer cases, the positive expression rate of hsp74 in bladder cancer tissues was 74.2%, which was similar to that in adjacent normal tissues (x = 0.063, 0.75 but there was a significant difference in the expression intensity distribution between the two groups (x = 21.86, p <0.005). In most cases, the expression intensity of hsp74 in bladder cancer tissues was higher than in normal bladder tissue (table 1, figure 6). Bladder cancer is a prevalent disease that causes substantial morbidity and mortality . Despite the continued refinement of surgical techniques, namely, radical cystectomy, the prognosis of muscle - invasive bladder cancer has remained unchanged for the past 30 years with five - year survival rate remaining disappointingly low at 40% . Understanding the biology mechanism underlying tumorigenesis and tumor progression of bladder cancer is essential for improving the capacity to diagnose and treat the disease . Unraveling the biological complexity underlying these processes is expected to provide novel tools of predictive nature and to enable identification of therapeutic targets by selecting those molecular targets significantly and differentially expressed in bladder tumors . Numerous markers that correlate to some extent with bladder cancer stage and prognosis have been identified . However, the ability of most markers in predicting the clinical outcome of individual tumors is limited, and alternative markers are still needed for detection of the disease and for predictive purposes . Recent advances in expression profiling of cancer cells by proteomic technologies, high - resolution 2-dimensional electrophoresis (2-de), and mass spectrometry have made it possible to identify candidate proteins as tumor markers in various cancers . In bladder cancer, celis et al . Performed proteome analysis by extensive 2-de and developed a comprehensive 2-de database for bladder cancer that includes profiles of both transitional and squamous cell carcinoma . Therefore we used 2-dimensional electrophoresis and mass spectrometry to carry out exploratory research in bladder cancer and identified hsp74 as potential target . This result gives us the bladder cancer intracellular localization of hsp74 in the preliminary concept . Hsp74 belongs to a member of the family heat - shock (stress) proteins (hsps). In the face of thermal, chemical, or physiological stresses that cause unfolding of proteins, cells preferentially express hsps, a process called the heat - shock (or stress) response . Related researches on hsp can draw the following conclusions: (1) hsp has relevance with cancer diagnosis; (2) detection of hsp can help identifying abnormalities in carcinogenesis; (3) hsp is associated with some tumor differentiation degree; (4) hsp has significant correlation with some specific molecule . So the detection of hsp has important significance in the program of individualized cancer therapy, regular followup, and avoidance of excessive use of cytotoxic cancer drug . It is common for a constitutively expressed hsp70 cognate (hsc70) to be present in cells under all conditions, where it functions as a chaperone during normal protein synthesis . Mucous membrane of urinary bladder is exposed for longtime to the urine which contains various kinds of mutagenic activity materials and toxic metabolites . Elevated hsp74 expression hsp74 was associated with keratin 1 as determined by coimmunoprecipitation from bladder cancer cell line blz211 . Keratin 1 belongs to keratin family and is a specific marker of mammal epithelium terminal differentiation since keratin 1 protein mainly locates in epithelial prickle cell layer and epithelial granular cell layer . Monoclonal antibodies to keratin 1 carboxy terminal (synthetic peptide) provide an important means of examining keratin expression in epidermal tumors and keratinizing disorders . Cytokeratins 1, 7, and 14 immunoexpression is helpful in the diagnosis of basaloid squamous carcinoma . It remains to be determined whether the binding of hsp74 to keratin 1 is dependent on a linear sequence of keratin 1 or a conformation of keratin 1 tetramers or polymers . Furthermore, although our data suggest a direct interaction of hsp74 with keratin 1, we cannot exclude the possibility that this interaction may be mediated by one or more additional proteins that form a large complex . To detect tumor mark by monoclonal antibodies is a kind of classical method . Using a combination of techniques including 2-de, co - ip, western blot, lc - ms / ms, immunofluorescence, and immunohistochemistry, we find the expression intensity of hsp74 in bladder cancer tissues is higher than that in normal bladder tissue . The results of this research suggest that hsp74 might be a potential marker of bladder cancer, although further validation is still needed . To use this antigen to detect tumor has more problems which need to be resolved . The relevance of hsp74 expression with clinical pathological characteristics and survival time is worth to follow - up study . Specifically, in tumor formation the interaction mechanism of keratin 1 and hsp74 need to be clarified further.
Comorbidity indicates the presence of one or more additional medical disorders co - occurring with a primary disease within the same individual . The prevalence of disease rises with age; therefore, comorbidity becomes increasingly more common over a person's life span . With the aging of the general population and advancements in medical care, the prevalence of chronic diseases has doubled between 1985 and 2005, and the proportion of patients with 4 chronic diseases has increased 3-fold 1 . A large cross - sectional study from the scotland medical database reported in 2007 that 42% of all patients had at least one comorbidity, while 23% had> 2 comorbidities 2 . Comorbidity is not only a feature of old age, but is also particularly associated with people who experience poor social support, high levels of socio - economic deprivation, and mental health disorders 2 . In addition, comorbidity is often associated with a decline in functional reserves and an increasing prevalence of frailty . Therefore, patients with comorbidities are more likely to have suboptimal outcomes and experience more treatment - related toxicities . Previous studies have suggested that cancer patients with multiple comorbidities are associated with a delay in cancer diagnosis 3, increased risk of surgical complications 4, 5, higher postoperative mortality rates 6 - 8, and a greater usage of medical resources 9 . For example, a population - based cohort study from sweden demonstrated that patients with comorbidities have an increased risk of all - cause mortality after curative surgery for esophageal cancer of 1.24-fold compared to those patients without comorbidities 10 . Furthermore, a review study found that patients with comorbidities have poorer survival outcomes (5-year mortality hazard ratios of 1.1 - 5.8 across various types of cancer) than those without comorbidities 11 . Unfortunately, in spite of the knowledge of the importance of comorbidities in cancer patients, it remains unclear whether the negative influence of comorbidity is attributable to cancer- or noncancer - specific mortality rates . Cancer - related mortality has been recognized as the leading cause of death in taiwan since the national registry of death database was made available in 198112, 13 . The proportion of cancer - related deaths has increased annually, accounting for 25% of the total number of deaths in taiwan in 2014 12, 13 . The population older than 70-years of age in taiwan has increased 2.4-fold between 1989 and 2014 14 . With the aging of the general population and increasing incidence of cancer worldwide, there is an urgent need to improve our clinical understanding of the influence of comorbidity on postoperative outcomes in cancer patients . The aim of this study, therefore, was to examine the effect of comorbidity on survival outcomes, in particular cancer- and noncancer - specific survival rates, after surgery in patients with solid cancers . In total, 37,288 patients who underwent radical surgery for solid cancers at four affiliated hospitals of the chang gung memorial hospital (linkou, keelung, chiayi, and kaohsiung) between january 2007 and december 2012 were included in this study . Patients with pathologically or radiographically suspicious malignancies underwent radical resection of their primary cancers with curative intent . Patients who underwent palliative resection or bypass surgery were excluded, as were patients with skin cancers and superficial urinary bladder cancers . All patients were stratified into subgroups, according to comorbidity using the charlson comorbidity index (cci) 15, for postoperative survival analysis . This study was approved by the institutional review boards at all four affiliated hospitals of the chang gung memorial hospital, in compliance with the helsinki declaration fourth revision (1996). Administrative and clinical data collected before cancer surgery included patient demographics (age, gender, eastern cooperative oncology group [ecog] performance status, admission mode [elective or emergency], prior history of cancer, cancer site by anatomical location, histological grade of tumor differentiation, and clinical tumor stage), american society of anesthesiologist (asa) physical status, and cci scores . Demographic information was recorded by primary care clinicians using a prospectively formulated electronic record form provided by the institutional cancer center with the intent of improving the quality of care for cancer patients in taiwan since the implementation of the 2006 cancer prevention and treatment act . Data quality was maintained for completeness and accuracy by individual multidisciplinary teams and well - trained cancer center personnel . Tumor stage was recorded as localized, regional, advanced, and unclassified using the surveillance, epidemiology, and end results program summary stage classification 16 . Asa scores were provided at preanesthetic evaluation by anesthesiologists while cci scores were calculated from tabulated electronic record forms using the international classification of diseases ninth revision coding . A modified cci that excluded scores for patient age and type of cancer was used in this study . Patients with a diagnosis of more than one tumor within the study period were analyzed from the date of surgery of the first tumor . Patients receiving multiple surgeries for their primary tumors within the study period were analyzed from the date of the first surgery . Overall survival was determined from the time of surgery to any cause of death or the date last known to be alive . Cancer- and noncancer - specific mortalities were defined as death due to cancer and any cause of death other than cancer, respectively . All dates of death were obtained from either the institutional cancer registry or taiwan cancer registry . Differences in the distribution of clinical variables among different age groups were compared using the chi - squared test . The effect of cci scores on survival was assessed using the cox proportional hazards model . Adjusted hazard ratios for different cci subgroups were estimated by performing a multivariate cox regression after adjusting for gender, age, prior history of cancer, ecog scale, asa score, admission mode, primary cancer site, tumor stage, and tumor grade . These nine clinicopathological variables were selected, according to our previous study 17, with the intent of minimizing potential confounding effects on postoperative survival outcome in cancer patients . Statistical analyses were conducted in statistical package for the social sciences (spss) for windows software version 17.0 (spss inc ., in total, 37,288 patients who underwent radical surgery for solid cancers at four affiliated hospitals of the chang gung memorial hospital (linkou, keelung, chiayi, and kaohsiung) between january 2007 and december 2012 were included in this study . Patients with pathologically or radiographically suspicious malignancies underwent radical resection of their primary cancers with curative intent . Patients who underwent palliative resection or bypass surgery were excluded, as were patients with skin cancers and superficial urinary bladder cancers . All patients were stratified into subgroups, according to comorbidity using the charlson comorbidity index (cci) 15, for postoperative survival analysis . This study was approved by the institutional review boards at all four affiliated hospitals of the chang gung memorial hospital, in compliance with the helsinki declaration fourth revision (1996). Administrative and clinical data collected before cancer surgery included patient demographics (age, gender, eastern cooperative oncology group [ecog] performance status, admission mode [elective or emergency], prior history of cancer, cancer site by anatomical location, histological grade of tumor differentiation, and clinical tumor stage), american society of anesthesiologist (asa) physical status, and cci scores . Demographic information was recorded by primary care clinicians using a prospectively formulated electronic record form provided by the institutional cancer center with the intent of improving the quality of care for cancer patients in taiwan since the implementation of the 2006 cancer prevention and treatment act . Data quality was maintained for completeness and accuracy by individual multidisciplinary teams and well - trained cancer center personnel . Tumor stage was recorded as localized, regional, advanced, and unclassified using the surveillance, epidemiology, and end results program summary stage classification 16 . Asa scores were provided at preanesthetic evaluation by anesthesiologists while cci scores were calculated from tabulated electronic record forms using the international classification of diseases ninth revision coding . A modified cci that excluded scores for patient age and type of cancer was used in this study . Patients with a diagnosis of more than one tumor within the study period were analyzed from the date of surgery of the first tumor . Patients receiving multiple surgeries for their primary tumors within the study period were analyzed from the date of the first surgery . Overall survival was determined from the time of surgery to any cause of death or the date last known to be alive . Cancer- and noncancer - specific mortalities were defined as death due to cancer and any cause of death other than cancer, respectively . All dates of death were obtained from either the institutional cancer registry or taiwan cancer registry . Differences in the distribution of clinical variables among different age groups were compared using the chi - squared test . The effect of cci scores on survival was assessed using the cox proportional hazards model . Adjusted hazard ratios for different cci subgroups were estimated by performing a multivariate cox regression after adjusting for gender, age, prior history of cancer, ecog scale, asa score, admission mode, primary cancer site, tumor stage, and tumor grade . These nine clinicopathological variables were selected, according to our previous study 17, with the intent of minimizing potential confounding effects on postoperative survival outcome in cancer patients . Statistical analyses were conducted in statistical package for the social sciences (spss) for windows software version 17.0 (spss inc ., baseline characteristics of the patients included in this study are summarized in table 2 . Among the 37,288 patients who underwent cancer surgery between 2007 and 2012, 69.1%, 21.4%, 6.4%, 1.8%, 0.8%, and 0.4% exhibited cci scores of 0, 1, 2, 3, 4, and 5 - 8, respectively . A significantly greater proportion of patients with comorbidities were male who presented with older age, greater admission from emergency departments, poorer ecog scales, higher asa scores, more extensive histories of cancer, more poorly differentiated grades of tumor, and greater numbers of primary tumor sites within intra - abdominal organs as compared to those patients without comorbidities . After a median follow - up duration of 38.9 (interquartile range, 22.8 - 60.4) months, 9,704 patients (26.0%) had died after cancer surgery at the date of study censor . Overall mortality rates of patients with cci scores of 0, 1, 2, 3, 4, and 5 - 8 were 22.9%, 29.5%, 38.2%, 43.2%, 50.2%, and 56.4%, respectively (figure 1). Figure 2 represents a histogram of the cancer- and noncancer - specific mortality rates for each cci subgroup at the date of study censor . In general, patients with higher cci scores were associated with both increased cancer- and noncancer - specific mortality rates . Hazard ratios of univariate and multivariate analyses of overall, cancer-, and noncancer - specific mortality rates, as stratified according to cci subgroups, are shown in table 3 . Patients with increasing cci scores were associated with significantly poorer outcomes in terms of overall and noncancer - specific mortality rates, in both the univariate and multivariate analyses . Consideration of the cancer - specific mortality rates, however, revealed patients with increasing cci scores to be significantly associated with higher mortality rates in the univariate analysis, but only patients with cci scores of> 2 were found to be significantly associated with higher mortality rates in the multivariate analysis compared to those patients without comorbidities . Given many patients died within the first 2-years after cancer surgery, we calculated the mortality rates according to cci stratification within each quarter up to the end of the first 2-years after cancer surgery, with the intent of analyzing the impact of cci subgroups on cancer- and noncancer - specific mortality risk (figure 3). The cancer - specific mortality rates in each quarter were consistent (range, 1.7%-2.0%) during the first year after cancer surgery, and gradually declined from 1.5% in the fifth quarter to 1.1% in the eighth quarter after cancer surgery, in the overall population . By contrast, the overall noncancer - specific mortality rates were shown to be consistent (range, 0.6%-0.9%) in each quarter up to the end of the first 2-years after cancer surgery . In general, patients with increasing cci scores were associated with higher cancer - specific mortality rates compared to those patients without comorbidities . However, differences in cancer - specific mortality rates between different cci subgroups were diminished until the sixth quarter after cancer surgery . Again, patients with cci scores of> 2 were associated with higher noncancer - specific mortality rates after cancer surgery . However, differences in noncancer - specific mortality rates between patients with cci scores of 0 - 2 and> 2 were only observed in the first two quarters after cancer surgery . In this study, we evaluated the influence of comorbidity on survival outcomes in patients with solid cancers undergoing potentially curative resections in multiple centers within a 6-year period . Through the integration of prospectively collected electronic medical records 17 and deceased data obtained from the taiwan cancer registry, we were able to analyze patient demographics, cancer types, histories of previous cancers, tumor stages, admission types, and associated medical comorbidities in a non - biased and quantitative manner with minimal absent data . Accordingly, our study identified that patients with increasing cci scores were associated with a greater postoperative mortality risk, including increased overall, cancer-, and noncancer - specific mortality rates . Most importantly, our study also demonstrated that patients with multiple comorbidities (especially cci scores of> 2) were most vulnerable to noncancer - related deaths within the first 6 months after cancer surgery . However, surgery can result in substantial morbidity and mortality in cancer patients, especially in those who are medically unfit or frail to begin with 18 - 22 . Consistent with previously published reports, our findings have demonstrated that patients with increasing numbers of comorbidities are associated with poorer postoperative survival outcomes, both in terms of cancer- and noncancer - specific mortality rates . However, differences in survival outcomes among patients within different cci subgroups were less distinct after adjusting for other clinicopathological characteristics . Our results remind us that patients with comorbidities are associated with a higher prevalence of poorer clinicopathological features; therefore, the true impact of comorbidities on survival outcomes may be more predictable after adjustment or concurrent consideration of those negative predictors . For example, a recent study of ours has demonstrated that surgical mortality risk was highest among older patients with multiple concomitant comorbidities and functional limitations (50% postoperative 3 month mortality rate compared to a 1% postoperative 3 month mortality rate in patients with no comorbidities and normal functional status 23). The high risk of postoperative mortality for extremely frail patients should be weighed against the potential benefits in order to determine their eligibility for surgery that may assist in the decision - making process as well as the design of alternative treatment options 24 . Previous studies have suggested that the presence of comorbidities increases the risk of all - cause mortality in patients with cancer 25 - 28 . The impact of comorbidity on survival outcomes in cancer patients is undoubtedly apparent, through increasing postoperative complications 4, 5 and poor adherence to adjuvant treatments 29 - 31 . Beyond inadequate treatments for cancer, several studies have suggested that cancer survivorship is associated with an increased likelihood of substandard care across a broad range of chronic medical conditions 32, 33 . In contrast, a survey that compared primary care physicians and oncologists, with respect to their practices for the care of breast and colon cancer survivors, reported that primary care physicians were less likely than oncologists to endorse routine use of recommended tests for detecting cancer recurrence 34 . Therefore, a shared care model by coordination between the oncologist and primary care physician may offer an innovative approach for the monitoring of cancer survivors 35, 36 . Traditionally, postoperative 30 - 90 day mortality rates have been used as a measurement for predicting surgical risk 37 . However a significant number of patients live for longer than 30 days after surgery but die within a year 38 . The increment in postoperative one - year mortality risk was especially prominent in elderly patients, those with comorbidities, and those who received emergent surgeries 18 . Consistent with these findings, our study showed a steady proportion of patients dying due to cancer in each quarter during the first year after cancer surgery . Furthermore, our study also demonstrated that patients with cci scores of> 2 were most vulnerable to noncancer - related deaths within the first 6 months after cancer surgery . There were no significant differences in noncancer - specific mortality rates between patients of different cci subgroups, who lived beyond 6 months after cancer surgery . Discrepancies between mortality rates at different postoperative time points, among patients with multiple comorbidities, highlights the importance of identifying patients at increased risk of death after surgery and refinement of postoperative care in cancer patients with multiple comorbidities . In addition to well - known clinical features, including older age, poorer ecog scales, and higher asa scores among patients with increasing cci scores, our study has also demonstrated that these patients are characterized by higher proportions of male gender, poorly differentiated grades of tumor, emergency department admissions, advanced tumor stages, and primary tumor sites within the stomach and hepato - biliary - pancreatic tract . In a report studied over 1.5 million people from scotland the association of male gender and poorly differentiated grade of patients with increasing cci scores in our study possible second to higher proportion of advanced tumor stage and visceral cancer types in these patients group . Previous studies reported that patients with increasing comorbidities are particularly associated with lower socioeconomic status 2 and advanced stage 3, which often resulted in suboptimal cancer screening, treatment and follow - up . Fleming et al found women with cardiovascular and musculoskeletal diseases to be at a lower risk of receiving a diagnosis of advanced breast cancer 3 . One possible explanation is that patients with certain comorbidities (e.g., heart / lung diseases) are associated with a higher utilization of screening mammograms and greater likelihood of diagnosis of early stage breast cancer 39 . Since there is a distinct lack of specific clinical presentations and effective screening methods for the early diagnosis of visceral cancers, these reasons may possibly explain the disparities between the basic characteristics of patients with and without comorbidities in our study . Following the installment of cancer prevention and treatment arc in taiwan in 2006, all newly diagnosed cancer cases are reviewed by a cancer - specific multi - disciplinary team before any antitumor treatment in our institution . At chang gung memorial hospital, all patients with previous known diseases, the primary care physician will review the patient's medical record, medication and blood works to determine the status of the disease . For those found of comorbidities along with cancer diagnosis, a consultation of each specialist will be obtained as part of the preoperative evaluation and followed at an as - needed basis during the perioperative period . The goal of treating other comorbidities during the perioperative period is to prepare the patient for cancer surgery and reduce surgical risks . Once the patient is ready to be discharged, follow - up appointment is usually made to visit the specialist alongside with post - operative follow - up at an outpatient setting . If a patient preferred to find another specialist at his / her own discretion, a summary of treatment during hospitalization would be given . The patient is advised to bring his / her current medication record at each follow - up . Because the easily accessible healthcare system, in taiwan, a patient is often followed by a specialist of each specific disease . The strength of our study was the large patient numbers included in the design from across multiple institutes in taiwan over a 6-year period . Our study evaluated the impact of comorbidities both on cancer- and noncancer - specific survival outcomes after cancer surgery . Additionally, several important clinicopathological variables that can potentially influence postoperative outcomes were prospectively identified and adjusted for to minimize their potential confounding effects on survival analysis therefore, our study is capable of representing the true impact of comorbidity on survival outcomes after cancer surgery . Firstly, our study only included patients who underwent cancer surgery, and there was a selection bias towards patients who were offered and had received surgical treatment . Therefore, the true postoperative mortality risks may be underestimated in patients with solid cancers and comorbidities . Secondly, since this study calculated cci scores from medical records using the international classification of diseases ninth revision coding it was not possible to evaluate the relationship between the severity of medical illnesses and survival outcomes . Thirdly, due to the heterogeneity of different cancer types, pathological variables, and differences in postoperative organ reserves, we did not include postoperative treatments (e.g., adjuvant chemotherapy or radiation therapy) in our statistical analysis; as such, treatment options were less available to patients with multiple comorbidities and might have had a confounding effect on survival outcome . In conclusion, our study has shown that patients with increasing numbers of comorbidities are associated with poorer survival outcomes in terms of overall, cancer-, and noncancer - specific survival rates in patients with solid cancers who underwent cancer surgery . Patients with multiple comorbidities (especially cci scores of> 2) were most vulnerable to both cancer- and noncancer - specific deaths within the first 6 months after cancer surgery . Our results suggest that for both the patient and clinician, it should be taken into consideration about cancer surgery when dealing with multiple comorbidities . Research involving human participants: all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards.
Falls are the leading cause of injury among older adults and the risk of falls and subsequent fall - related injury increases with age . Falls are not only associated with morbidity and mortality in older populations, but are also related to immobility, decreased quality of life, fear of falling, functional dependency, early admission to long - term care facilities, and increased medical costs . Accordingly, identifying older adults at an increased risk of falls and effective fall prevention interventions have the potential to reduce fall risk among older individuals, as well as fall - related disability and medical costs . In the united states, around one - third of people aged 65 years or older living in the community fall every year, and about half of those who fall do so repeatedly . Although not all falls lead to injury, about 10% result in a major injury such as a fracture, serious soft tissue injury, or traumatic brain injury requiring medical attention . In 2012, 2.4 million nonfatal falls among older adults were treated in emergency departments and more than 722,000 of these patients were hospitalized . Accordingly, the direct medical costs of falls, adjusted for inflation, is estimated at $30 billion . The incidence of falls among chinese older people is approximately half of that among caucasian populations . A systematic literature review of studies conducted in china, hong kong, singapore, and taiwan revealed that the annual fall rate ranges from 14.7% to 34% (median, 18%). In four prospective studies, injuries were reported by 60% to 75% of those reporting falls, with fractures accounting for 6% to 8% of all injuries . Few studies have explored the prevalence and medical burden of falls among korean older adults . One study of 351 individuals aged 65 years or older found that 42% reported at least one episode of falling in the previous 12 months, 38% of whom required either the attention of a physician or hospitalization . In another study, 13% of 828 community - dwelling older korean adults experienced falls during the last year . Additionally, a questionnaire - based interview survey of 2,295 older adults living in rural communities revealed that 32% had suffered from fall - related injuries during the previous year . The direct costs of these injuries were calculated to be 596,466,000 won (us $1 = 1,140) and the total socioeconomic costs were estimated to be 1,054,547,000 won . When the above calculated socioeconomic cost for the 2,295 subjects is applied to the 1,067,262 korean rural older adults in 2009, the socioeconomic costs due to fall - related injuries can be estimated as 343,614,988,000 won . Accordingly, falls are one of the most common, and most important, medical problems among korean older adults . The greatest risk factors for falls include previous falls, strength, gait and balance impairments, visual impairment, arthritis, disability, depression, and cognitive impairment . Additionally, polypharmacy, anti - hypertension or psychotropic medications, arrhythmia, and parkinson s disease are reportedly risk factors for falls [12 - 16]. The risk of falls increases with the number of risk factors, and comprehensive multifactorial assessment and risk factor interventions can decrease the risk of falls and improve the health status of older adults . A steering committee included the president and cabinet members of the korean association of internal medicine (kaim); it was responsible for establishing a strategy for guideline development and approving a budget . A working group was responsible for development and writing the guidelines; it included members of the clinical practice guidelines committee of kaim, one methodology expert (a member of korean cochrane), and two coordinators . The guidelines were developed via multidisciplinary involvement by the kaim and the korean geriatrics society, with a focus on rehabilitation medicine, family medicine, orthopedic surgery, and neurology . The nominal group technique was applied to select the final recommendations, with a panel selected from members of the participating societies . Finally, an external review committee consisted of representatives of the participating societies who were not involved in the nominal group technique . The working group met 14 times, the external review group met once, and an additional seven workshops were held to engage in the adapte process . A methodology expert from korean cochrane (h.j.k .) Participated in the development of the guidelines to help develop a scientific and standardized method . Clinical questions were designed based on pico (population, intervention, comparison, and outcomes). Candidate guidelines were obtained from the following databases on february 20, 2014; medline, embase, cochrane library, koreamed, korean medical database, korea education and research information service, national guideline clearinghouse, international guideline library, and turning research into practice (appendix 1). Target studies about guidelines published between january 1, 2009 and february 20, 2014 were reviewed . Inclusion criteria were as follows: (1) evidence - based; (2) written in korean or english; (3) generated through expert consensus and external review; and (4) latest revised version . Guidelines for hospitalized patients, outdated versions, and guidelines that were generated using the adaptation process were excluded (fig . Two independent reviewers from the working team performed the literature review and finally four guidelines were selected . Appraisal of guidelines for research and evaluation ii (agree ii) was performed for the selection of seed guidelines . Two reviewers evaluated seed guidelines based on the korean - agree ii developed by the steering committee for clinical practice guideline of korean academy of medical science; this was validated through a formal consensus, and its practicality was supported through the actual guideline assessment . Agree ii includes six domains (scope and purpose, stakeholder involvement, rigor of development, clarity of presentation, applicability, and editorial independence) and is comprised of 23 structured key items and two items for general assessment . Rigor of development was considered the most important selection criteria, and finally four guidelines were selected with a score greater than the scaled final score of 60% adapte (fig . Data extract tables were created to extract recommendations for each subheading with reference literatures (appendix 2). We also performed a literature review with a de novo method for searching for updated findings for each clinical question (pico). The level of evidence for the planning method, quality, and consistency of each study was evaluated based on grade (grades of recommendation, assessment, development, and evaluation) criteria for high overall quality of evidence across outcomes (table 1). The first draft recommendations were made by the working group, and the final recommendations were selected using the nominal group technique . Consensus was reached by a panel of experts who were selected from the participating societies; they included experts in the fields of internal medicine, family medicine, neurology, rehabilitation medicine, and orthopedics . A facilitator presented the first draft recommendations and asked the experts to brainstorm ideas; during this process, participants did not consult each other or discuss their ideas . Participants then presented each idea through a round robin process, and group discussions generated new ideas or eliminated irrelevant ideas . Final recommendations were confirmed by a voting process (table 2) [21 - 24]. A peer review was performed by three reviewers who were members of the korean geriatrics society (e.j.l . ), korean academy of rehabilitation medicine (j.y.y . ), and korean cochrane (h.j.k . ). The guidelines for fall prevention were announced publicly at an annual program for clinical medical education attended by general practitioners, gastroenterologists, and family doctors (february 15, 2015). Attendees suggested that supplementary material or tools would be helpful to evaluate the risk stratification for falls and to determine appropriate exercises for fall prevention . The kaim financially supported the development of the guidelines and there was no other external financial support . The guideline committee of the kaim is an independent organization, and there were no internal and external influences . A steering committee included the president and cabinet members of the korean association of internal medicine (kaim); it was responsible for establishing a strategy for guideline development and approving a budget . A working group was responsible for development and writing the guidelines; it included members of the clinical practice guidelines committee of kaim, one methodology expert (a member of korean cochrane), and two coordinators . The guidelines were developed via multidisciplinary involvement by the kaim and the korean geriatrics society, with a focus on rehabilitation medicine, family medicine, orthopedic surgery, and neurology . The nominal group technique was applied to select the final recommendations, with a panel selected from members of the participating societies . Finally, an external review committee consisted of representatives of the participating societies who were not involved in the nominal group technique . The working group met 14 times, the external review group met once, and an additional seven workshops were held to engage in the adapte process . The guidelines were developed using an adaptation process based on evidence - based medicine . A methodology expert from korean cochrane (h.j.k .) Participated in the development of the guidelines to help develop a scientific and standardized method . Clinical questions were designed based on pico (population, intervention, comparison, and outcomes). Candidate guidelines were obtained from the following databases on february 20, 2014; medline, embase, cochrane library, koreamed, korean medical database, korea education and research information service, national guideline clearinghouse, international guideline library, and turning research into practice (appendix 1). Target studies about guidelines published between january 1, 2009 and february 20, 2014 were reviewed . Inclusion criteria were as follows: (1) evidence - based; (2) written in korean or english; (3) generated through expert consensus and external review; and (4) latest revised version . Guidelines for hospitalized patients, outdated versions, and guidelines that were generated using the adaptation process were excluded (fig . Two independent reviewers from the working team performed the literature review and finally four guidelines were selected . Appraisal of guidelines for research and evaluation ii (agree ii) was performed for the selection of seed guidelines . Two reviewers evaluated seed guidelines based on the korean - agree ii developed by the steering committee for clinical practice guideline of korean academy of medical science; this was validated through a formal consensus, and its practicality was supported through the actual guideline assessment . Agree ii includes six domains (scope and purpose, stakeholder involvement, rigor of development, clarity of presentation, applicability, and editorial independence) and is comprised of 23 structured key items and two items for general assessment . Rigor of development was considered the most important selection criteria, and finally four guidelines were selected with a score greater than the scaled final score of 60% adapte (fig . Data extract tables were created to extract recommendations for each subheading with reference literatures (appendix 2). We also performed a literature review with a de novo method for searching for updated findings for each clinical question (pico). The level of evidence for the planning method, quality, and consistency of each study was evaluated based on grade (grades of recommendation, assessment, development, and evaluation) criteria for high overall quality of evidence across outcomes (table 1). The first draft recommendations were made by the working group, and the final recommendations were selected using the nominal group technique . Consensus was reached by a panel of experts who were selected from the participating societies; they included experts in the fields of internal medicine, family medicine, neurology, rehabilitation medicine, and orthopedics . A facilitator presented the first draft recommendations and asked the experts to brainstorm ideas; during this process, participants did not consult each other or discuss their ideas . Participants then presented each idea through a round robin process, and group discussions generated new ideas or eliminated irrelevant ideas . Final recommendations were confirmed by a voting process (table 2) [21 - 24]. A peer review was performed by three reviewers who were members of the korean geriatrics society (e.j.l . ), korean academy of rehabilitation medicine (j.y.y . ), and korean cochrane (h.j.k . ). The guidelines for fall prevention were announced publicly at an annual program for clinical medical education attended by general practitioners, gastroenterologists, and family doctors (february 15, 2015). Attendees suggested that supplementary material or tools would be helpful to evaluate the risk stratification for falls and to determine appropriate exercises for fall prevention . The kaim financially supported the development of the guidelines and there was no other external financial support . The guideline committee of the kaim is an independent organization, and there were no internal and external influences . 1 . Primary care physicians should be able to identify community - dwelling elderly at an increased risk for falls by asking about a history of falls and performing gait or balance tests . Grade of recommendation: 1 primary care physicians should be able to identify community - dwelling older adults at a high risk for falls . Previous studies have identified independent risk factors of falls or fall - related injuries, and fall risk increases as the number of risk factors increase . However, it is challenging to translate these findings into a strategy for primary care physicians to reliably identify older adults that require fall interventions . Any positive answer to the screening questions puts the person screened in a high - risk group that warrants further multifactorial fall risk evaluation . A history of falling is most commonly used to identify an increased risk for future falling and has generally been considered concurrently or sequentially with other key risk factors, particularly gait and balance . Previous studies confirmed that fall history is the most common risk factor other than age . The definition of fall history varies, with a history of at least one fall during the previous 12 months or a history of more serious falls that required medical attention . Gait and balance deficits should be evaluated in older individuals reporting a single fall as a screen for identifying individuals who may benefit from a multifactorial fall risk assessment . Older adults at higher risk of falling, as identified by screening, should be assessed for known risk factors . According to the national institute for health and care excellence (nice) guidelines, older people in contact with healthcare professionals should be asked routinely whether they have fallen in the past year and asked about the frequency, context, and characteristics of the falls . Older people reporting a fall or those considered at risk of falling should be observed for balance and gait deficits and considered for their ability to benefit from interventions to improve strength and balance . Although the united states preventive services task force did not find evidence for frequent brief falls risk assessments, they recommended that primary care physicians could consider the following factors to identify older adults at an increased risk of falls: a history of falls, a history of mobility problems, and poor performance on the timed get - up - and - go test . In contrast, the american geriatrics society / british geriatrics society (ags / bgs) guidelines recommend that all older adults under the care of a health professional should be asked at least once a year about falls, frequency of falling, and difficulties in gait or balance . For individuals who screen positive for falls or fall risk, evaluation of balance and gait should be part of the multifactorial fall risk assessment . Commonly used tests of gait or balance include the timed up and go test, the berg balance scale, and the performance - oriented mobility assessment . Accordingly, despite the controversy about the frequency of falls risk assessment and tools for gait and mobility evaluation, we recommend that primary care physicians should be able to identify older adults at an increased risk for falls by asking about a history of falls and performing gait or balance tests such as the timed up and go test, the berg balance scale, and the performance - oriented mobility assessment (fig . Multifactorial fall risk assessments to identify multiple risk factors for falls can reduce the risk of falls and improve the health status of older adults at an increased risk for falls using screening tests such as fall history and abnormality in gait or balance tests . Grade of recommendation: 1 comprehensive multifactorial fall assessments and interventions include assessment of multiple risk factors for falls and providing medical and social care to address factors identified during the assessment . A multifactorial fall risk assessment followed by interventions to modify any identified risk factors is considered a highly effective strategy to reduce falls among older adults, as this can address the risk factors of falls, and is expected to lead to more reductions in fall risks than dealing with each risk factor separately . Multifactorial fall risk assessments are a comprehensive geriatric assessment or falls - focused assessment, generally including two or more of the following assessments: vision, gait, mobility, muscle strength, medication use, cognitive impairment, orthostatic hypotension, and environmental risks . A multifactorial fall risk assessment should be performed for community - dwelling older persons who report recurrent (2) falls, difficulties with gait or balance, or who seek medical attention or present to the emergency department because of a fall . According to the nice guidelines, multifactorial assessments should include fall history, assessment of gait, balance, mobility, and muscle weakness, assessment of osteoporosis risk, assessment of the older person s perceived functional ability and fear of falling, assessment of visual impairment, assessment of cognitive impairment and neurological examination, assessment of urinary incontinence, assessment of home hazards, cardiovascular examination, and medication review . According to the ags / bgs guidelines, multifactorial fall risk assessments can identify factors associated with the increased risk of falling and the most appropriate interventions . They list the following components of multifactorial fall risk assessments; medication, visual acuity, neurological impairment, muscle strength, heart rate and rhythm, postural hypotension, feet and footwear, and environmental hazards . Some research has reported that some combination of multifactorial fall risk assessments and interventions in a select population can provide benefits . However, the characteristics of a comprehensive multifactorial assessment and intervention have not been clearly defined, and different approaches to classification may lead to different results . Additionally, there has been statistical heterogeneity and uncertainty regarding the optimal combination of multifactorial risk assessments . The uspstf does not recommend automatically performing an in - depth multifactorial risk assessment in conjunction with comprehensive management of identified risks to prevent falls in community - dwelling older adults because the likelihood of benefit is expected to be small . They recommend that clinicians should consider the balance of benefits and harms based on the circumstances of prior falls, medical comorbid conditions, and patient values in determining whether this service is appropriate in individual cases . However, for older adults at an increased risk for falls based on screening tests such as fall history and abnormalities in gait or balance tests, multifactorial fall risk assessments and interventions can decrease the risk of falls and improve health status . Therefore, we recommend that primary care physicians perform multifactorial fall risk assessments to identify multiple risk factors for falls and improve the health status of older adults at an increased risk for falls using screening tests such as fall history and abnormalities in gait or balance tests (fig . 3 . The use of combined vitamin d and calcium supplementation may be recommended to prevent fractures in community - dwelling elderly who are at an increased risk for falls . Vitamin d supplementation may be recommended to prevent falls in community - dwelling older people who have low vitamin d levels . Grade of recommendation: 2 vitamin d is known to play an important role in bone tissue . The effects of vitamin d on intestinal absorption of calcium and bone mineralization increase bone mineral density and decrease the risk of fracture . Several lines of clinical evidence suggest the existence of a link between vitamin d and muscle or nerve function [26 - 28]. Vitamin d deficiency or insufficiency may result in metabolic bone disease, may increase the risk of fall, and is associated with increased risk for several health conditions including cardiometabolic diseases, infection, and autoimmune diseases . A major reason for vitamin d deficiency is the lack of cutaneous production of vitamin d by ultraviolet due to an indoor lifestyle . Thus, vitamin d deficiency is very common in western countries, as well as korea . Therefore, determining whether to replenish vitamin d in community - dwelling older people to reduce falls or fractures is important . Many studies have analyzed the effect of vitamin d on falls and fractures in the relatively healthy elderly, but the results have been inconsistent . This could be due to potential confounders such as the characteristics of participants, vitamin d alone or the combination of vitamin d and calcium, the dosage and form of vitamin d, treatment period of vitamin d, or the baseline level of vitamin d. thus, each set of guidelines includes slightly different recommendations . Preventive services task force recommends vitamin d supplementation to prevent falls in community - dwelling adults aged 65 years or older at an increased risk for fall or older persons with proven or suspected vitamin d deficiency . In contrast, the nice has made no firm recommendations about the use of vitamin d for this indication, because of uncertainty regarding the relative contribution to fracture reduction and the dose and route of administration required . The recommendations in these guidelines are derived from meta - analysis of randomized controlled trials . A meta - analysis of 14 trials (n = 28,135) that evaluated the efficacy of fall prevention by supplementation with vitamin d, either alone or with calcium co - supplementation, did not reveal statistically significant differences in rate of falls, risk of falling, or risk of fracture . Analysis limited to the elderly at higher fall risk revealed no significant differences in either rate of falls or risk of falls . However, vitamin d supplementation to older people with lower vitamin d levels significantly reduced the rate of falls (relative risk [rr], 0.57; 95% confidence interval [ci], 0.37 to 0.89) and risk of falls (rr, 0.70; 95% ci, 0.56 to 0.87). The effects of vitamin d on fractures differ depending on the characteristics of the participants and co - supplementation with calcium . In contrast, administration of both vitamin d and calcium was associated with a significant reduction in the incidence of hip fracture (rr, 0.84; 95% ci, 0.74 to 0.96), non - vertebral fracture (rr, 0.86; 95% ci, 0.78 to 0.96), and any fracture (rr, 0.95; 95% ci, 0.90 to 0.99). In a subgroup analysis by residential status, these effects for fracture reduction were observed only in institutional residents, and were not significant in community - dwelling older people (hip fracture: rr, 0.91; 95% ci, 0.77 to 1.09; any fracture: rr, 0.96; 95% ci, 0.91 to 1.01). However, there was a small increase in the risk of gastrointestinal symptoms (rr, 1.05; 95% ci, 1.01 to 1.09) and a significant increase in renal calculi and renal disease (rr, 1.17; 95% ci, 1.03 to 1.34), especially for vitamin d plus calcium supplementation . Hypercalcemia was more common in people receiving calcitriol, a vitamin d analogue, compared to those receiving placebo or control (rr, 4.41; 95% ci, 2.14 to 9.09). Other systemic reviews have found an increased association of cardiovascular disease with calcium and/or vitamin d supplementation, particularly in people with a higher dietary calcium intake . Additionally, it should be considered that daily calcium intake in the korean population is very low (511.0 7.0 mg) compared to that of participants enrolled in these studies . In conclusion, routine supplementation of vitamin d for fall and fracture prevention for community - dwelling healthy older people is not recommended . However, vitamin d supplementation for elderly people who have lower vitamin d levels may prevent falls . Combined vitamin d and calcium supplementation may prevent fractures in elderly people at an increased risk of fall or fracture . A small but significant increase in gastrointestinal symptoms and renal disease therefore, supplementation with vitamin d and/or calcium for older people living in the community should be individualized . 5 . Supplementation with vitamin d may be recommended for elderly people residing in long - term care settings for the prevention of falls . Grade of recommendation: 2 elderly residing in long - term care settings are at high risk for falls and fracture and are frequently deficient in vitamin d. effective important interventions are required to reduce falls in this specific high - risk group . Two trials were conducted: oral vitamin d3 plus calcium or oral vitamin d2 plus calcium versus a control group supplied with calcium . These two trial results both revealed a statistically significant reduction in the rate of falls . However, another trial involving oral vitamin d3 (800 iu plus calcium 1,200 mg vs. matching placebo control group) revealed no significant reduction in fall risk . After hospital discharge, neither vitamin d supplementation nor a home - based program of quadriceps resistance exercise improved the risk of fall compared to a control group, but patients in the exercise group were at an increased risk of musculoskeletal injury . An intervention group (oral vitamin d 800 iu plus calcium 1,200 mg) was compared with a control group (calcium 1,200 mg). Although there were fewer falls in the vitamin d group, neither the mean number of falls or time to first fall differed between groups . Elderly people residing in long - term care settings were randomly assigned to receive one of four doses of vitamin d (200, 400, 600, 800 iu) or a placebo control for 5 months . The highest vitamin d group (800 iu) had fewer fallers and a lower incidence rate of falls (72%) than the placebo control group . In vitamin d2 (2.5 mg) or control groups living in care homes, no significant reductions in risk of falls or fractures were observed . The effects of multivitamin supplementation, which included oral vitamin d3 400 iu and calcium 360 mg, were investigated for 6 months . There was a statistically significant reduction in rate of falls, but not in risk of falls . According to the ags / bgs clinical practice guideline for prevention of falls in older persons (2010), vitamin d supplements of at least 800 iu daily should be provided to elderly people residing in long - term care settings with proven or suspected vitamin d insufficiency . Additionally, vitamin d supplements of at least 800 iu daily should be considered for elderly people residing in long - term care settings who have gait or balance disorders or who are at high risk for falls . 6 . We recommend regular exercise to prevent falls and fall risk in community - dwelling elderly people . Grade of recommendation: 1 elderly people who have healthy living habits, avoid sedentary lifestyles, and undergo physical exercise such as walking and other muscle exercises often maintain their health and have an independent daily life . Falls may cause fractures, which can make it impossible for elderly people to live independently . Therefore, many studies have focused on fall prevention, including the benefits of physical exercise in improving the functional capacity of frail elderly people . Compared the fall incidence rate between two groups of people who engaged in regular exercise or did not engage in regular exercise for a period of 1 year in 2009 . The regular exercise group had an average fall incidence of 0.6, whereas the non - exercise group had an average fall incidence of 0.8, and the difference between the two groups was statistically significant . In 2010, clemson et al . Reported that those who regularly performed balance exercises and muscle strengthening had a significantly lower fall rate than those who did not . One group of elderly people living in the community performed exercises for 30 to 90 minutes with one 10-minute break two to three times every week . One study investigated fall incidence rates among those who engage in multiple forms of exercise (muscle strengthening, balance training, endurance training, and cooperation training) compared to rates among those who do not exercise, and reported that those who engaged in multiple exercises had a significantly lower fall incidence rate . Performed a meta - analysis of 79 studies, and found that elderly people who engage in muscle strength or endurance exercises two to three times every week have a significantly lower fall incidence rate than those who do not . The fall prevention guidelines proposed by the us / uk geriatrics society in 2010 strongly recommend that regular and multi - layered exercises be included in fall prevention programs for the elderly . The nice guidelines recommend muscle strength and balance exercises to prevent falls among elderly people living in local communities, especially those who have experienced repeated falls, and suggested that the exercise be ordered and managed by experts . Uspstf suggests that fall risk can be reduced when exercise and physical therapy are applied to high fall - risk groups of elderly patients living in local communities, and reported that this can lead to a 13% decrease in falls . In conclusion we recommend balance training, strengthening exercise, aerobic exercise, or resistance exercise to prevent falls and fall risk among community - dwelling elderly . Grade of recommendation: 1 regular exercise can prevent falls among community - dwelling elderly . Exercise among community - dwelling elderly is classified as group - based exercise or homebased exercise . Home - based exercise has advantages such as being less expensive and allowing long - term performance . Regular exercise in home - based programs improves physical function, prevents falls, maintains bone mineral density, and is feasible within daily life . However, home - based programs also have limitations in terms of lower compliance and being less effective than groupbased exercise . Static and dynamic balancing exercises can be used to improve balance among community - dwelling elderly . Balance exercises may include sit - to - stands, tandem standing, tandem gaiting, unipedal standing, knee bends, change in direction, catching / throwing a ball, and tai - chi [48,55 - 58]. Strengthening exercises may include ankle cuff weights, thera bands, and various resistance exercises . They may also include walking, exercise on stationary cycles, and knee and hip extensions performed with a oneleg press in a sitting position . The appropriate amount and type of exercise depends on individual health status . In conclusion, community - dwelling elderly persons at high risk of falling should undergo balance training, strengthening exercises, aerobic exercises, and/or resistance exercises.
The pursuit for a global and self - consistent conceptual, mechanistic, and theoretical framework within which to discuss the denaturing properties and behaviors of cosolvents such as urea and guanidinium chloride (gdmcl) continues to garner a significant amount of scientific curiosity and effort . The quest for a fundamental understanding of protein denaturation has a long and rich history, to which the reader is referred . Based on recent experimental and molecular simulation studies, the notion of direct interactions of denaturants with proteins in solution has come to be accepted in consensus . Since common denaturants used in practical situations are needed in significantly high concentrations, i.e., 5 m urea for instance, the notion that there are no direct interactions between denaturant and protein becomes less justifiable . Within the context of direct interactions, one of two major mechanisms for denaturation involves the lessening of the hydrophobic effect as it relates to the formation of a compact prefolded ensemble of states where protein hydrophobic surface exposure to solvent the idea is that by associating with hydrophobic regions of the protein (specific residues, clusters of residues forming extended topographical surfaces, hydrophobic side chains, etc . ), denaturant molecules can shield the hydrophobic surface area even in unfolded or extended configurations of the peptide / polymer . This chemical denaturation mechanism naturally involves direct interaction of the cosolvent molecule with regions of the protein surface . A particular aspect of this interaction deals with the precise nature of association geometries and the associated free energetics; specifically, molecules such as urea, and more so guanidinium cation (gdm), can present several predominant relative orientations to the protein surface through which the interaction is mediated . In general, it is proposed that a dominant interaction of urea with surface groups in protein simulations involves hydrogen bonding with polar side - chain functions, while the unique hydration properties of the gdm support alternative interaction modes involving stacking with side - chain planar and hydrophobic groups . However, we should note that the nature of the relative orientations would be dictated in part by the nature of solvation and hydrophobic effects as they pertain uniquely to each denaturant molecule . Understanding of the precise geometrical and associated free energetic properties of denaturant protein interactions is important as a piece in a more complete understanding of the denaturation process from a molecular perspective . Previous studies have shown that cosolvents such as gdm adopt orientations relative to flat, model hydrophobic surfaces that are planar . Vapor interface, flat hydrophobic plate, and hydrophobic polymer surface . However, there is a lack of direct evidence for similar orientational behavior of gdm upon approaching more complex aqueous protein interfaces . The inherent chemical and topographical heterogeneity of protein surface makes it difficult to find a qualitatively rigorous approach to evaluate the relative orientation between the surface of gdm and the protein . To fill this gap, we apply molecular dynamics simulations investigating the association of gdm cation with a specific protein with a relatively flat surface region consisting of hydrophobic residues . In the context of chemical denaturation via direct association, we ask here about the orientations that gdm and urea adopt when interacting with hydrophobic regions of proteins . The combination of this analysis addresses ideas of direct interaction as well as hydrophobic effects as they pertain to the denaturation process . Furthermore, there is sentiment in the literature demonstrating the importance of solvent fluctuations and their relation to what is called the hydrophobic nature of solutes . Found that water density near the surfaces of self - assembled monolayers (sams) with hydrophobic head groups (cf3, ch3) shows a poor distinction from that of sams with hydrophilic head groups (oh, conh2). However, differences arise when considering the fluctuations of water density near the two regions . Enhanced fluctuations reflected by the broad probability distributions of water number density are observed around hydrophobic surfaces compared with the bulk solution and hydrophilic surfaces . Moreover, the enhanced density fluctuations around hydrophobic surfaces are further characterized by more compressible hydration shells and increased cavity formation, indicating that the nature of hydration shells around hydrophobic surfaces is softer and more flickering than near hydrophilic ones . Since the long - ranged solute - induced perturbations of aqueous protein interfaces involve the coupling of local hydration shells of the solutes with distant hydration shells around protein surfaces, the natures of both would affect the extent of induced interfacial fluctuations . It would be interesting to compare the interface height fluctuations as gdm / urea approaches the hydrophobic / hydrophilic protein regions . We note that the interface height fluctuations we are pursuing here are conceptually different from the density fluctuations of refs (32, 33, 36, and 37), though both reflect the nature of hydration water around the protein surfaces . It is natural here to investigate the nature of induced fluctuations of the solvent at the protein water interface via consideration of the fluctuations of the height of this interface (once defined in a well - controlled manner) upon approach of a denaturant molecule to a hydrophobic protein region as well as when the denaturant resides at very close separation to the protein . The particular protein on which we are focusing in this study is hydrophobin - ii (hfbii), a small protein expressed by filamentous fungi . The protein is known for its ability to form hydrophobic coatings on surfaces and self - assembles into monolayers on hydrophobic / hydrophilic interfaces such as the water / air interface . Mapped the effective hydrophobic regions and effective hydrophilic regions of hfbii by considering the density of small probe hydrophobic solutes around each region of the protein . They selected three regions with different hydrophobicity based on this and further monitored the density fluctuations in the vicinity of these regions . Their calculation shows that the largest density fluctuations occur around the most hydrophobic region whereas the least density fluctuations are detected around most hydrophilic region . This particular observation suggests hydrophobins as useful candidate proteins for comparing behaviors at hydrophobic and hydrophilic interfaces as denaturant molecules approach . We note that the purpose of this study is to demonstrate the specific denaturant s stability and orientational preference around regions with different hydrophobicity of the protein with implication of the direct interaction as well as hydrophobic effects for the association between denaturant and the protein . The aim of this study does not focus on the denaturation process by these denaturants, so we use the totally fixed protein in the simulation along with quite low concentration of denaturants (1 m and an extreme case, single solute) compared with the significant high concentration (up to 5 m) in the actual denaturation experiments . We further emphasize that by using the single solute in this study, it is possible for us to systematically distinguish underlying characters of stability for different species (gdm and urea) and orientational preference for different orientations (parallel and perpendicular relative to the regions of interest). Ii we discuss the simulation protocols and computational details of the liquid vapor interface and aqueous protein interfaces . We begin with discussion of potentials of mean force (pmfs) and interfacial fluctuations as single gdm / urea cross the aqueous liquid vapor interface . We consider gdm / urea density distributions around the aqueous hfbii hydrophobic interface in 1.0 m solutions in the second part . We further investigate the pmfs and interfacial fluctuations as single gdm / urea approach this aqueous protein hydrophobic interface, demonstrating the resemblance between liquid vapor interface and hydrophobic protein interface in terms of solute specific effect and orientational preferences . We finish this section by examining single gdm / urea approaching another region, which is considered a hydrophilic patch compared with the hydrophobic region we initially study . All the simulations in this study were performed with md program namd 2.9b3, using charmm22 all - atom force fields with cmap backbone torsion correction term . Simulations of single gdm / urea approaching the liquid vapor interfaces were performed in the nvt ensemble . The simulation cell was rectangular with dimensions 40 40 150, in which z is the direction normal to the liquid vapor interface . The system contained one single gdm / urea and 1977 nonpolarizable tip3p water model water molecules . A rigid water geometry is enforced using shake constraints, and an integration time step of 1.0 fs was used . The temperature was kept constant at 300 k by applying the langevin friction force scheme with a damping coefficient of 5 ps . A switching distance of 10, nonbonded real - space cutoff of 12, and pair list generation distance of 14 were used for the van der waals interactions, and the particle mesh ewald (pme) method was employed for the calculation of conditionally convergent electrostatic interactions . The grid size of pme in x - dimension is 40, in y - dimension is 40, and in z - dimension is 150 (as close to a 1 grid point separation as possible). In order to obtain the pmf for transferring single gdm / urea from bulk aqueous environment to the liquid vapor interface, we define a collective variable, which is based on the cartesian z - component of the separation between the water slab center of mass and single gdm / urea central carbon, describing this pseudochemical reaction path . To enhance sampling of the distribution of configurations where the collective variable holds a particular value, relevant restraint potentials were introduced on the collective variable in order to prevent it from moving outside of the desired range . In this case windows with width 1.0 . In each window, central carbon of single gdm / urea was restrained to z - positions from 0 to 30 relative to the water slab center of mass using a harmonic potential urestraint(z; zrelative, ref) = /2k(z zrelative, ref) with the force constant of 4 (kcal / mol)/ . To consider the orientational dependence of gdm around interface, we further desired to compare the free energetics of single gdm / urea transferring from the bulk with two distinctive orientations: the planar ring of gdm / urea parallel to the liquid vapor interface and perpendicular to the liquid vapor interface . Here the orientations were defined based on identical definitions from previous publications in which the angle between the vector normal to the molecular ring and the z - axis was computed . Gdm / urea was considered as parallel (as shown in figure 1a) and perpendicular (as shown in figure 1b) to the liquid we note that for the parallel orientation the normal vector of the molecular ring is along z - direction; for the perpendicular orientation, the normal vector of the molecular ring can either be along x or along y direction . Because of the homogeneous nature of liquid vapor interface and the identical setup in x and y dimensions in the simulation, here we only need to consider one (when the normal vector is along y direction) of these two configurations in the perpendicular orientation case . In these two sets of simulations, initially, the parallel and perpendicular configurations of gdm / urea were selected as starting structures, and the orientations were maintained by restraining the directions of central carbon nitrogen vectors . Based on the definition of orientations above, gdm with parallel configuration nitrogen vectors in the plane of xy, with the magnitude along z - direction being zero . Therefore, harmonic potentials with force constant k = 1000 (kcal / mol)/ were applied to keep the magnitudes of z components of two of the three central carbon, we can ensure the parallel orientation of single gdm with respect to the liquid vapor interface . For gdm with perpendicular orientation, all three central carbon nitrogen vectors are in the plane of xz . To maintain this orientation, harmonic potentials with force constant k = 1000 (kcal / mol)/ were applied to restrain the magnitudes of y components of the carbon these restraint protocols were also applied to single urea molecule by considering only the two central carbon nitrogen vectors . Apart from the orientational restraints, identical choice of collective variable and setup of simulation windows were applied . The total sampling time for each window was 15 ns for all the simulations and properties were calculated from all but the initial 1 ns, which was treated as equilibration . (a) representative snapshot of single gdm with parallel orientation to the liquid vapor interface . (d) single gdm with parallel orientation to the hfbii protein solvent interface . (e) single gdm with perpendicular y orientation to the hfbii protein (f) single gdm with perpendicular x orientation to the hfbii protein solvent interface . Simulations of hfbii in 1.0 m concentration of gdmcl / urea aqueous solutions were performed in the npt ensemble using a cubic cell with a box size 60 60 60 . The protein structure was based on the ultrahigh resolution structure of hfbii, with pdb code 2b97, and it was constructed using charmm - gui . Monomer of this hfbii protein, which is composed of 70 residues, was placed in the center of the box and fully solvated with 6481 water molecules, along with 116 pairs of gdmcl or 116 urea molecules . The initial structure of the protein was arranged in a way that its largest hydrophobic patch, consisting of amino acid residues val 18, leu 19, leu 21, ile 22, val 24, val 54, ala 61, leu 62, and leu 63 (the three letters representing the amino acid types and the number representing the position of the amino acid in the primary sequence), was nearly perpendicular to the z direction (further verification is in table s1 of the supporting information). The protein was rigidly fixed at the original configuration during the simulation while other system components were unrestrained . Temperature was maintained by langevin bath at 300 k, and the pressure was kept constant by langevin pressure control at 1 atm . A switching distance of 10, nonbonded real - space cutoff of 12, and pair list generation distance of 14 were used for the van der waals interactions . For the grid size of pme setup, the values are changed to 60 in all dimensions, corresponding to the cubic simulation cell in this case . Six different replicates were applied for each system, and properties were computed based on at least 10 ns of production run for each replicate . A representative snapshot of the simulation system can be found in figure 1c . Furthermore, in order to illustrate the molecular details of orientation and free energetics of gdm / urea around protein interfaces, we simulated a system with single gdm / urea approaching the hydrophobic aqueous protein interface with different orientations . We use an identical protein starting structure as in the 1.0 m solution case, with the hydrophobic interface of the protein nearly perpendicular to the z direction . In this way, vapor interface case, the relative orientations between single solute and protein interface can be defined in a straightforward way: when the normal vector of gdm / urea ring is along z direction, the solute is considered to be parallel to the hydrophobic protein interface as shown in figure 1d; when the normal vector is along y direction (figure 1e) or x direction (figure 1f), the solute is considered to be perpendicular to the hydrophobic protein interface . Because of the asymmetry of hydrophobic protein interface, differences arise between these two perpendicular configurations . For the convenience of discussion, we denote the orientations in figures 1e and 1f as perpendicular y orientation and perpendicular x orientation, indicating that the normal vector is along y direction and x direction, respectively . Here, we note that although the hydrophobic protein patch is commonly considered as flat, it still has some curvature . Hence, strictly speaking, speaking of an actual parallel or perpendicular orientation of gdm plane relative to the protein patch is not rigorous . However, in this work, we aimed to study the contrasting hydration properties and surface fluctuations induced by different orientations of gdm (water - depleted flat faces versus the more strongly water - associated ring (edge - on) side of the cation) with respect to the hydrophobic patch of the protein . Therefore, in this convention, parallel orientation simply indicates that gdm has more overlap with the protein patch in terms of their projections to the xy plane, relative to the perpendicular orientation . The whole protein was fixed during the simulation with center of mass located at (x = 0, y = 0, z = 0). A fixed cl (at x = 0, y = 0, z = 15) was added as the counterion to neutralize the positive charge in the case of gdm . Similar to the liquid vapor interface situation, for calculation of pmf, we use the cartesian z component of the separation between the center of mass of protein and central carbon of single gdm / urea as the collective variable . Select configurations of single gdm / urea with parallel, perpendicular y, and perpendicular x orientation were used as starting structures with central carbon of the molecule located at x = 0 and y = 0 . X component and y component of the solute s central carbon were restrained at this original position x = 0 and y = 0 during the simulation using namd s selectconstraints infrastructure, with sufficiently large force constant k = 1000 (kcal / mol)/ . Vapor interface cases mentioned above . In this case, single gdm / urea will approach the specific spot on the patch (x = 0 and y = 0) with particular orientation while still keeping some rotational degree of freedom by using the normal vector to the molecular ring as rotation axis . We just centered on one specific region on the patch due to the fact that the interface is heterogeneous, resulting in the differences of the extent of inherent interface fluctuations at various locations (to be discussed further below). For a meaningful discussion of the molecule - induced fluctuation (fluctuation in addition to the level inherent in pure water) as it approaches the hydrophobic interface, one representative spot with fixed position and unchanged inherent fluctuation had to be defined . For more discussion of the situations of the single solute with other restraint conditions (totally rigid solute or restrained in a specific sampling volume), the reader is referred to the supporting information, section s1 . In this case, along the positive z direction, 49 continuous windows with width 0.2 the spans of the windows going from interfacial region to bulk region (in) were [15.4:15.6], [15.6:15.8], [15.8:16.0],..., [24.4:24.6], [24.6:24.8], [24.8:25.0]. In each window, a harmonic restraint potential with force constant of 10 (other simulation conditions remain the same as that of the system of protein in 1 m concentration of gdmcl / urea aqueous solution . The first 2 ns was allowed for equilibration before a total of 20 ns production data was generated for each window . For a complete understanding of the influence of the hydrophobicity of the protein patch on the orientational preference of gdm solute, we considered another system in which single gdm approaches a more hydrophilic protein region which consists of residues asp 25, cys 26, lys 27, thr 28, ala 58, asp 59, and gln 60 . The simulation conditions remained identical except that the protein was posed in a different way in the simulation cell with the selected hydrophilic interface almost perpendicular to the z direction . The window setup ranged from [14.0:14.2], [14.2:14.4], [14.4:14.6].. to [24.4:24.6], [24.6:24.8], [24.8:25.0] for a total of 56 windows . It has been previously explored by willard and chandler that one can construct a coarse - grained solvent density field from atomic coordinates in individual snapshots of an md simulation . The interface related to the solvent is defined as a constant density surface for the coarse - grained field in space . Specifically, in this work, we are interested in the water vapor interface and water protein interface; we thus use the water oxygen atom as a reference point for constructing coarse - grained interfaces . The oxygen atom in most, including the present, force fields is sufficiently large that its size represents the majority of the size of the model molecule; thus, using only the water oxygen atom to construct the interfaces is a justifiable approximation . The water oxygen density field is constructed as follows: we set up series of spatial grid points and compute the corresponding coarse - grained densities at space - time point r, t, represented as (r, t) by eq 1.1where ri(t) is the ith water oxygen atom s position in space . The summation over all water molecules density contributes to the coarse - grained density at a particular grid point . Each oxygen atom s density contribution is coarse - grained via a gaussian function in eq 2.2where r is the magnitude of r, is taken as 3.0, and d stands for dimensionality (3 in this case). The final d - dimensional density field will be constructed by acquiring each grid point s density . The interface is determined as the (d 1)-dimensional manifold with a constant value c. differences arise when constructing the liquid thus, we select a cartesian coordinate system to construct the liquid vapor interface and spherical coordinate system for the protein water interface . The liquid vapor interface is constructed by connecting grid points where (x, y, z) = bulk/2 . This instantaneous surface is denoted as (ht(x, y), at time t). We can average these instantaneous surfaces to obtain the mean surface h(x, y). Subtracting the mean values from the ht(x, y), we obtain ht(x, y) as surface height and the height fluctuations h(x, y). For the protein water interface, grid points in space are defined by (r,,) and each (,) coordinate set in the spherical system defines a radial vector . We use a different constant value c = 0.6bulk here compared with the liquid vapor interface case because this choice results in a more unambiguous construction of a protein solvent interface . Correspondingly, instantaneous protein interface can be expressed as (ht(,)), mean surface as h(,), surface height as ht(,), and height fluctuation as h(,). This analysis provides a reference context within which to discuss the results at a hydrophobic protein interface later . To first address solute orientational propensities as they vary along the order parameter, we compute orientationally resolved probability distribution profiles along the z - axis as a single gdm / urea approaches the liquid vapor interface as shown in figure 2a, c . Here, in a statistical manner, we consider the probability of the single solute at position z with orientation, which is defined based on the following eq 3:3where n(z,) denotes the solute number count at position z with orientation . The numerator represents the number of solutes in a slab from z z/2 to z + z/2, with select orientation within the range of cos(/2) and cos(+ /2). The denominator represents the total number of solutes in the slab region z z/2 to z + z/2; this is used to normalize the probabilities in the relevant slab whose boundaries along the order parameter are z the limits cos() = 1 and cos() = 0 represent gdm orientations that are parallel and perpendicular to the liquid vapor interface, respectively . In bulk region with z <13, the probabilities of gdm / urea with different orientations are identical, indicating no orientational preference, while in the interfacial region (15 z 20), single gdm manifests a higher tendency to adopt the configuration that is parallel to the liquid vapor interface . This observation is consistent with the result in our previous publication on an identical system using a polarizable force field (tip4p - fq) and wernersson et al.s work using 1.1 and 5.3 m gdmcl solutions . Single urea also displays a marginal orientational preference for a parallel configuration as well; urea s propensity for the parallel orientation is lower than that of gdm based on the lower intensity of the corresponding region in figure 2c . We note that this higher probability of parallel orientation of single solute around interfacial region suggests a lower free energy of this configuration relative to the perpendicular . To further explore this difference, we consider potentials of mean force for single gdm / urea from bulk through liquid vapor interface being restrained at particular orientations as shown in figures 2b and 2d for gdm and urea, respectively . In both panels, black lines represent conditional pmf profiles for single solute with parallel orientation; blue dashed lines represent conditional pmf profiles for single solute with perpendicular orientation . The orientation - averaged pmf profile (with no restraints on the orientation) the weighted histogram analysis method (wham) was used for generating the final pmf in all cases . The standard error was estimated by using the block averaging method obtained from each consecutive 0.5 ns time block in the production run of each umbrella sampling window . This selection will ensure the block size was significantly larger than the correlation time in each window . The pmf is defined to be zero when the solute is in the bulk, which is determined by window z = 0 . To better compare the interface stability among different orientations, in the large graph of panels b and d, we emphasized the pmfs around the interfacial region while the entire pmf along the collective variable can be found in the inset . For single gdm, the parallel orientation shows a minimum of roughly 0.4 kcal / mol, with uncertainty about 0.1 kcal / mol, prior to the gds at the separation of z = 16.5, while the perpendicular orientation displays no surface stability at all . Overall, when there is no orientational restraint applied on the gdm, as shown by the green line, no surface stability is found near the interface . The pmf is less repulsive in the case with no orientational restraint compared to the perpendicular orientation scenario; this is consistent considering that it is an average result from the contributions of all possible configurations . The pmf for no orientational restraints (green dotted line) shows a slight shoulder around z = 16, indicating the effect of the parallel orientations . However, since the stability of the parallel orientation is rather small, and configurations differing from the parallel geometry are associated with significantly higher free energies at the interface, the overall effect leads to a pmf displaying no apparent interfacially stable state . For single urea, the parallel orientation pmf shows a slight minimum of 0.04 0.07 kcal / mol . Considering the uncertainty here, whether parallel orientation of urea shows surface stability or not is debatable . However, we notice that, in contrast to the perpendicular orientation, the parallel orientation is more free energetically favorable, although this trend is not as obvious as the case for gdm . All these pmf results are consistent with the probability distributions of orientations as discussed above . Vapor interface may be related to the hydration structure of gdm as previously studied by mason et al . And cooper et al . The hydration around gdm is anisotropic . In the molecular plane, the n h group can serve as hydrogen bond donor, interacting with water molecules as demonstrated in the gas phase, while above or below the planar face, it is inadequate to serve either as hydrogen bond donor or acceptor . Vapor interface with parallel orientation, it is easy for desolvation to occur, which is free energetically favorable . For the structurally analogous molecule urea, it still can serve as hydrogen bond donor above or below the planar face, so it is less facile for the parallel urea molecule to desolvate compared with that of gdm . (b) pmf of single gdm from bulk transporting through liquid vapor interface with parallel orientation, perpendicular orientation, and no orientational restraint . (c) orientationally resolved probability map of single urea around liquid vapor interface . (d) pmf of single urea from bulk transporting through liquid vapor interface with parallel orientation, perpendicular orientation, and no orientational restraint . For clarity, in (b) and (d), no orientational restraint profiles are shifted by 1 kcal / mol; perpendicular profiles are shifted by 2 kcal / mol . The gds positions are denoted as orange dashed lines in (b) and (d). Recent studies have demonstrated an interesting connection between liquid vapor interfacial stability of chemical species and the extent to which the presence of these molecular species in the vicinity of the interface induces collective fluctuations of the interface in addition to the level inherent in pure water (absence of the solute) due to thermal motion; these studies have focused on monovalent inorganic ions as initial test systems . Initially, it is found that the species demonstrating an interfacial stability (e.g., i) as demonstrated by free energy minima in the region of the gds as evaluated via potentials of mean force, appear to enhance liquid vapor interfacial fluctuations, while those that show no interfacial stability (e.g., cl) induce no or lesser extent of fluctuations . The differences in induced interfacial fluctuations by two representative ions, cl and i, has been attributed to these two types of ions presenting distinct hydration shell environments . The nature of the solvent structure around i determines that it is more amenable to inducing fluctuations of the interface as a consequence of a greater disruption of the solvent structure on approach to the interface . Inspired by this, we consider that differences in induced interfacial fluctuations should arise as the parallel and perpendicular orientations of gdm approach the liquid vapor interface since these two configurations display distinct hydration shell environments with parallel orientation presenting a more malleable solvent environment and perpendicular orientation showing a more rigid hydration environment due to the more effective hydrogen bonding of water in the plane of the ring . The two orientations are associated with dramatically different free energetic profiles at the liquid vapor interface . The surface height fluctuations were computed with the protocol of section ii.b . The mean surface height and surface height fluctuation when single gdm resides at the position of z = 14 is shown in figures s1a and s1b of the supporting information . Both the mean surface profile and surface height fluctuation profile are radially symmetric, with the largest value at the position where the gdm is just approaching the point (x = 0, y = 0). For convenience, we use this representative value h(x = 0, y = 0) to compare the magnitude of interface fluctuations when the solute is restrained at different z - positions, with the result shown in figure 3 . Fluctuation profiles for gdm and urea with distinct orientations are presented in panels a and c, respectively . In the case of pure tip3p water without the existence of solute, the inherent fluctuation for current system size is around 1.32 . Using this value as a normalization factor, normalized fluctuation profiles were obtained, presented in panels b and d for gdm and urea, respectively, which will display solute - induced contribution in a direct way . Hl2> 1 indicates the surface height fluctuation is enhanced relative to pure water; with hl2 <1, it denoted that the surface height fluctuation is damped . In the gdm case, the parallel orientation induces a large fluctuation, with the maximum normalized fluctuation value around 2.2 at the location of z = 14, which is around 3 prior to the position of the free energy minimum . In stark contrast, no obvious enhancement of surface fluctuations is associated with perpendicular orientation . These trends are expected considering the distinct hydration structures for the parallel and perpendicular orientation of gdm . The nature of the malleable hydration shell around parallel orientation gdm is similar to that of single, low charge density anions, like i. the solvent structure is more amenable to inducing larger fluctuations due to the fact that it is more easily disrupted as the solute approaches the interface . On the other hand, the perpendicular orientation of gdm showing no interface stability is more like cl, with more rigid solvation structure around the periphery of the ring due to the existence of hydrogen bonding . These results using a nonpolarizable force field are consistent with our previous work using the tip4p - fq polarizable force field, indicating a force field independence of the fundamental, underlying physical origin of this correlative phenomenon . Previously, it has been pointed out the importance of considering polarizability in the ion - specific effect . By neglecting the polarizability in the force field, larger anion i may not show significant surface stability, giving a poor distinction with respect to cl . However, our results indicate that in the case of gdm the orientational preference is pronounced enough even in the nonpolarizable force field . In light of this, in the following section for the discussion of gdm around the protein surface we also notice that differences in induced fluctuations also exist in the case of urea with dissimilar orientations . For the parallel orientation, the largest induced normalized fluctuation value is around 1.85, which is still larger than the fluctuation from perpendicular orientation, 1.45 . Interestingly, for the parallel orientation, the induced fluctuations from gdm is larger than that from urea, corresponding to gdm s greater free energetic stabilization; the perpendicular orientations show a reverse trend as the induced fluctuation from gdm is smaller than that from urea, which correlates with the pmf trend that gdm is more repulsive in this case . Again, the smaller differences in interfacial stabilization and induced fluctuation between parallel and perpendicular orientation is related to the spatial location of hydrogen bonding network, either below or above the planar face, leading to the closer solvation structure of the parallel oriented and perpendicular oriented urea . Overall, the differences in orientational preference around liquid vapor interface, interfacial stability, and induced fluctuation between gdm and urea may possibly be connected to the efficiency of these two solutes as denaturants via direct interactions with hydrophobic side chains and surface regions of proteins . For a further understanding of this, we attempt to extend this investigation from the ideally hydrophobic aqueous liquid vapor interface to a somewhat more realistic and more complex aqueous protein hydrophobic interface . (a) surface height fluctuation for liquid vapor interface at (x = 0, y = 0) as a function of position of single gdm . Vapor interface at (x = 0, y = 0) as a function of position of single gdm . Vapor interface at (x = 0, y = 0) as a function of position of single urea . (d) normalized surface height fluctuation for liquid vapor interface at (x = 0, y = 0) as a function of position of single urea . Before we consider the free energetics of single gdm / urea approaching the hydrophobic protein interfacial region, we provide a general overview of distributions of solute orientation relative to the hydrophobic protein patch . To probe this, generally, we define a sampling volume in the cartesian space corresponding to the hydrophobic patch around the protein . This sampling volume is shown in figure 4a, within the range of 8 x 8, 8 y 8, and 12 z 25, roughly including residues val 18, leu 19, leu 21, ile 22, val 24, val 54, ala 61, leu 62, and leu 63 . Orientationally resolved probability distribution of gdm around this defined region is shown in figure 4b . The probability at position z with orientation in this case is defined in the same way as that in liquid we note that this marked tendency has previously been noticed in proximity to hydrophobic surface . . Found that gdm accumulates in the vicinity of flat hydrophobic plate in a roughly parallel way . Also mentioned the gdm has a preference for parallel stacking with the hydrophobic polymer surface from the snapshots of their simulations . Here, using a simple approach, we defined the relative orientations between gdm solute and hydrophobic protein surface and showed the orientational preference of gdm around the hydrophobic patch of hfbii . Furthermore, we consider orientationally resolved probability distributions of urea in an identical probe volume in figure 4c . Parallel oriented urea is also preferred compared with the perpendicular configuration around the hydrophobic protein surface . However, this trend is less intense in the case of urea compared with gdm, which is similar to the situation at the liquid vapor interface . This is consistent with the previous report that urea, compared with gdm, displays more orientational diversity around hydrophobic plate - like surfaces . So far, we only concentrated on solute distributions around the hydrophobic region of the protein and attempted to connect this with the similar observation around the liquid vapor interface, which is one model of an ideal hydrophobic interface . A complementary study would be focusing on another distinct region around the protein surface with different hydrophobicity . Therefore, we define another sampling volume corresponding to the hydrophilic patch of the protein (including residues asp 25, cys 26, lys 27, thr 28, ala 58, asp 59, and gln 60) in cartesian space within the range of 8 y 8, 8 z 8, and 12 x 25 . The volume of this sampling region remains the same as that defined for the hydrophobic region, but the position of the probe region in cartesian space is different . In figure 4d is shown the orientationally resolved probability distribution for gdm around this hydrophilic region . Overall, no preference for parallel oriented gdm is observed in this case, although this preference can be detected in a small portion of the map with the separation of z = 14 . Compared to the orientationally resolved probability maps for gdm around hydrophobic (panel b) and hydrophilic area (panel d), it is safe to claim that around more hydrophobic regions there is a stronger tendency for the parallel stacking of gdm with protein surface, which is also been noticed in flat plate systems previously . We note that these general trends are robust as an identical patch with different sampling volume, as defined in the supporting information figures s2 and s3, further verifying this orientational preference around hydrophobic / hydrophilic regions . (a) representative of sampling volume for probing orientational resolved probability of solute around certain region of protein interface . (b) orientational resolved probability distribution of gdm around hydrophobic protein interface in 1.0 m gdmcl solution . (c) orientational resolved probability distribution of urea around hydrophobic protein interface in 1.0 m urea solution . (d) orientational resolved probability distribution of gdm around hydrophilic protein interface in 1.0 m gdmcl solution . The pmf profiles for single gdm / urea approaching the hydrophobic protein surface region are shown in figure 5 . Panel a shows the pmf of single gdm with parallel orientation (solid red line), perpendicular y orientation (dotted green line), and perpendicular x orientation (dashed blue line) moving toward the hydrophobic patch region from the bulk, which is located at z = 25 in this case . The pmf profiles were generated by postprocessing umbrella sampling md trajectories with wham, and the standard errors were estimated by using the block averaging method obtained from each consecutive 0.5 ns time block in the production run of each umbrella sampling window . The parallel configuration gives rise to a pmf minimum of 2.85 0.04 kcal / mol as it nears the hydrophobic patch at a separation of z = 15.7; a shallow second minimum can be observed at a separation of z = 19 . A free energetic barrier can be observed between these two minima, which may be related to the dramatic change of the number of water molecules within the first hydration shell of the solute as shown in figure s4 of the supporting information . 0.04 kcal / mol around z = 17 for the perpendicular y orientation gdm, while for perpendicular x gdm, a monotonically repulsive trend was observed . This difference may be determined by the exact composition and local spatial arrangement of the residues on and near the hydrophobic patch as shown in figures s5a and s5b of the supporting information . When gdm with perpendicular x orientation approaches the patch, there is a repulsive interaction between nh groups of gdm and side chains of residues ile 22 and leu 63 on the patch . More importantly, we notice that compared with pmfs for perpendicular oriented gdm showing marginal or no stability, pmfs for parallel orientation gdm are much more free energetically favorable . This further echoes the result shown in figure 4b, indicating that gdm prefers to associate with the hydrophobic protein patch with its more hydrophobic, easily desolvated, parallel orientation . This preference is explained by england et al . As hydrophobicity - driven stacking interaction in their study using hydrophobic plate . Because of the inability of hydrogen bond formation between hydrophobic surface and water molecules, the hydrophobic surface has a stronger tendency to minimize the exposed area in the aqueous environment . This can be achieved by the face - on coating by gdm of the surface, which is free energetically favorable . The observed stacking mode of self - association among gdm can also be considered as hydrophobically driven interaction . Instead of association with large hydrophobic plate or protein surface, in this case, gdm pairs with another gdm by maximize the overlapping of their hydrophobic planar rings . We further verify this by considering the pmfs of single gdm approaching another gdm with different relative orientations . The details of this can be found in the supporting information, section s2 . In figure 5b, pmf profiles for urea with different orientations moving toward the identical hydrophobic patch are presented . Again, parallel oriented urea molecule shows the most free energy stability with a value around 2 kcal / mol compared with the two perpendicular orientations . However, comparing parallel urea association free energy around hydrophobic protein patch with that of parallel gdm, we find that it is less favorable, which is due to the lower hydrophobicity of urea s planar surface as discussed earlier . These behaviors are consistent with the results from orientation distribution maps in figure 4 . (a) pmf for single gdm with parallel orientation, perpendicular y and perpendicular x orientation from bulk approaching the hydrophobic protein solvent interface . (b) pmf for single urea with parallel orientation, perpendicular y, and perpendicular x orientation from bulk approaching the hydrophobic protein the stability of a single solute at the protein interface correlates with the induced interfacial fluctuations as the solute approaches . Solvent interface was constructed based on the protocol mentioned in section ii.b . A representative average protein solvent interface is shown in figure s1c, and the corresponding height fluctuation is shown in figure s1d . Overall, the contour of the mean interface is reasonable considering that the shape of the protein is globular . The magnitude of interface fluctuations can be judged by the color scale in panel d, in which case a single gdm is located at z = 18 right above the position x = 0, y = 0 . We consider this point as a reference point since it displays the largest induced fluctuations compared with other regions on the protein surface as indicated by the bright ring in figure s1d . To better illustrate the change in interface fluctuation magnitude as single gdm / urea approaches the patch, the induced fluctuation at this reference point, h(x = 0, y = 0), as a function of z position of the central carbon of the solute is plotted in figure 6 . In figure 6a, at large separations of the single gdm from the hydrophobic patch region, none of the configurations of gdm solvent interface solely comes from the inherent, thermal fluctuation . As the restrained gdm with distinct orientations approaches the hydrophobic patch, the induced fluctuation profiles exhibit striking differences . Parallel - oriented gdm induces large fluctuations of the interface (0.95) at the separation of z = 18, which is around 5 times that of the inherent interfacial fluctuation (0.19). In the case of perpendicular - orientated gdm, maxima in the fluctuation profiles can also be found at the same separation of z = 18 . However, the extent of the induced interfacial fluctuation is smaller compared with that of the parallel orientation, with perpendicular y giving a value of 0.4 (2 times of inherent fluctuation) and perpendicular x giving a value of 0.35 (1.8 times of inherent fluctuation). Vapor interface showing that interfacially stable parallel configurations of gdm induce larger interfacial fluctuations than the perpendicular, less interface - stable orientations of gdm . As expected, parallel orientations urea induces a larger extent of fluctuation (around 0.75) than the perpendicular ones (around 0.45), corresponding to the greater free energy stability of the parallel configurations around the interface shown in figure 5 . Comparing the induced fluctuation values between parallel configurations of the two solutes, the more hydrophobic and more surface stable gdm gives a higher level of enhanced fluctuation . These results support the argument that the more hydrophobic nature of the parallel - oriented gdm makes the hydration shell weakly bound and less ordered, so that it has more tendency to break and couple with the hydration water in the vicinity of hydrophobic protein patch region, which will cause a large perturbation of the protein solvent interface in addition to the level present in pure water . According to the previous studies, this enhanced fluctuation represents an increase of interface entropy, which may contribute to differentially stabilizing configurations where the parallel orientation gdm is closer to the interface compared to other configurations of the solute . (a) surface height fluctuation for hydrophobic protein interface at (x = 0, y = 0) as a function of restrain z position of single gdm with parallel, perpendicular y, and perpendicular x orientation . (b) surface height fluctuation for hydrophobic protein interface at (x = 0, y = 0) as a function of restrain z position of single urea with parallel, perpendicular y, and perpendicular x orientation . To close this discussion about induced interfacial fluctuations, one may ask whether the instantaneous coarse - grained interface we construct can artificially pass through the solute, thus giving rise to artificially large fluctuations . To explore this, we plot the difference in the z - position of the central carbon of gdm and the z - position of the interface (zinterface) as gdm moves toward the protein patch along the z - axis . Here, the z - position of the interface is equal to the value of the surface height of the interface at the point (x = 0, y = 0, zinterface).> 0 means the interface is between single gdm and the protein patch; while = 0 indicates that interface just passes through the gdm; <0 implies that the gdm resides between the interface and the protein . We will show that even when all the zinterface values are distributed on one side of the solute with value constantly being larger or smaller than zero, the distribution of zinterface is not necessarily small . This would suggest that the induced fluctuations are nonartifactual, and the higher fluctuation values are not due to the combination of three different scenarios (> 0, = 0, and <0). Figure 7 displays the values of and the distributions of as single gdm is located at three representative positions: z = 16, 18, and 19.5 . Panels a and d correspond to the parallel configuration; panels b and e correspond to the perpendicular y configuration; panels c and f correspond to the perpendicular x configuration . At a separation of z = 18, where the gdm induces a large interfacial fluctuation, we observe that is always larger than zero for all the three cases, indicating that the interfaces will always reside between the protein and the solute, so there is no artifact where the surface passes through the solute . The same applies to the situation that gdm is located at z = 19.5 as indicated by the blue line . Although at the separation of z = 16 there are some values less than zero, at this point the interfacial fluctuation is suppressed by the presence of the solute . This suggests further that enhanced fluctuations are not influenced by the interface fluctuating on both sides of the solute . Furthermore, at closer separations of gdm and the protein water interface, the meaning of the local interface becomes ambiguous perhaps, but this is not a serious issue as the major differences in interface effects occur well before the solute arrives at the interface . An additional point worth addressing is that at a separation of z = 18 we observe that the parallel configuration of gdm exhibits a wider distribution of values as shown in the panel d green line . This is consistent with the earlier result of figure 6a that parallel orientations of gdm induce larger fluctuations of hydrophobic protein solvent interface compared to the perpendicular ones . In a recent study, patel et al . Discussed that water near hydrophobic surfaces can be described as being near a phase transition characterized by enhanced fluctuations in relevant order parameters . The relevant order parameter is solvent density in their work, the distribution of which varies from unimodal (when two hydrophobic interfaces are far apart) to bimodal at separations where the volume between surfaces fluctuations between wet and dry states to unimodal once the intersolute space is completely dry (post - dewetting transition). In this work, as solutes approach the hydrophobic surface as a perturbation to the interfacial water, a different order parameter based on the interfacial height is considered . Ideally, this interfacial height should display the same signatures of bimodal distribution as the solute reside at the position that induces the largest surface fluctuation . From previous discussion since zgdm is almost constant (this is the fixed position of the solute), the distribution profiles of and zinterface should be identical except the shift along the x - axis . A fat tail in the distribution profile in figure 7d is observed as the solute is located at a separation of z = 18, which is near the position of largest fluctuation . Furthermore, the distribution profiles of for gdm with parallel orientation at the separation of z = 17.5 and z = 17.7 are shown in figure 8a . Interestingly, at the exact separation where solute induces the largest fluctuation, z = 17.7, a prominent bimodal distribution is observed, which is consistent with the view that at this position, there would be transitions between a wet and dry region between the solute and the protein water interface . Figure 8b shows the log probability of versus, analogous to figure 3c in patel et al . The present probability distributions for the interface position recapitulate the results of patel et al . In a rather dramatic fashion . This further speaks to the notion that water near hydrophobic interfaces, even on the smaller scales of specific regions of biomolecules, is poised close to phase transitions, which upon perturbation by external potentials (in this case, a solute approaching the interface and perturbing the solvent density near the protein surface as a consequence of the nature of the solute s hydration shell) undergoes a transition . (a c) differences between single gdm position and surface position (surface position is defined by the surface height at position x = 0, y = 0) for gdm with different restrained orientations locating at various positions: (a) parallel orientation, (b) perpendicular y orientation, and (c) perpendicular x orientation . (d f) probability distributions of for gdm with different restrained orientations locating at various positions: (d) parallel orientation, (e) perpendicular y orientation, and (f) perpendicular x orientation . (a) probability distributions of for gdm with parallel restrained orientation locating at various positions close to the peak of largest fluctuation . (b) probability distributions (log scale) of for gdm with parallel restrained orientation locating at various positions close to the peak of largest fluctuation . Finally, we consider pmfs of single gdm approaching a hydrophilic region on the protein surface . Figure 9a shows the pmf profiles of a single gdm approaching the hydrophilic protein patch with parallel orientation (red), perpendicular y orientation (green), and perpendicular x orientation (blue). A slight free energy stabilization is observed in all the cases, which may due to the electrostatic interaction between the nh group of gdm and side chain of hydrophilic residues (like asp 25) on the patch as shown in figure s5c of the supporting information . However, compared with the free energy of gdm with most favorable parallel configuration approaching the hydrophobic protein region (3 0.15 kcal / mol), the free energetic advantages from gdm with all three configurations approaching the hydrophilic patch are quite small (around 0.5 0.15 kcal / mol). Furthermore, all three configurations show little to no difference in free energy, suggesting that the orientational preference of gdm around certain types of surfaces is highly dependent on the effective hydrophobicity of the region, with significant orientational preference of gdm occurring around the more hydrophobic surface regions . Furthermore, the induced interfacial fluctuation profiles of single gdm with these distinct orientations approaching this hydrophilic region are shown in figure 9b . Previously, we have reported that for the protein in pure water regions with different hydrophobicity will display dissimilar inherent interface height fluctuations . The larger magnitude of fluctuations are related to the malleable nature of the water and facile cavity formation around hydrophobic patches . When gdm is located far from the patch, in all three cases, an inherent fluctuation value of 0.07 is detected, which is lower compared with the inherent fluctuation value around hydrophobic protein region 0.18 . As gdm moves closer to the hydrophilic interface, both parallel and perpendicular orientation have an inappreciable effect on hydrophilic interfacial fluctuations . Although parallel configuration may induce a little larger fluctuation compared with the perpendicular one, the difference is quite small, around 0.02 . Such negligible differences in inducing fluctuations among these configurations corresponds to the marginal differences of free energies around hydrophilic interface . (a) pmf for single gdm with parallel, perpendicular y, and perpendicular x orientation from bulk approaching the hydrophilic protein solvent interface . (b) surface height fluctuation for hydrophilic protein interface at (x = 0, y = 0) as a function of restrain z position of single gdm with parallel, perpendicular y, and perpendicular x orientation . (a) gdm number density map around hfbii protein (hydrophobic side). (b) gdm number density map around hfbii protein (opposite side). (c) representation of hydrophobic protein patch of hfbii with orange highlighting each hydrophobic residue on the patch . In this article, we continue to explore and demonstrate a connection between interfacial stability and induced interfacial fluctuations as interfacially stable solutes approach ostensibly hydrophobic aqueous the context in which we consider the present work is relevant for discussion of the nature of direct chemical interactions between typical chemical denaturants of proteins, gdm and urea, specifically at hydrophobic regions of a model protein, hfbii . Our calculations of potentials of mean force indicate that gdm and urea exhibit nontrivial stability at the aqueous hydrophobe interface as indicated by figure 5 . Furthermore, we observe a richer subdivision of the contributions to the total free energy arising from two relative orientations of the solute that we have chosen to study: the parallel and perpendicular orientations as defined relative to the surface of the protein . Though the protein surface is not quite parallel to the axis chosen as our order parameter for calculations of potentials of mean force, the selected definitions, we feel, suffice for the current purposes . With respect to the orientation - free energy correlation, our calculations indicate that the orientations of both solutes in which the solute is parallel to the interface are associated with stronger free energy minima compared to configurations where the solutes approach in a perpendicular orientation . These two orientations appear to envelope the total free energy profiles (though we cannot say with certainty what contributions intermediate orientations would offer; however, we stress that in this study our aim is to demonstrate the self - consistency of the free energy profiles computed via the potentials of mean force with the orientation probability densities determined from free, solute - unrestrained md simulations of the solutes in solution with the protein). Furthermore, we find that the correlative behavior between solute orientation and free energy stability (using the current force field combinations for water, solute, protein, and ions) mimics that observed at the aqueous liquid vapor interface (figures 2 and 4, probability distribution maps). Our results for both the protein water interface and the pure liquid vapor interface are in agreement with previous studies . Recent simulations have highlighted the unique nature of hydrophobic interfaces as it relates to the fluctuations induced in solvent density vicinal to the interface (refer to garde et al.s work). Hydrophobe interfaces as simpler atomic species (monovalent ions) and slightly more complicated molecular species approach such interfaces . Both these approaches ostensibly define a further characteristic property associated with hydrophobic solutes (and perhaps the hydrophobic effect in general). The present calculations indicate that associated with interfacial stability of the chosen chemical denaturants is an induced fluctuation of the interface upon approach of the solute to the interface . We stress that the induced fluctuations of the interface formed between the hydrophobic region of the protein and solvent occur before the solute resides directly at the interface . This is an important detail, as it speaks to the somewhat long - ranged nature of the effects generated by certain solutes prior to any direct interaction being realized . That solutes can affect an interface from a distance is a subtle though non - negligible effect we suggest . Moreover, the present results suggest that denaturant orientations that are parallel to the interface (vis - - vis, display interfacial stability) are the orientations that induce the largest fluctuations of the interface (and hence the solvent density). The relation between solute orientation and induced fluctuations is related to the nature of the solvation shells of the solute presented toward the interface upon approach of the solute . In the case of gdm approaching the interface in a parallel orientation, the solvation shell presented is a more malleable one, where the solvent is more labile and free to rearrange . This leads to greater solvent density fluctuations and hence higher interfacial induced fluctuations . In the case of the perpendicular orientations of gdm and urea, the tighter hydrogen bonding patterns of water (as demonstrated in previous studies) create a more rigid, well - defined solvation shell that is not easily disrupted . This translates to lower solvent density fluctuations and hence lower induced fluctuations (or even suppression of interfacial fluctuations). The present results are thus consistent with recent work and provides yet another example of the relation of hydrophobic effect, solvent fluctuations, and interfacial stability . This relationship appears to be common across a series of atomic and molecular species as well as encompassing charged, polar, and nonpolar characteristics of the solutes considered . These observations suggest that molecular ions, such as gdm, as well as polar molecules with heterogeneous charge distributions (at least in the context of empirical molecular mechanics force fields) inherently have built into them regions of high and low charge density . The dependence of local solvation structure on this heterogeneous (or asymmetric) charge density is to a large extent involved in determining the propensities of the modalities involved in specific association of molecules with specific types of interfaces . Observations based on classical simulations as well as recent dft - based calculations thus suggest an intriguing fundamental underlying theme . These ideas call for further study regarding specific details about the nature of the relationship between fluctuations, degree of solute hydrophobicity, solute solvation / hydration shell properties, and interfacial stability . Finally, figure 10 shows the number density of gdm molecules in the vicinity of the canonical hydrophobic region of hfbii as well as on the side opposite to this hydrophobic patch (the opposite side not being hydrophobic to any significant extent). Our analysis of simulation data from 1 m gdm solutions with no restraints demonstrates a propensity for the gdm to the hydrophobic region.
The leucine - rich, glioma inactivated 1 (lgi1) gene was identified at the brake point of a reciprocal chromosome translocation t(10;19)(q24;q13) in the glioblastoma cell line t98 g . Lgi1 protein includes domains presumably involved in protein - protein interaction such as the leucine - rich repeats flanked by cysteine rich regions and the seven - bladed beta - propeller domain [24]. Lgi1 gene is predominantly expressed in brain, and it is associated with autosomal dominant lateral temporal epilepsy (adlte) [5, 6]. Although the specific function of lgi1 remains to be defined, it was suggested to form complexes with kv1.1 potassium channels, essential regulators of synaptic transmission in brain and also to be a ligand of the trans - membrane receptor adam22, a protein highly expressed in brain whose function is still uncertain [7, 8]. The loss of lgi1 expression in most high - grade gliomas supported the function of tumor suppressor gene . In agreement with this hypothesis the reexpression of lgi1 in glioma cells, lacking endogenous lgi1, impaired cell growth and migration capacity . These phenotypic changes were linked with the downregulation of matrix metalloproteinase (mmp) genes by lgi1-mediated inhibition of the erk / mapk pathway . A role of lgi1 in the development of malignant brain tumors was substantiated by the discovery of somatic missense mutations in lgi1 gene associated with high- and low - grade gliomas . The ability of lgi1 to suppressor growth of neuroblastoma cells was suggested by the results of lgi1 overexpression, which was shown to impair proliferation and to induce apoptosis through the inhibition of the pi3k / akt pathway [12, 13]. Further support to the tumor suppressor function of lgi1 was provided by a comprehensive study on malignant esophageal tumors (barrett's - related adenocarcinomas) showing that expression of lgi1 was consistently downregulated . The aim of this study was to establish whether lgi1 was capable to suppress growth of cancer cells different from glioblastoma and neuroblastoma . For this purpose we performed stable expression of lgi1 in hela cell, which is derived from a human cervical adenocarcinoma and expresses the human papillomavirus proteins e6/e7 (hpv-18). Essentially these viral proteins sustain cell proliferation and survival through the inactivation of tumor suppressors p53 and prb, which are implicated in cell cycle arrest and induction of apoptosis [16, 17]. For these features conferring remarkably vigorous growth the results showed that expression of lgi1 inhibited the rate of hela cell proliferation and increased cellular death . Furthermore lgi1 altered expression of key regulators of apoptosis such as the antiapoptotic b - cell lymphoma 2 gene (bcl2) and of the proapoptotic b - cell lymphoma 2-associated x protein gene (bax). The lethal effect produced by lgi1 expression in hela cells suggests that the proposed role of tumor suppressor might be extended to adenocarcinoma - derived cells . Hela cells (atcc number: ccl-2) were cultured in dulbecco's modified eagle medium (dmem, low glucose) with 10% heat inactivated fetal calf serum (fcs). For transfection cells (1.5 10) transfection with plasmid pclgi1 including the human lgi1 cdna (2075 bp) or with empty vector pcdna3 (10 g / ml each; invitrogen, groningen, the netherlands) was performed by the dna ca - phosphate coprecipitation . Cells were cultured for 48 hours after transfection; then they were replated at low density . Total rna was isolated from subconfluent cells by the geneelute mammalian total rna kit (sigma chemical co., st louis, mo, usa). The cdna synthesis was performed by the reverse transcriptase improm - ii starting from random primer examer (promega, madison, wi usa). For quantification of lgi1 mrna, a cdna segment of (236 bp) was amplified with primers: 5-tgtaaactgaaatggctagtggaa, and 5-agtaaaaggctgagcgatgactac . Amplification of a bcl2 segment (258 bp) was carried out with primers: 5-ggtcatgtgtgtggagagcgt, and 5-acttcacttgtggctcagataggc . A bax fragment (539 bp) a portion of the glyceraldehyde-3-phosphate dehydrogenase (gapdh, 909 bp) was amplified using primers: 5-gaccccttcattgacctcaactaca and 5-ggtcttactccttggaggccatgt (clontech, palo alto, ca usa). For the semiquantitative rt - pcr analysis different number of pcr cycles (<25) and serial cdna dilutions were employed to establish conditions of linear amplification . The pcr products were separated by agarose gel electrophoresis; then the net intensity of ethidium bromide stained bands was determined using the kodak 1 d image analysis software . The following antibodies (santa cruz biotechnology, santa cruz, ca usa) were used to probe blots: antibody c-19 directed to the c - terminal peptide of lgi1 (60 kda), affinity purified polyclonal antibody (bax 21) against bax protein (23 kda), and monoclonal antibodies to bcl-2 and gapdh proteins (28 kda and 36 kda, respectively . The immunoreactions were revealed by the ecl reagent (pharmacia amersham biotech, little chalfont, uk) and quantified by the kodak 1 d image analysis software by evaluating the net intensity of the bands . An elisa immunoassay based on brdu incorporation was employed to measure cell proliferation according to the instruction procedures (roches diagnostic, germany). Cells were plated in quadruplicate on 96 well cluster plates (100 cells / mm) and cultured for 24 hours . Absorbance at (450690 nm) was measured using a plate reader . To determine the fraction of dead cells, the lactate dehydrogenase (ldh) released in the medium and that present in intact cells was measured using the cytotox non - radioactive cytotoxicity assay (promega, madison, wi usa). Cells were plated on 96-well plates (100 cells / mm) and cultured for 24 hours . One half of the culture medium was transferred to a fresh well to determine ldh spontaneously released by dead cells . Then the conversion of tetrazolium salt (int) into the red formazan product by ldh activity was measured (absorbance 490 nm). The fraction of dead cells was calculated as follows:% cytotoxicity = [spontaneously released ldh 2 100]/total ldh . The apo - one homogeneous caspase-3/7 assay (promega) cells were seeded on 96-well plates (100 cells / mm), and the caspase-3/7 substrate z - devd - r110 was added 24 hours later . The reaction was carried on for 6 hours, and the measurement of fluorescence was then performed by a plate spectrofluorimeter (485 nm excitation wavelength, 530 nm emission wavelength). Ldh assays were performed on replicated cell samples to determine the number of living cells, which was used to normalize caspase activity . Experimental data were expressed as the mean sd . To determine the significance of variations, values were analyzed by student's t - test; levels of p <.05 were considered to be significant . Stable transfection of hela cells with lgi1 cdna was performed to establish whether expression of lgi1 might affect cell viability . The level of lgi1 expression was evaluated in three cell clones designated he - lgi1 - 1, he - lgi1 - 2, and he - lgi1 - 3, respectively (figure 1). The results of the quantification of lgi1 mrna was quite in agreement with the estimated values of lgi1 protein (table 1). Although lgi1 protein was detected in whole cells sample, the possibility that it might be secreted as it occurred in 293 t cells cannot be excluded, since it might be bound to the cell surface . The endogenous expression of lgi1 in nontransfected hela cells and in cell clones stably transfected with empty vector was undetectable by rt - pcr and western blotting (see he - pcdna3; figure 1 and table 1). The absence of lgi1 expression in human uterine tumors and in normal uterine tissue is supported by the analysis of expressed sequence tags (ests) reported by unigene and is in agreement with lack of lgi1 expression in mouse uterus . The brdu incorporation procedure was employed to evaluate the influence of lgi1 expression on the rate of hela cell proliferation . All cell clones expressing lgi1 exhibited a lower proliferation rate in comparison with control cells transfected with empty vector (figure 2), which were similar to nontransfected hela cells (not shown). With he - lgi1 - 1 cell clone, in which lgi1 expression is very little, decrease of cell proliferation was on average 30% . With he - lgi1 - 2 cells, expressing the highest lgi1 levels the decrease was 80%, whereas it was 70% with helgi1 - 3 cells, showing intermediate levels of lgi1 expression (figure 2). The results showed that the inhibitory effect of lgi1 on cell proliferation was proportional with the levels of lgi1 expression . To establish whether expression of lgi1 also might affect survival of hela cells we measured the amount of ldh spontaneously released in the medium as a consequence of cell death . The fraction of dead cells was significantly greater in cell clones expressing lgi1 in comparison with control cells (figure 3). In particular, the percentage of he - lgi1 - 1 dead cells was on average 7%, slightly but significantly greater than that of he - pcdna3 cells, which was 5% . The lethal effect was much greater with he - lgi1 - 2 and he - lgi1 - 3, whose fractions of dead cells were on average 26% and 21%, respectively . The decline of cell survival was clearly correlated with the levels of lgi1 expression, likewise inhibition of cell proliferation . Since significant effects were obtained with cell clone helgi1 - 1 expressing very low levels of lgi1, it can be suggested that the impairment of cell proliferation and survival was a consequence of the proper lgi1 activity rather than the outcome of artificial overexpression . Furthermore, these effects on cells of epithelial origin exclude the requirement of interactions with proteins specifically expressed in brain for the ability of lgi1 to contrast cell growth and survival . To gain information on the process of hela cell death caused by the expression of lgi1 we measured the activity of the apoptosis effectors caspase-3 and -7 in the experimental conditions employed to monitor cell death (figure 4). The relative fluorescence emitted by the specific caspase-3/7 substrate upon cleavage was particularly enhanced with he - lgi1 - 2 (7.4-fold) and he - lgi-3 cells (4.2-fold), whereas a modest increase was measured with he - lgi1 - 1 (figure 3). The results supported the possibility that expression of lgi1 might trigger apoptosis of hela cells . To substantiate the induction of apoptosis of hela cell expressing lgi1 we measured the level of the antiapoptotic bcl2 and of the proapoptotic bax gene expression, since the relative proportion of these proteins plays a central role in the control of cell survival . A significant decrease of bcl2 mrna and protein was measured in cell clones expressing lgi1 in comparison with control cells, whereas the expression of bax was consistently increased (figure 1; table 1). In particular, the upregulation of bax expression was proportional with the levels of lgi1, with a maximum increase of 4.6-fold of bax protein in he - lgi1 - 2 (table 1). The results showed that the antiapoptotic bcl2 gene was downregulated and that of its opponent proapoptotic bax was upregulated as a consequence of lgi1 expression . A decreased ratio of bcl2/bax favors the release of apoptogenic molecules from mitochondria, which activate the intrinsic pathway of apoptosis in various cancer cells, including hela [2126]. Thus lgi1 might increase the susceptibility of hela cells to spontaneous apoptosis, similarly to what occurred in neurobalstoma cells . Apparently lgi1 was capable to counteract efficiently the oncogenicity of hpv, specifically of the viral protein e6, which prevents apoptosis by stimulating the ubiquitin - mediated degradation of p53, a transcriptional regulator capable to enhance the expression of bax and to repress that of bcl2 [16, 2729]. Thus the balance of these apoptotic factors imposed by e6 to sustain cell survival might be destabilized by lgi1 in favor of apoptosis . Although the mechanism of cell death induced by lgi1 has not been completely elucidated, it seems possible that lgi1 might interfere with central survival pathways such as the pi3k / akt, as it occurred in neuroblastoma cells, because the prosurvival effects of this pathway includes transcriptional regulation of bcl2 and bax genes [30, 31]. In conclusion, this study supports the possibility that lgi1 might act as tumor suppressor of adenocarcinoma - derived cells, in line with the downregulation of lgi1 reported in barrett's - related adenocarcinomas and with previous studies on glioblastoma and neuroblastoma cells [914].
A medication error can be defined as a failure in the treatment process that leads to, or has the potential to lead to, harm to the patient. The use of the term failure signifies that the process has fallen below an attainable standard . As per the us institute of medicine's report (1999) to err is human, every year 7000 preventable adverse drug reactions in usa were due to medication errors . To the best of our knowledge, this is the first report on the adverse event of intradermal hypersensitivity testing with cloxacillin resulting in pain and skin necrosis as a result of medication error . We describe a case report of cloxacillin - induced skin necrosis which occurred following intradermal skin testing for screening of hypersensitivity response . Intradermal skin testing for diagnosing hypersensitivity response to cloxacillin was done for three patients who were admitted in the surgery ward of a tertiary care hospital . This included a 12-year - old boy diagnosed with cryptorchidism and admitted for inguinal exploration, a 28-year - old male admitted for bilateral breast mass reduction, and a 52-year - old male who was admitted for inguinal mass exploration . The test dose was given in the forearm of each patient, one after the other, using the same diluted preparation of cloxacillin . The nurse who prepared the test dose solution had apparently mixed 500 mg of cloxacillin sodium in 2 ml of distilled water, and had used 0.1 ml of the solution for intradermal injection in each of the three patients . However, the institute protocol for intradermal skin testing was only 0.5 mg per test dose . All three patients complained of intense pain, itching and rash over the site of injection within 30 min of receiving the test dose intradermal . This was followed by darkening of skin over the injected area which progressed to necrosis within 6 to 12 h and was associated with persistent pain [figures 1 and 2]. After 12 h of receiving the test dose, pain subsided spontaneously in all three patients . Skin necrosis at the site of intradermal test dose skin necrosis at the site of intradermal test dose adverse drug reactions are one of the important causes for significant morbidity and mortality among patients . Among various causes for adverse drug reactions, medication errors include administration of a drug which is inappropriate for the condition or at an inappropriate dose . A study by kopp et al ., on medication errors in intensive care patients showed that around 17% of medication errors resulted in adverse drug reactions which were preventable . Further, 83% of medication error had the potential to cause adverse drug reactions . In a study by bates, approximately 28% of adverse drug events were due to medication errors which were preventable . Further, studies had also shown that the most common cause of medication error (36%) was due to overdose followed by selection of wrong drug and wrong route of drug administration . This article highlights the seriousness of the reaction which could potentially occur with cloxacillin due to medication error . Since the drug was given at 50 times the dose recommended for intradermal testing as per the institute protocol, the patient faced a serious risk of developing serious adverse events such as anaphylaxis . There had been reports of patients developing anaphylaxis even with the low dose used for penicillin hypersensitivity intradermal skin test . Many of the medication errors occurred due to oversight or negligence during the process of prescribing, dispensing, storage and administration of drug . In a hospital setup, this could occur due to oversight by the nursing staff responsible for administering the drug . Some of the common reasons could be overtime working, under trained staff, unsupervised nursing interns, complicated and unclear protocols, interpersonal communication gap between health care professionals and also poor availability of ideal resources . In this report, the drug was diluted in a wrong way by an unsupervised nursing intern . There is an important role for pharmacovigilance centers that can contribute to the detection and prevention of medication errors and increasing the safety of patients . These centers must alert health care professionals about the importance of reporting medication errors through bulletins and journals.
Human cells show a different ddr during mitosis than in other cell cycle phases . During most of the cell cycle, after cells reach the point of no return, however, dsbs on mitotic chromosomes do not trigger cell - cycle delay or arrest; and the cells rather proceed through mitosis even if they contain unrepaired dsbs or fragmented chromosomes. (32) indeed, the sensitivity to ionizing radiation is higher in mitotic cells than in interphase cells. (33,34) moreover, dsb introduction into mitotic cells by etoposide treatment induces massive chromosome aberrations in the next cell cycle in a cancer cell line (fig . Thus, dsbs during mitosis are very toxic to cells because of the induction of severe genomic instability . Effect of etoposide - induced double - strand breaks (dsbs) during mitosis on genomic instability of mitotic chromosomes . Representative images of chromosome spreads from etoposide - treated (+ etoposide) and non - treated (etoposide) in hct116 human colon cancer cells arrested in mitosis . During mitosis, dsbs activate pikks to induce phosphorylation of h2ax, and mdc1 as well as the mrn complex are recruited to the dsb sites, as seen during interphases (fig . However, the recruitment of rnf8, rnf168, 53bp1, and brca1 to dsb is largely suppressed during mitosis (fig . 1b, d, right). (30,33) the mechanism of the suppression and its purpose were not well understood . Two groups recently showed that localization of rnf8 and 53bp1 to dsbs is inhibited during mitosis. (35,36) the first work done by orthwein et al . (2014) showed that, in human cells, rnf8 and 53bp1 are phosphorylated during mitosis by cdk1 and that inhibition of the phosphorylation restores their localization to dsb sites (fig . Orthwein et al . Also identified t198 on rnf8 as a cdk1 phosphorylation site and showed that rnf8-t198a, a phosphorylation - defective protein, can localize to mitotic chromatin after dsb introduction . Rnf8 prepared from mitotic extracts cannot bind to mdc1 in vitro, but rnf8 prepared from cdk1 activity - inhibited cells interacts with mdc1 in vitro . They also found that t1609 and s1618 on 53bp1 are phosphorylated during mitosis (fig . 3a) and that 53bp1-t1609a / s1618a double mutant protein can localize to mitotic chromatin and restores dsb repair in cells expressing rnf8-t198a . Their study confirmed that t1609 is phosphorylated by cdk1, whereas s1618 is a target of plk1 in vitro . These show that there are at least two distinct mechanisms to regulate the recruitment of these ddr effector proteins. (35) domain structure of 53bp1 and comparison of cdk1 and plk1 sites among xrcc4 orthologs . (b) domain structure of xrcc4 and conservation of phosphorylation sites among various species . S326 in the c - terminus of human xrcc4 (hs) is a potential cdk1 phosphorylation site . The gray and black boxes show xlf and dna ligase iv binding sites, respectively . Phosphorylation of cdk1 or plk1 sites are shown in mouse (ms), chicken (gg), zebrafish (de), and budding yeast (sc). Dna ligase iv and xlf binding sites in mouse, chicken, and zebrafish were predicted by aligning with the dna ligase iv and xlf binding sites of human xrcc4 using t - coffee software . (2014) indicated that t1609 and s1618 of 53bp1 are hyperphosphorylated in the absence of pp4c phosphatase in human cell lines. (36,37) they analyzed the function of pp4c phosphatase during ddr and found that these sites are phosphorylated specifically during mitosis. (36) these mitosis - specific phosphorylation sites are located in the ubiquitin - dependent recruitment motif of 53bp1, a recognition site of ubiquitinated h2a, which is required for the localization of the protein to dsb sites. (1517) both of the reports(35,36) showed that dna damage during mitosis in cells expressing phosphorylation - defective 53bp1-t1609a / s1618a mutant, which restores its localization to dsb sites, leads to increased micronuclei formation . This suggests that restoration of dsb repair during mitosis causes defects in proper chromosome segregation and that inhibition of dsb repair during mitosis serves to maintain genomic stability . In addition to micronuclei formation, orthwein et al . Showed that restoration of accumulation of rnf8 and 53bp1 to damaged mitotic chromosomes increases telomere fusions, which seems to be mediated by dsb repair such as c - nhej. (35) therefore, suppression of dsb repair by phosphorylation of rnf8 or 53bp1 by cdk1 or plk1 may protect the genome during mitosis . In particular, suppression of rnf8- and 53bp1-dependent dsb repair during mitosis may prevent telomere fusions . Two groups showed that inhibition of the dna - pkcs suppresses micronuclei formation induced by mitotic dsbs in 53bp1-t1609a / s1618a - expressing or 53bp1-t1609a / s1618a rnf8-t198a - expressing cells, suggesting and that the core c - nhej complex plays a role in the segregation defects. (35,36) recently, we showed another dsb repair suppression mechanism by modulating the c - nhej core complex during mitosis. (38) dsb repair is largely suppressed during mitosis, but a substantial level of repair still occurs. (38) c - nhej causes formation of anaphase bridges, which are bridge - like dna structures that span daughter chromosomes and frequently induce genomic instability through inappropriate chromosome segregation. (39,40) in our study, severe chromosome aberrations such as dicentric and fragmented chromosomes are induced by mitosis - specific dsb introduction by transient treatment with etoposide (fig . 2). (38) this observation raises a possibility that anaphase bridges are caused by dicentric chromosome formation by interchromosomal connections between telomeres or other chromosomal loci . These results suggest that there is a process to connect sister chromatids or individual chromosomes in ddr during mitosis . Dna ligase iv - dependent c - nhej contributes to dicentric chromosome formation through telomere fusion in cells with dysfunctional telomeres,(41) which act as dsb ends. (42) therefore, we hypothesized that c - nhej is involved in the formation of anaphase bridges . Both xrcc4 and xlf are regulatory subunits of the dna ligase iv complex and are essential for its activity. (4345) anaphase bridge formation is reduced after xrcc4 knockdown, suggesting that c - nhej contributes to the formation of some of these bridges during mitosis. (38) knockdown of the hr - specific factor xrcc3 has almost no effect on anaphase bridge formation, indicating that c - nhej is more critical than hr for the formation of anaphase bridges in mitotic cells . Our studies strongly suggest that inappropriate use of the c - nhej pathway causes chromosome bridges, which lead to chromosome aberrations from mitotic dsbs . A phosphoproteomics study revealed that many sites are phosphorylated during mitosis, including those on xrcc4,(46) so we evaluated cell - cycle regulation of xrcc4 phosphorylation. (38) this mitosis - specific phosphorylation of xrcc4 contributes to the suppression of anaphase bridge formation after induction of dsbs during mitosis. (38) the mitosis - specific phosphorylation of xrcc4 depends on both cdk1 and plk1 activities. (38) s326 is a putative cdk phosphorylation site, and s326a substitution substantially reduces mitosis - specific phosphorylation of xrcc4 (fig . 3b). (38) interestingly, the s326 phosphorylation residue overlaps with the polo box recognition motif. (47) both s256 and s326 are phosphorylated during mitosis. (46) because s256 resides in a putative plk1 phosphorylation site (fig . 3b), cdk - dependent phosphorylation of s326 might prime the mitosis - specific phosphorylation of xrcc4 by plk1 . Although the ortholog of xrcc4 has not yet been identified in nematode or fission yeast, most of the dna ligase iv subunits have been identified in various species (table 1). We previously analyzed cell - cycle regulation of lif1p, which is the s. cerevisiae ortholog of xrcc4. (4850) lif1p is phosphorylated by cdks from s phase through mitosis and this phosphorylation plays a role in suppressing c - nhej in s to m phases through the pathway dependent on sae2, the s. cerevisiae ortholog of ctip. (49) these findings prompted us to assess the conservation of the phosphorylation sites among xrcc4 orthologs . Although the locations of the phosphorylation sites are different, both s. cerevisiae lif1p and human xrcc4 have one putative target site for cdk1 and multiple sites for plk1 (fig . Both the cdk1 and plk1 sites near the c - terminus were conserved between human and mouse (fig . 3b). In chicken, there are two cdk1 sites near the c - terminus and one plk1 site . However, in zebrafish, although the cdk1 site near the c - terminus was not conserved, there are four cdk1 sites in other locations . Three of them are located downstream of the dna ligase iv binding site and overlap with plk1 sites (fig . All of the xrcc4 orthologs have cdk1 or plk1 phosphorylation sites downstream region of the dna ligase iv interaction domain . Thus, the function of mitosis - specific phosphorylation of xrcc4 is likely to be conserved among many species . We showed that xrcc4 phosphorylation - defective mutant (xrcc4-s326a) restores rapid repair of mitotic dsbs associated with more anaphase bridge formation. (38) this suggests that mitotic xrcc4 phosphorylation is involved in suppressing c - nhej to prevent chromosome instability in human cells . Ctip promotes dsb end resection in hr and a - nhej. (51) because anaphase bridge formation is increased in ctip - depleted cells,(38) ctip may have a function to suppress anaphase bridges, possibly through the a - nhej pathway . Brca1, which is important for recruitment of ctip to dsb sites, does not localize to mitotic dsb sites. (33) however, only the function of ctip in hr, but not in a - nhej, is dependent on interaction with brca1. (52) moreover, in xenopus m - phase extract, ctip can bind mitotic chromatin. (53) thus, ctip may function as an a - nhej factor in suppression of genomic instability during mitosis . Alternatively, ctip - dependent end resection may have functions to suppress anaphase bridge formation . Conservation of dna ligase iv complex subunits among species although dna ligase iv (catalytic subunit) localizes to mitotic chromosomes, xrcc4 does not,(54) suggesting that the dna ligase iv complex function during mitosis is different from those in other cell cycles . Because complex formation of the dna ligase iv with xrcc4 is believed to be essential for its activity, an activity of the dna ligase iv complex may be modified during mitosis . We confirmed the difference in the localization of dna ligase iv and xrcc4 during mitosis. (38) like wild - type protein, xrcc4 phosphorylation - defective mutant protein also fails to localize to mitotic chromosomes, showing that the phosphorylation of xrcc4 is not responsible for inhibiting the localization to chromosomes during mitosis . On the basis of these findings, we propose that there may be two mechanisms to suppress c - nhej: the reduced recruitment of c - nhej - specific factors to mitotic chromosomes, and mitosis - specific phosphorylation of xrcc4 by modulating the function of the dna ligase iv complex . Taken together, these results suggest that xrcc4, a key regulatory subunit of the dna ligase iv complex, is required not only for c - nhej in interphase but also for suppression of c - nhej during mitosis to prevent genomic instability in human cells . Cells prevent carryover of dna lesions at the g2/m checkpoint because dna damage as well as the dna repair process are toxic during mitosis . As described above, there are several types of dsb repair suppression systems that occur during mitosis . However, mitotic cells do not undergo the second level of ddr: neither 53bp1 nor brca1 localizes to dsb sites . Mitosis - specific phosphorylation of 53bp1 and rnf8 prevents their localization to dsb sites. (35,36) because 53bp1 and brca1 localization to dsb sites is important for nhej and hr, respectively, dsb repair pathways should be largely suppressed on mitotic chromosomes (fig . We found that the third level of ddr is also suppressed or modified by mitosis - specific phosphorylation of xrcc4, a component of the core c - nhej complex. (38) even if dsb repair pathways are largely suppressed by mitosis - specific phosphorylation of 53bp1 and rnf8, considerable levels of dsb repair still occur and cause genomic instability during mitosis . Phosphorylation of xrcc4 during mitosis slows dsb repair. (38) thus, xrcc4 phosphorylation has some functions to modulate dna ligase iv complex activity (fig . In addition, xrcc4 fails to localize to mitotic chromatin. (54) the failure of xrcc4 to localize to mitotic chromosomes also may modify the composition of the dna ligase iv complex . Ctip, but not rad51, is recruited to mitotic chromatin. (53) this also suggests that there is the third level of suppression mechanisms in hr and that ctip has some functions to prevent genomic instability without the requirement of brca1 localization . (a) double - strand break (dsb) induces histone h2ax phosphorylation (h2ax) by atm . (b) cdk1 and plk1 phosphorylate rnf8 and 53bp1 to inhibit 53bp1 and brca1 localization of dsb sites . (c) cdk1 and plk1 phosphorylate xrcc4, a regulatory subunit of the dna ligase iv complex, to suppress canonical non - homologous end joining (c - nhej) activity . Ctip - dependent alternative non - homologous end joining (a - nhej) may prevent anaphase bridge formation . Cells use multiple safeguards to prevent genomic instability by suppression or modification of dsb repair activities during mitosis, through the activation of mitotic kinases, cdk1 and plk1 . The molecular mechanisms of mitosis - specific phosphorylation and the change in localization of the dna ligase iv component xrcc4 are still unknown . Analyzing how dna ligase iv activity in mitotic cells differs at the molecular level from during other cell - cycle phases is critical to understand the mechanisms of these controls . Another c - nhej component, dna - pkcs also phosphorylated by plk1, is dephosphorylated by pp6 and is required for accurate chromosome segregation. (55) it is also interesting to analyze how those c - nhej factors are involved in accurate mitosis. (56) active dsb repair during mitosis affects chromosome segregation, which often results in apoptosis, aneuploidy, or other chromosome aberrations . Thus, studies of dsb repair control during mitosis are important to understand the origin of genomic instability, which causes cell tumorigenesis . In addition, activation of c - nhej during mitosis causes severe damage to growing cells like cancer cells, but the activation of nhej by itself would not be detrimental to interphase cells . This property would be useful for the development of anticancer drugs in the future.
The expenditure series (tables 1 - 9) initially was conceived as a national accounting (income and product accounts) series meant to satisfy two criteria: quasi - comprehensiveness of the underlying identity and adherence to rigorous economic classification principles . The identity ensures that all costs incurred for a stated purpose or function are added up . The components are private consumption of medical care hospital care, physician services, pharmaceuticals, therapeutic appliances, other insurable benefits (except those related to sickness benefits paid in cash, which belong to an income maintenance function)and general government outlays on health care, including public health preventive services, administration and regulation, etc . The qualification quasi is used for several reasons . For one, there is a grey area related to those services that contribute to the health status of the population but are classified differently in various countries: under agriculture, under health, or elsewhere . Because detailed information is not readily accessible, it has not been possible to reallocate these services systematically . Whenever detailed information was available and permitted it, the reallocation has been effected for armed forces health services, prison health services, school health services, and publicly funded medical research and development, which are treated as auxiliaries to defense, justice, education, or science support outlays in the national accounts . In the national accounts, expenditure for health on private business premises is treated as an intermediate outlay, although the same services rendered to civil servants are final expenditure . Eye tests for airline pilots and similar examinations are necessary for rendering services such as safely flying passengers, but mostly the primary purposes of these services are preventive screening, which implies that also, gross capital outlays have been added to the aggregate outlays . In some countries, such as france, a depreciation allowance is included in the pricing (for example, the price of hospital services) and has to be deducted to avoid double counting; this was not always possible . The estimated size of the gap or the inclusion of unwanted categories of expenditure, errors, and omissions may be in the range of 96 to 102 percent of the desired amount, much less for many countries . Another major problem is that certain national accounting systems still are rudimentary . In public debates in a few countries, such as belgium however, in france, a 1987 benchmark reevaluation led to a substantial downward revision, which is still only partly documented . Underreporting in the basic national systems from which the data are extracted regularly leads to small reevaluations of the estimates . The publication of the tables in this issue of the health care financing review is likely to generate further changes in ongoing accounting methodologies . Countries, such as switzerland, that had no health accounts have established a working party to create such a system . Department of health and human services publishes detailed national health accounts data (which, with only a 1-percent difference, nearly correspond to the national income and product account health outlays data, compiled by the bureau of economic analysis in the u.s . Health and welfare canada publishes data according to the same accounting rules as those used in the united states . Details published in the dutch accounts or made available by the central bureau of statistics suggest that the boundaries and economic classification principles used in the netherlands are close to those adopted in north america . In france, germany, and the united kingdom, the accounting system has gained in reliability over time . However, the international compilation for countries is still crude . It is hoped that greater cross - comparability will be reached in the 1990s through international harmonization of boundary concepts and classification principles . The use of international classifications other than those proposed by the oecd should be mentioned . Estimates from these other classification systems do not include purely private medical outlays, and a fair number of public health outlays included here are also omitted . The heterogeneity of sources for the segments of the expenditure series is another cause of gaps in the identity . Total expenditure equals institutional care plus ambulatory care plus pharmaceutical purchases plus therapeutic appliances plus collective services (such as armed forces medical care) plus public health programs plus biomedical research and development outlays plus administrative outlays . Detailed expenditure flows are only one prerequisite of the accounting substrata needed for health policy analyses . To obtain a measure of real resources devoted to health care across countries or over time requires controlling for price trends . Have the resources devoted to health care genuinely decreased in some countries? Have they merely been stabilized in most, as exhibited by the simple ratio of total health expenditure to gross domestic product (gdp) or gross national product (gnp)? The answers provided rest on the evidence collated or estimated by statisticians regarding prices . (schieber and poullier briefly address the relationship between economic growth and the rate of consumption of health services in their article in this issue .) The methods of elaboration of the implicit price indexes of gdp and gnp are well documented; those of the numerator (health care consumption) are less so . In a dynamic economic process, individual prices vary as a reflection of new scarcities, gains in productivity, market power, and a host of other demand and supply factors; if an aggregate price index for medical consumption gdp deflator or consumer price index (cpi)is used, specific changes occurring in inpatient care, ambulatory care, pharmaceutical production, therapeutic appliances, and government - supplied services are not captured . The oecd health data file includes distinct price measures (shown as 1980 = 100 in the accompanying tables) for each consumption function . The indicators that are accessible are mostly details of private consumption price trends, and these often fail to reflect the impact of changes in real factor costs . Input price measures, however, also are gradually collated . At a more aggregate level, a new total medical consumption price index is presented in table 13 . This replaces the measure of private consumption trends previously presented in measuring health care . The old measure was a fair indicator for belgium, switzerland, and the united states, where private consumption constitutes from two - thirds to more than four - fifths of total consumption . For some countries (australia, austria, denmark, finland, greece, iceland, italy, japan, norway, sweden, and the united kingdom), the new measure is a weighted index of private and general government consumption of medical care and health services . For other countries (belgium, germany, ireland, luxembourg, spain, switzerland, and the united states), the price index shown reflects only private consumption; this can be a source of serious bias . For canada, france, and the netherlands, total health consumption indexes are calculated by the national authorities, but the underlying methodology is not given along with the published figures . The canadian price index is a weighted sum of the following: physician services (i.e., fee - for - service increases in medical care insurance plans, not salaries), which are calculated for each province and tabulated by health and welfare canada; dental care and optometrist services, which are measured by consumer prices; average wages and salaries in hospitals (80 percent) combined with hospital supplies (20 percent), which is information collected by statistics canada; and prescribed and nonprescribed drugs, which are two consumer price series collected by statistics canada . The french index appears to be equally divided between a weighted price measure of the consumer price components and a cost index for public institutional care . Information on both wage costs and supplies is obtained in an annual survey conducted since the early 1980s . Consumer prices were the only deflators available for public hospital costs in the earliest years . The netherlands annually publishes current and constant price data for a range of inpatient care institutions, several ambulatory services, medical goods, and health administration services . The estimates allow calculation of a measure of total costs since 1980; provisional figures were calculated for the period 1972 - 80 (the estimation for past years is not yet completed); and consumer price figures were derived from several national accounting versions prior to 1972 . The final australian private consumption expenditure deflator is a weighted measure of doctors' and dentists' fees for a range of services paid for by consumers; hospital services (weighted the same as the general government index, described next); and hospital and medical fund administrative expenses (fixed wage for the salary component; cpi trends for meals, transportation, and telephone services; and producer prices for paper and printing). For the general government index, wages and salaries for hospital, medical, and ancillary staff are weighted by fixed - weight wage and salary rates, and supplies are weighted by the corresponding input prices . Finland also uses separate deflators for intermediate consumption and each component of value added, summing up the costs . Private health services arc mainly based on social security schedules for paying patient bills, the physician fees and laboratory billing serving as control measures for a range of services . Luxembourg uses consumer price data for dental care, inpatient bed days, laboratory tests, and pharmaceutical products when evaluating market goods and services . Spain deflates nonmarket services through a weighted index of wages and supplies, with detailed calculations based on previous year = 100 . (this index is not published and is not included in the estimates shown here .) The united states deflates physician, dentist, and other professional services and care in for - profit hospitals by the relevant cpi components . Nonprofit hospital and nursing home prices are composite input prices established by the health care financing administration . Drug preparation and sundries are deflated by the relevant medical care commodities cpi components, and ophthalmic and orthopedic appliances are deflated by the relevant eye care cpi components . Medical and hospitalization insurance is deflated by the weighted average of physician and hospital services based on estimated benefits composition; it is not separated from income loss insurance and workers' compensation insurance in the published details, the latter two being deflated by the total cpi index . (this information is not published and is not included in the estimates shown here .) Fixed investment is also deflated by a range of relevant measures for medical and surgical instruments, hospital furniture, hospital construction, etc . Only a limited amount of input cost data is available, so the series shown does not, in principle, include adjustments for productivity . The headings used for tables 10 - 13 reflect common acceptance of pricing trends in health economics, but strictly defined they are conventional deflators rather than input price indexes . For example, physicians may be committed by law to apply one level of fees to insured patients but may have some freedom to apply another price to private patients . Where two or more sets of prices and price trends exist for given commodities or services, the level of observation does not permit differentiation . The statistician is compelled to make decisions on the basis of partial knowledge, which is only gradually incremented . The data presented reflect a provisional state of the art that is believed to be superior to use of cpi, gdp, or gnp aggregate price trends . The widespread use of separate indexes for wage components and supply components suggests that, notwithstanding the extreme variety in the basic parameters observed (e.g., in inpatient care institutions), the underlying deflation approach is reasonably comparable across countries . The documentation of differences across countries in the level of social protection is essential to the understanding of variations in the private - public mix in the financing of health services . The oecd health data file constitutes an attempt to translate such descriptive documentation into a series of indicators reflecting the extent to which social preferences have moved countries toward coverage of the population by a public scheme and toward substituting collective for individual financing of medical benefits . The indicators of public coverage constitute an entitlement index . In countries with universal access to publicly funded health services, for all others, an interpretation is required of institutions and regulations that, at times, are fairly detailed . An index of social security coverage only is insufficient in the netherlands and spain, for example, specific schemes run by entities that are legally private exist for specific groups of public employees . Since the schemes are compulsory for the groups concerned, the outlays have been treated as public, and so should the potential beneficiaries . For instance, in the netherlands, a universal tax - funded scheme for long - term hospitalization has been run since 1968, but one - quarter of the population has not been subject to compulsory insurance for shorter hospitalization and other medical benefits . Ireland has a three - tier system under which, in mid-1987 (but not in the first two decades monitored), public hospital accommodation was accessible to all . The population with voluntary health insurance, or vhi (private coverage) is estimated to have increased from 17.3 percent in 1976 to 29.3 percent in 1987 . The benefits of vhi are mainly in the ambulatory care and pharmaceutical functions (plus physician services in hospitals for category i). However, a measure of the covered population is not simply the difference between 100 and the percent of population with vhi, because a special entitlement program for pharmaceutical benefits exists, notably for chronic care . The estimates shown in tables 15 - 17 are, for most countries, best estimates from the oecd secretariat . These are likely to be revised as more knowledge is gained of the 24 health systems under study . Several of these systems, in turn, comprise distinct subsystems . The appropriate index for a country cannot be a simple weighted average for that country, as each subsystem exhibits a number of exemptions and special cases . An estimate of the average level of public cost sharing is still more difficult to establish . The nomenclatures of services accessible without charge (full reimbursement), those for which there is nominal cost sharing (partial reimbursement), and those for which gratuitous supply or reimbursement is denied (such as some cosmetic surgery, services supplied by physicians working outside a social contract framework, and medicines listed as nonreimbursable) sometimes comprise several hundred entries, and no detailed spending data are available . Pensioners are exempt or pay lower rates in some countries, such as belgium; children are entitled to free dental care in several countries of northern europe; war victims are entitled to zero cost sharing in france; the unemployed and/or other underprivileged groups have access to specific programs in some countries, such as the united kingdom and the united states . The generosity index, as it is sometimes labeled, is not a simple ratio of public - to - total expenditure . In the nearly three decades spanned by the observations, most countries have liberalized access to or are partly subsidizing reproductive services (family planning, prenatal and postnatal care, abortion, etc .) Older social care services aimed at the killer diseases of the period before world war ii (tuberculosis, poliomyelitis, etc .) Have been displaced by programs aimed at a new killer acquired immunodeficiency syndrome (aids)and by a range of treatment options made possible by advancing medical technology (such as treatment of end stage renal disease and the prevention of congenital malformations). In the opposite direction, the development of thousands of new medicines has forced many governments to moderate or even slightly reduce the level of pharmaceutical benefits . In actuality, this range of trends is still ill - translated in tables 18 and 19 . However, data from these tables can be used to find signals about turning points (years during which public schemes increased or decreased benefit levels) and to generate awareness about the typically modest size of patient cost - sharing requirements in most oecd countries . In many public debates, the public effect of cost - sharing techniques health planning has long been conducted in terms of ratios, such as hospital beds per 1,000 population, with better endowed neighboring countries serving as targets . Underutilized beds contribute little to raising the health status of populations, and the differences in countries' occupancy rates are startling . The oecd health data file has thus been designed as a measure of usage in addition to a measure of actual inputs . Efforts to standardize definitions so as to include similar inpatient institutions or active medical personnel have yielded only partial results to date . Time series are more reliable than cross - sections, although this is not always the case . For example, the data on inpatient care beds in norway matches the expenditures reported in tables 3 and 4 only from 1980; earlier data seriously underreport the input into the norwegian health system . Tables 20 - 30 also differ from other classic presentations in attempting to portray an annual average (obtained from a daily census or other recording system) or a midyear position . For instance, in some countries, physicians are allowed to charge their patients for diagnostic and prescriptive advice given by phone; elsewhere, they are not the frequency of doctor consultations may be correlated with payment methods (as discussed by sandier in this issue). The trends shown in table 23 are probably more reliable than cross - country comparisons . Hospital outlays on pharmaceuticals are excluded . In some countries, medicines delivered in outpatient wards physicians in rural areas of some countries are allowed to dispense medicine, a source of minor data distortion, in japan, one of the largest medicine consumers of all oecd countries, physicians in urban areas can dispense medicine as well . The number of units of medicine reported in table 24 reflects only residual sales in pharmacies . In most countries, records also vary across the spectrum of countries with respect to renewable items for chronic treatments: should six boxes of beta - blockers prescribed for a hypertensive patient over one - half year be entered as one prescription or six? In compiling international data, one lacks the necessary details to ensure consistent treatment across countries . The literature on the impact of cultural and other factors on medical consumption in adjacent catchment areas and on the appropriateness of care in similar socioeconomic conditions has become sizable . Smaller efforts have been devoted to highlighting similarities across countries, whether tied to cultural factors or to prevailing socioeconomic incentives . Some facets of these large area variations are illustrated in tables 31 - 49 . In tables 31 - 37, mean length - of - stay is shown by discharge category using the 18 classes of the international classification of diseases (icd) adopted under the auspices of the world health organization and used by all oecd countries . However, several countries do not yet record all their hospital admissions exhaustively and systematically, as indicated in the enthoven article in this issue . In principle, the surveys on which these tables are based cover the entire nation for which data are reported . However, data for australia relates only to the most populous state (new south wales), and data for the united kingdom come from a one - tenth sample based on a british classification developed by the office of population and census . (this classification is also used in ireland .) By and large, the figures reported for the 1970s are based on the eighth revision of the icd . Use of the ninth revision spread only slowly across the spectrum of countries, so the figures for the 1980s are partly eighth revision and partly ninth revision . This factor is even more important when considering the three - digit entries in tables 38 - 44 . In some countries, the entries for mental disorders in table 31 are the least comparable, and this affects the overall total . The occasional reallocation of services inside a nation's hospital structure influences total length - of - stay; this is the case with sweden beginning in 1984 . The data for switzerland are for a hospital federation covering more than four - fifths of all beds . The data for austria and germany are only for patients belonging to the largest health insurance schemes . The average length - of - stay figures for new zealand are weighted for private and public institutions data on all staff employed by hospitals (table 45) are not completely comparable because the extent of subcontracting differs among countries although there are persistent conceptual differences, considerable efforts are brought to bear to reduce the comparability gap that occurs when national series are simply collated . By and large, the trends are consistent, and no amount of statistical massaging would crush the important intercountry variations observed . Although some progress has been achieved, oecd countries do not monitor the outputs of their health systems satisfactorily because appropriate indicators are few . International organizations have limited their collection of data to a small set of standard indicators . The data on life expectancy at birth and at various ages (tables 50 - 57) and infant and perinatal mortality rates (tables 58 and 59) originate from national yearbooks and are sometimes supplemented by demographic journals . Mortality rates (table 60) are supplied from the annual oecd publication labour force statistics . Part of the progress in a longer life span in the 1950s and 1960s is attributable to a sharp drop in infant and perinatal mortality . New diagnostic techniques to cope with high - risk pregnancies and the advent of sophisticated gynecology - obstretrical centers to cope with difficult deliveries have been essential factors in accelerating the downward trend in perinatal mortality and, by limiting complications at birth, have contributed to the improved well - being of both mothers and babies . The death rates for infectious diseases have dropped in oecd countries by about nine - tenths in the past half - century . However, in macro - policy terms, these measures are usually held to be of limited value . They may reflect advances in medical knowledge and the diffusion of certain medical procedures, but they do not lend themselves to the measurement of the outcome of public health programs and policy . International comparisons require ratios or the use of a common numeraire to deal with differences in population sizes and currency units . In this data compendium, tables 1 - 9 and selected other entries are supplied in their original national currency expression and without reduction for population sizes . The required denominators or multipliers are published, though not always at hand when needed for analytical purposes . The principal source for these data is volume 1 of national accounts 1960 - 1987, published by oecd in 1989 . This source was occasionally supplemented by the underlying corresponding national publications . The oecd national accounts and accompanying demographic data reflect guidelines that have been gradually established by the statistics profession in a harmonization process started in the early 1950s . Differences observed in, for example, hospital beds per 1,000 population or pharmaceutical outlays per person when using national sources or the oecd data are not necessarily attributable to the numerator but may be caused by the denominator . Although an effort has been made to massage the numerator entries, the harmonization process of working from details to publish the most comparable aggregates is still in its infancy, whereas the body of principles on which the denominators are based is considerable . International agencies have opted to publish gross domestic product (gdp) estimates, not gross national product (gnp) or gross national expenditure (gne) estimates; the difference net factor income from abroad is typically not very large and is not the reason why oecd gdp and domestic gnp figures are often at great variance . Canada, the united kingdom, and the united states, to mention three large countries, adopted estimation methods in the 1940s which have not been fully incorporated in the standardized national accounts of the united nations, the oecd, and (with some specific developments) the statistical office of the european community . The standardized approach is reported here, the data being calculated by the concerned national statistical offices and meeting with their official approval . The implicit gdp price indices, shown as 1980 = 100, are originally established in each country's own base year prices, then rebased at a subaggregate level by the oecd secretariat whenever the base was not 1980 . The subperiods are linked by a chain index . Typically, past international comparisons converted national currencies at exchange rates (table 66). The vagaries of foreign exchange markets lead to grievous underestimates or overestimates of the variables analyzed . Purchasing power parities are presented in table 67 . Indices representing the average prices in each country relative to the average international prices to purchase the same market basket of goods and services were calculated for most oecd countries for 1980 and 1985 . Partial estimates also existed for 1950, 1955, and 1970 . From these base years, and with national accounting details, the series has been retropolated and extended beyond 1985.
Double male syndrome is a rare sex chromosome aneuploidy condition characterized by presence of two extra x and y chromosomes with an incidence of 1:18,000 - 1:40,000 male births . This syndrome is clinically manifested later in life with developmental delays, learning disabilities, behavioral problems, and delayed or incomplete puberty . Many cases go undiagnosed as most of the above mentioned abnormalities do not develop until early puberty . The term taurodontism was coined by sir arthur kein to describe the bull - like appearance of the teeth . Taurodontism can be defined as a morphological variation of a tooth in which the pulp chamber is vertically elongated and the roots are reduced in size . The etiology of taurodontism is attributed to the failure of hertwig epithelial root sheath diaphragm to invaginate at the proper horizontal level . Based on the severity, taurodontic teeth can be classified ashypo-, meso-, and hypertaurodont forms . Hypotaurodont expresses the least pronounced changes, while mesotaurodont expresses moderate variation, and hypertaurodont is characterized by the most severe variation in which the roots bifurcate or trifurcate close to the apices . This is the first reported incidence of the endodontic management of a hypertaurodontic mandibular molar tooth in a patient diagnosed with 48, xxyy syndrome . A 19-year - old male reported to the postgraduate department of endodontics of our dental school, for management of multiple carious teeth . During history taking, it was elicited that the patient was a school dropout due to difficulty in coping with the educational curriculum . It was also evident, that the patient had below average communication skills with low self - esteem . (b and c) postsurgical photograph after bilateral sagittal split osteotomy and genioplasty intraoral examination revealed dental caries in relation to teeth 15, 37, and 47 and multiple missing teeth (14, 13, 22, 23, and 36). An orthopantomograph revealed multiple taurodontic teeth in relation to 37, 46, 47, 17, 26, and 27 [figure 2a]. The taurodontic teeth exhibited large pulp chamber extending beyond the cervical area and reaching the furcation suggestive of hypertaurodontism . The tooth was nonsensitive to percussion, and radiographic interpretation was suggestive of asymptomatic apical periodontitis . (c) a 2-year follow - up post endodontic restoration in 37 the complete endodontic management of the case was performed by one of the author, while the other author took care of post - endodontic restoration and clinical follow - up with department of oral surgery . The patient was anesthetized with 1.7 ml of 4% articaine with 1:100,000 epinephrine (septocaine, septodont, new castle, de, usa) and access opening was performed under rubber dam isolation . Endodontic treatment in taurodontic teeth has been described as difficult as the complex morphology could hamper the location of the root canal orifices . The access opening and canal tracing was performed with the help of adental operating microscope (g6, global microscope, usa). Careful inspection and exploration revealed a large coronal pulp chamber extending 8 - 10 mm below the cervical margin (cementoenamel junction (cej)). The presence of two canals (mesial and distal) bifurcating 3 - 4 mm short of the radiographic apex was identified . An electronic apex locator (root zx, morita, tokyo japan) was used to determine the working length . The shaping and cleaning of all canals was done using nickel - titanium rotary instruments, k3 (sybron endo, orange, ca, usa). Irrigation was done with 5.2% sodium hypochlorite solution (naocl) during shaping and cleaning of the root canal system . Passive ultrasonic irrigation (pui) activation was employed using #20/0.00 taper ss noncutting ultrasonic tip (irrisafe, satelec, acteon, merignae, france) followed by 1 min flush of 17% ethylenediaminetetraacetic acid (edta; smearclear, sybronendo, ca, usa). The canals were dried using sterile paper points and dressed with intracanal calcium hydroxide medicament and the tooth was sealed temporarily with an intermediate restorative material (cavitg, 3m - espe, seefeld, germany). The patient was asked to return after 2 weeks . At the second appointment, copious irrigation was done in order to remove the calcium hydroxide and the canals were made dry with sterile paper points . Following radiographic control of the fit of gutta - percha mastercone (guttapercha, sybronendo, ca, usa), the apical part of the main root canals was obturated with vertically compacted warm gutta - percha using the touch&heat 5004 device (sybron endo, ca, usa). The coronal portion of the main canal was backfilled with thermoplasticized gutta - percha using the obtura iigun (obtura - spartan corp, fenton, mo, usa). The access cavity was sealed with dualcuredcomposite resin (paracore, coltene / whaledent, nj, usa). The patient was then referred to the department of oral surgery for surgical correction of prognathic mandible . The first stage involved a bilateral sagittal split osteotomy followed after 3 months by a second stage surgical advancement genioplasty procedure . Follow - up was at 6-months and 2-year intervals [ure 1b and c]. A normal individual has 22 pairs of autosomes (numbered chromosomes) and one pair of autosomes and a single x and y chromosome, making a karyotype or chromosomal makeup of 46, xy . Similarly, a normal female has 22 pairs of autosomes and two x chromosomes, making a karyotype 46, xx . 48, xxyy syndrome is characterized by an extra x chromosome and an extra y chromosome . This, in addition to the 22 pairs of autosomes, males with 48, xxyy syndrome has four sex chromosomes, making a karyotype 48, xxyy . Successive meiotic nondisjuctional events during spermatogenesis, resulting in a xyy sperm have been attributed as the most probable etiological mechanism of 48, xxyy syndrome . The presence of extra sex chromosomes may lead to altered deoxyribonucleic acid (dna) methylation at various loci in genome leading to altered gene expression and subsequent phenotype variation . According to gardner and girgis, a patient with multiple taurodontic teeth without known association of any systemic syndrome should be consulted for chromosomal analysis, as there is a higher association of taurodontic teeth with x - chromosome aneuploidy syndromes . Hence, we referred our patient to the department of human genetics for chromosomal analysis . Chromosome analysis with gtg banding with a resolution of 450 - 550 bp, showed a numerical abnormality in all cells analyzed, with the presence of an extra x and an extra y chromosome resulting in 48 chromosomes and a diagnosis of 48, xxyy syndrome was made [figure 3]. Chromosome analysis with gtg banding showing numerical abnormality in all cells with presence of extra x and y chromosome the prevalence of taurodontism ranges from 2.5 to 11.3% of the human population . Taurodontism can occur either as an isolated manifestation or in association with various systemic syndromes . A literature search for relevant articles regarding endodontic management of taurodontic tooth associated with systemic syndromes was performed using ovid medline, cochrane database of systemic reviews, embase, and pubmed . Table 1 summarizes the chronological order of case reports of taurodontism associated with systemic syndromes . Chronological reports of systemic syndromes associated with taurodontism this case showing 48, xxyy karyotype is recognized as a distinct clinical and genetic entity . This syndrome was previously considered to be a variant of klinefelter syndrome (47, xxy); however, it is now considered to be a distinct phenotype with more significant cognitive and psychological impairments . The characteristic differences between these two syndromes have been well documented by tartaglia et al . Comparison of 47, xxy klienfelter syndrome and 48, xxyy syndrome (adapted from tartaglia et al ., 48, xxyy, 48, xxxy, and 49, xxxxy syndromes: not just variants of klinefelter syndrome, acta paediatr: 2011; 100(6):851 - 860) knowledge regarding 48, xxyy syndrome and its dentofacial manifestations is important for early diagnosis and long - term predictable medical and dental health management . An early recognition of such a disorder is crucial in improving the psychological as well a dental quality of life in affected patients.
It is a phospholipase with little triacylglycerol lipase activity and plays a significant role in the metabolism of highdensity lipoprotein (hdl). Decreased hdl cholesterol and phospholipid levels have been shown in mouse models overexpressing el, whereas inhibition of el activity by antibodies or gene knockout leads to an increase in hdl cholesterol and phospholipid levels . In humans, increased plasma el concentration has been associated with a less favorable lipid profile with elevated plasma triglyceride and apolipoprotein (apo) b as well as smaller lowdensity lipoprotein (ldl) particles size, and not all studies have shown an association with hdl . There are data suggesting that el might be proatherogenic . An increased level of plasma el has been associated with visceral obesity, metabolic syndrome, type 2 diabetes mellitus and inflammation . It has been shown that el is expressed in endothelial cells, macrophages and smooth muscle cells in atherosclerotic lesions of human coronary arteries, and an association between plasma el concentration and coronary artery calcification score has been reported in a large crosssectional study . El is involved in the remodeling and catabolism of hdl particles in the plasma compartment and el efficiently cleaves phospholipids in hdl and releases fatty acids . In addition to its lipase activity, strauss et al . Have shown that el also facilitates hdl particle binding and uptake as well as selective uptake of hdl cholesteryl ester in hepg2 cells by its bridging function . Modulation of hdl by el might affect the capacity of hdl to act as extracellular acceptors of free cholesterol in reverse cholesterol transport . Have shown that el modification alters chemical composition and physical properties of hdl, resulting in its decreased binding capacity to scavenger receptor class b type i (srbi) and a diminished ability to mediate srbidependent cholesterol efflux . In contrast, qiu et al . Have reported that el promotes apolipoprotein (apo) a1mediated cholesterol efflux in thp1 macrophages . Overexpression of el in human apoa1 transgenic mice led to a large reduction in hdl cholesterol and in serum phospholipids / apoa1 ratio . As a result, the efflux potential of serum through srbi decreased by 90% and adenosine triphosphate (atp)binding cassette transporter a1 (abca1)mediated efflux increased by 63% . Atpbinding cassette transporter a1 mainly mediates cholesterol efflux to apoa1 and prehdl, whereas srbi mainly mediates cholesterol efflux to mature hdl . Because serum el concentration is increased in patients with type 2 diabetes, we have investigated whether increased el is associated with changes in the serum capacity to induce cholesterol efflux and cardiovascular risk in diabetic patients . Cardiovascular risk was assessed noninvasively by measuring arterial stiffness, which provides useful information on vascular health and serves as a surrogate for cardiovascular morbidity and mortality risk . Patients with type 2 diabetes mellitus according to world health organization criteria were recruited from the diabetes clinics at queen mary hospital . Because we have previously shown that cellular cholesterol efflux to serum was impaired in diabetic patients with incipient or overt nephropathy, only diabetic patients with normoalbuminuria (urinary albumin excretion rate <30 mg / day) were recruited for the present study to avoid the potential confounding effect of nephropathy . Patients on insulin therapy or lipid lowering agents, or had a history of cardiovascular disease were also excluded . All subjects must have had stable glycaemic control with no change in antidiabetic therapy for the preceding 3 months . Fasting blood samples were taken for the measurement of glucose, glycated hemoglobin (hba1c), lipids, high sensitivity creactive protein (crp), cholesterol efflux and el . Arterial stiffness was assessed noninvasively by measuring aortic pulse wave velocity (pwv) using sphygmocor vx pwv system (version 7.0; atcor medical, sydney, australia). Pulse wave velocity was determined by measuring the velocity of the blood pressure waveform between the carotid and femoral artery sites using a singlelead electrocardiogram and a tonometer to measure the pressure pulse waveform sequentially in the two artery sites . The present study was approved by the ethics committee of the university of hong kong and informed consent was obtained from all subjects . Plasma total cholesterol and triglyceride were determined enzymatically on a hitachi 912 analyzer (roche diagnostics, mannheim, germany). Ldl cholesterol was calculated by the friedewald equation or measured directly if plasma triglyceride was> 4.5 plasma apoa1 and apob were measured by rate nephelometry using the beckman array system (beckman instruments, fullerton, ca, usa). Hba1c was measured in whole blood using ionexchange high performance liquid chromatography with the biorad variant haemoglobin testing system (biorad laboratories, hercules, ca, usa). Plasma high sensitivity crp was measured by a particleenhanced immunoturbidimetric assay (roche diagnostics) using anticrp mouse monoclonal antibodies coupled to latex microparticles . Serum el was measured by competitive elisa using a rabbit polycloncal antibody specific to human el (novus biologicals, littleton, co, usa) as described . The immunogen used for raising the antibody was an nterminal synthetic peptide made to the human el protein sequence . The antiel antibody was highly specific and there was no crossreactivity with hepatic lipase or lipoprotein lipase . Briefly, a 96well eia microtiter plate (costar, corning, ny, usa) was coated with antigen, the nterminal synthetic peptide of the human endothelial lipase (novus biologicals) in coating buffer overnight at 4c . The wells were then washed extensively five times and incubated with blocking reagent at room temperature for 2 h. after rinsing five times, equal volume of onequarter diluted serum samples plus antiel antibody (novus biologicals) was added . After another five washes, horseradish peroxidaseconjugated goatantirabbit secondary antibody was added and incubated for 1 h. the plate was finally measured at 450 nm by an elisa reader . Fu5ah rat hepatoma cells (a generous gift from dr gh rothblat) were used to measure the capacity of serum to induce cholesterol efflux by srbi and abca1 . Fu5ah rat hepatoma cells have a high expression level of srbi, but they lack functional atpbinding cassette transporters . We have previously shown that abca1 expression can be specifically induced in fu5ah cells by stimulating the cells with 22(r)hydroxycholesterol (22rhc)/9cisretinoic acid (9cra) and can be used to assay abca1mediated cholesterol efflux, and both srbi and abca1 expression was not affected by incubation with serum . The magnitude of abca1mediated cholesterol efflux in our assay is similar to the magnitude of abca1mediated cholesterol efflux reported in efflux assays using raw264.7 cells stimulated with cyclic adenosine monophosphate (camp) to induce abca1 expression . To measure cellular cholesterol efflux to serum, fu5ah cells were cultured in minimal essential medium (mem) containing 5% calf serum and labeled with [h]cholesterol for 48 h (1 ci / well; ge healthcare biosciences, piscataway, nj, usa). Abca1mediated cholesterol efflux was measured using [h]cholesterollabeled fu5ah cells treated with 22rhc (5 g / ml) and 9cra (10 5% diluted serum in mem was added and incubated at 37c for 4 h. radioactivity was measured in both medium and cells, and the radioactivity released to the medium was expressed as the fraction of the total radioactive cholesterol present in the well . Cholesterol efflux mediated by srbi, expressed as percent, was calculated as the amount of label recovered in the medium divided by the total label in each well in untreated cells . Cholesterol efflux mediated by abca1 was calculated as fractional cholesterol efflux from 22rhc/9cratreated cells minus fractional cholesterol efflux from untreated control cells . The interassay coefficient of variations for srbi mediated and abca1mediated cholesterol efflux were 8.0% and 4.5%, respectively . Results are expressed as means sd or as median and interquartile range if the data were not normally distributed . Comparisons between two different groups were carried out using an independent samples ttest, and skewed data were logarithmically transformed before analysis . Multiple linear regression analysis was used to simultaneously assess the relationship between el and various variables . Age, sex and smoking status were not significantly different between the two groups, but body mass index (bmi) and waist circumference were significantly higher in the diabetic patients than controls . In the present study, 32 patients were taking metformin, 23 were taking sulphonylurea and the rest were taking a combination of metformin and sulphonylurea . Fasting glucose, hba1c and crp were significantly higher in the diabetic patients than the controls . * p <0.05, * * p <0.01 vs controls . Bmi, body mass index; bp, blood pressure; crp, creactive protein; dm, diabetes mellitus; hba1c, glycated hemoglobin; pwv, pulse wave velocity . Plasma hdl cholesterol and apoa1 were reduced and triglyceride was higher in the diabetic group . Srbi mediated cholesterol efflux to serum was impaired in the diabetic patients, whereas no significant difference in abca1 mediated cholesterol efflux to serum was found . Because plasma hdl levels differed significantly between diabetic patients and controls, data were also analyzed after adjusting for hdl cholesterol level, and srbi mediated cholesterol efflux to serum remained impaired in the diabetic patients (p <0.01). There was no significant correlation between serum el and hdl in controls (r = 0.08) or diabetic patients (r = 0.05), whilst there was a weak trend between serum el and logcrp (controls r = 0.13, p = 0.08; diabetics r = 0.14, p = 0.07). No association between serum el and pwv was observed in either the diabetic patients or controls . In the diabetic patients, pwv correlated with logcrp (r = 0.20, p <0.05) and srbi mediated cholesterol efflux (r = 0.17, p <0.05) in addition to its strong correlation with systolic bp (r = 0.54, p <0.01). Neither the association between pwv and logcrp nor srbi mediated cholesterol efflux remained significant after adjusting for blood pressure . * p <0.05, * * p <0.01 vs controls . Abca1, adenosine triphosphatebinding cassette transporter a1; apoa1, apolipoprotein a1; apob, apolipoprotein b; dm, diabetes mellitus el, endothelial lipase; hdl, highdensity lipoprotein; ldl, lowdensity lipoprotein; srbi, scavenger receptor class b type i. univariate analysis was carried out to investigate which clinical parameters were associated with cholesterol efflux . In the control subjects, serum el correlated inversely with srbi mediated cholesterol efflux (figure 1a) but not with abca1 mediated cholesterol efflux . Srbi mediated cholesterol efflux to serum also correlated with hdl (r = 0.59, p <0.001), apoai (r = 0.60, p <0.001), waist circumference (r = 0.16, p <0.05), but not with logcrp . However, in the diabetic patients, there was only a trend towards an association between serum el and srbi mediated cholesterol efflux (figure 1b) and there was no correlation with abca1 mediated cholesterol efflux . In the diabetic cohort, srbi mediated cholesterol efflux to serum correlated with hdl (r = 0.62, p <0.001), apoai (r = 0.51, p <0.001), waist circumference (r = 0.19, p <0.05) and logcrp (r = 0.32, p <0.001), whereas abca1 mediated cholesterol efflux to serum correlated only with hdl (r = 0.23, p <0.01). Correlation between serum endothelial lipase and scavenger receptor class b type i mediated cholesterol efflux to serum in (a) controls and (b) diabetic patients . Srbi, scavenger receptor class b type i. to determine whether serum el was an independent determinant of srbi mediated cholesterol efflux to serum, multiple linear regression analysis was carried out including variables that had significant associations . In controls, age, sex, waist circumference, smoking status, hdl and serum el were entered into the model, and the results are shown in table 3 . Plasma hdl, serum el and waist circumference were the independent determinants of srbi mediated cholesterol efflux to serum in controls, accounting for 24%, 10% and 2% of the variability of cholesterol efflux respectively . In contrast, the major independent determinants of srbi mediated cholesterol efflux in the diabetic patients were hdl, logcrp and age (table 4), accounting for 26%, 6% and 2% of the variability of cholesterol efflux, respectively . Forcing serum el and/or hba1c into the model did not change the results . Efflux of free cholesterol from cells is an early step of reverse cholesterol transport and the efficiency of cellular cholesterol efflux is influenced by the concentrations of lipoproteins that act as cholesterol acceptors and the activities of serum proteins, such as lipases and lipid transfer proteins involved in the remodeling of lipoproteins . The capacity of whole serum or plasma to stimulate cholesterol efflux from cells has been used by a number of laboratories as a means to investigate reverse cholesterol transport, as whole serum provides integrated information on lipoproteins and serum components involved in promoting cholesterol efflux from cells . The capacity of serum to induce cholesterol efflux has been shown to be an independent predictor of coronary artery atherosclerosis in clinical studies . Serum capacity to induce srbimediated cellular cholesterol efflux is reduced in type 2 diabetic patients with or without diabetic complications and abca1mediated cholesterol efflux is also impaired in patients with nephropathy . Similar to previous studies, we have shown that serum capacity to induce srbimediated cellular cholesterol efflux is decreased in diabetic patients, whereas no significant reduction was seen in serum capacity to induce abca1mediated cholesterol efflux . In the present study, we have determined whether serum capacity to induce cellular cholesterol efflux was related to serum el level . As expected, plasma hdl level was the major determinant of serum capacity to induce cellular cholesterol efflux, be it mediated by srbi or abca1 . In healthy control subjects, there was a weak but significant inverse correlation between el and serum capacity to induce srbimediated cellular cholesterol efflux . This is consistent with animal studies showing that increased el was associated with a decrease in efflux potential of serum through srb1 . El can reduce srbidependent cholesterol efflux partly by altering the chemical composition and physical properties of hdl . El has both catalytic and noncatalytic functions and can mediate binding of lipoproteins independent of its enzymatic activity . Because the association between el and serum capacity to induce srbimediated cellular cholesterol efflux remains independent of hdl on analysis, whether this might be related to the noncatalytic function of el warrants further investigation . In contrast to the findings in healthy controls, only a trend towards an association between serum el and serum capacity to induce cellular cholesterol efflux was seen in type 2 diabetic patients . In these patients, crp turned out to be an important determinant of serum capacity to induce srbimediated cellular cholesterol efflux . Inflammation has a major effect on reverse cholesterol transport and our data would suggest that the influence of inflammation on serum capacity to induce srbimediated cellular cholesterol efflux might override that of el in diabetic subjects . Chronic subclinical inflammation has been shown in patients with type 2 diabetes mellitus . Although inflammation has been shown to activate el, this is unlikely to be the main mechanism whereby inflammation impairs serum capacity to induce cholesterol efflux in our diabetic patients, because we did not find any significant correlation between el and cholesterol efflux . Inflammation can influence serum capacity to induce cholesterol efflux by a number of mechanisms independent of el . Inflammation also activates secretory phospholipase a2 and inflammatory remodeling of hdl impairs its capacity to serve as cholesterol acceptor ex vivo and in vivo . In addition, inflammation also affects serum proteins that are involved in promoting cholesterol efflux from cells . In human subjects, attenuation of lecithin cholesterol acyltransferase and cholesterol ester transfer protein activity has been reported during infection and inflammation . Taken together, subclinical inflammation might play a role in reducing serum capacity to induce cellular cholesterol efflux in type 2 diabetes mellitus . However, the present study was limited by its crosssectional nature, and we could only show an association and not a causal relationship . Because el might be proatherogenic and plasma el has been associated with coronary artery calcification score in healthy individuals with a family history of premature coronary heart disease, we have also determined in the present study whether plasma el is correlated with arterial stiffness in diabetic patients . Vascular stiffness reflects both functional and structural changes in the artery wall that precede and accompany atherosclerosis . Pulse wave velocity reflects arterial stiffness and is regarded as a surrogate marker of severity of atherosclerosis . Experimental studies have shown that aortic pwv increases with the development of atherosclerosis in primates and there is a strong association between aortic pwv with intimamedia thickness and severity of plaques evaluated by ultrasonographic images . We have shown that arterial stiffness is increased in diabetic patients without overt cardiovascular disease, but we did not find an association between el and pwv in our diabetic patients . In conclusion, in patients with type 2 diabetes, serum el concentration was increased, but impaired serum capacity to induce cholesterol efflux in these patients was mainly related to low hdl and subclinical inflammation.
Conserved domains in proteins have crucial roles in protein interactions, dna binding, enzyme activity and other important cellular processes . With recently released predictions of the number of genes in the human genome being less than many previous predictions, interactions among protein domains protein domains are often conserved across many species and, as such, they offer an interesting dataset for analyzing how genomes maintain any given domain in relation to other conserved domains, as well as for analyzing the relationship of conserved domain occurrence to proteome size . Many groups have attempted to find, document and annotate these conserved domains . Whereas most groups use a form of hidden markov models for profiling, each group approaches the problem in a unique way, yielding a wide range of databases that can be used to verify each other . For this study i used the smart cd database to collect data on the number of genes containing each conserved domain in each genome . The study was restricted to the five eukaryote genomes sequenced so far: homo sapiens, drosophila melanogaster, arabidopsis thaliana, caenorhabditis elegans and saccharomyces cerevisiae . Results were confirmed using a repository of databases called the proteome analysis database (abbreviated here as pad). It has been possible to compare conserved domains across different genomes, and to validate the approach by using a repository of databases (pad) and one database from this group (smart). A close link is revealed between numbers of genes with a given conserved domain and the total number of genes in each genome . Data were gathered as follows: a perl script was written to submit requests to the smart database for the number of genes with each of 519 conserved domains in each genome . Information in pad is already in genome - specific columns for the 200 most frequent conserved domains in humans and was downloaded directly . The information was parsed and stored for each genome . From the smart database, 211 conserved domains were selected on the basis of the fact that they occurred at least once in each of the five genomes (see additional data files). From pad, 122 conserved domains were selected on the basis of the fact that they occurred at least once in each of the five genomes (see additional data files). My initial observation was that for many conserved domains, the ratio of the sum of genes in genome 1 containing the conserved domain to the total number of predicted genes in genome 1 was proportional to the ratio of the sum of genes in genome 2 containing the conserved domain to the total number of predicted genes in genome 2 . Given that: a = sum of proteins with given conserved domain (cd) in genome 1; b= sum of proteins with given cd in genome 2; e= sum of predicted genes in genome 1; f= sum of predicted genes in genome 2, then on average: upon rearranging equation 1, it was noted that for many conserved domains the ratio of the number of genes containing the given conserved domain in each genome accurately reflected the ratio of the total predicted number of genes ach genome . Or, given the variables in equation 1, then on average: to normalize the data i used a ratio of the sum genes with a given conserved domain in a genome to the sum genes with the given conserved domain in all five genomes . This was used to minimize the effect that the predicted number of genes may be significantly wrong for one of the genomes whereas the others may be more accurate . Equation 1 was rewritten to reflect this normalization . Given that a= sum proteins with given cd in genome 1; g= sum proteins with given cd in five genomes; e = sum predicted genes in genome 1; h= sum predicted genes for all five genomes, then on average: the sums of conserved domains in each equation 3 ratio range were depicted graphically for each genome, and are displayed in figure 1 (smart database) and figure 2 (pad). The average ratio for each genome was calculated and multiplied against the sum predicted genes of all five genomes, yielding a number close to the number of predicted genes in each respective genome (table 1). Equation 2 could be used to predict total genes in a genome given that the other variables are reasonably well known, such as from expressed sequence tag (est) data . More important, this suggests the possibility that these conserved domains are maintained in this ratio as a result of functional constraints on interacting domains . The fact that this ratio is maintained fairly well in all five eukaryotic genomes attests to its potential importance . Although there is much disagreement on the total number of genes for the different genomes, similar gene - finding methods were used for each of the five published eukaryotic genomes . It can therefore be assumed that ratios of predicted genes between the genomes will remain similar to present ratios, as the gene numbers for each genome are clarified . Likewise, neither smart nor pad claim to have found all occurrences of each conserved domain in each genome . However, because of similar strategies used for finding conserved domains in different genomes within each database, the ratio of total genes found with a given conserved domain in each genome is likely to remain near constant as gene prediction improves . An interesting finding from this research was that while the ratios for h. sapiens, a. thaliana, and s. cerevisiae related closely to the total predicted genes for each organism, both databases gave a peak ratio that exchanged total predicted gene numbers between d. melanogaster and c. elegans (figures 1,2). From figure 2 it can be seen that it is outlying conserved domain ratios that cause the average in table 1 to be shifted closer to actual predicted total gene numbers for c. elegans . While this exchange cannot be explained at present, it may offer insights into the distinctions between the genomes, and genes that remain unidentified . It is important to note that by mainly analyzing conserved domains occurring most frequently, conserved domains that occur only once in each genome are, for the most part, excluded from the analysis . It has been shown that conserved domains in proteins are maintained in proteome - specific ratio for the five eukaryotic genomes sequenced so far . The reasons for this ratio are unclear, but it would not be unreasonable to suspect that the functional interactions of these protein domains require that they be kept in a specific ratio . Further research may reveal that conserved domains outside of this ratio are critical to the organism's unique functions, and will be necessary to understand the reasons for, and universality of this ratio in eukaryotic genomes . The smart database was searched for conserved domains occurring at least once in each of the five genomes . For pad the search was restricted to those conserved domains listed in the top 200 domains occurring in humans for which there was at least one occurrence in each of the four other genomes . This strategy of limiting the study to more global conserved domains was used to increase the chance that the conserved domains were constructed correctly and to increase the statistical reliability of the results . The total number of predicted genes for each genome was as follows: h. sapiens, 35,000; d. melanogaster, 14,100; a. thaliana, 26,000; c. elegans, 19,100; s. cerevisiae, 6,300 . This yielded a total of 100,500 genes for all five genomes, and a total of 39,500 for d. melanogaster, c. elegans, and s. cerevisiae alone . The number of genes in each genome is approximate because it is an estimate that is continually being updated . Smart_cds.txt is a text, tab - delimited file containing all 211 conserved domain names from the smart database used in this study . For each conserved domain name, pad_cds.txt is a text, tab - delimited file containing all 122 interpro entry numbers for the domains in pad used in this study . For each interpro entry number, the corresponding number of genes containing the conserved domain in each genome is listed . Smart_cds - a text, tab - delimited file containing all 211 conserved domain names from the smart database used in this study . Click here for additional data file pad_cds - a text, tab - delimited file containing all 122 interpro entry numbers for the domains in pad used in this study . I thank those at tigr who reviewed the ideas presented here and b. parvizi and j. vamathevan for help with the writing and analysis . Thank you to s. malek for critical review of the manuscript . Sum of conserved domains (cds) in each ratio range of cds in a genome (see equation 3) compared to their occurrence in all five genomes (211 cds considered). Equation 3 was used for all cds for each genome . The number of cds in each ratio range for each genome was summed and graphed . It is apparent that the number of cds peaks at a particular ratio for each genome, with an average near the respective proteome size (multiply average ratio for each genome by 100,500 as in table 1). Sum of cds in each ratio range of cds in a genome (see equation 3) compared to their occurrence in all five genomes (122 cds considered). The number of cds in each ratio range for each genome was summed and graphed . It is apparent that the number of cds peaks at a particular ratio for each genome, with an average near the respective proteome size (multiply average ratio for each genome by 100,500 as in table 1).
The postpartum stage for women is marked by a feeling of contentment and pleasure while being stressful and problematic . Various studies undertaken in fields related to this experience indicate that becoming a mother is accompanied by prominent physical, social, and psychological changes which can affect not only mother's psychological healthiness, but also all other aspects of her personal and family life . Mother's views about herself and her social and family roles take a turn in this period . In fact, she should care for the child while adapting herself to the new situation; hence, she is confronted with numerous challenges through the process of adaptation . This increase in mother's gender roles brings about a role strain that is, in fact, mother's emotional responses to her postnatal experiences . According to the role strain theory, since everyone's time and energy is limited, any rise in the number of their social roles could expose them to more role conflicts which threaten both their physical and psychological healthiness . These changes leave profound impacts upon the quality of mothers lives, especially those of first - time mothers, and make this postnatal period, in a way, unbearable to them . Mothers distress in this phase endangers both their psychological healthiness and the integration of their maternal role; it also hinders their ability to do the affairs and weakens the quality of their relationships and social engagement while giving them a sense of anxiety, incompetence, loneliness, added to a feeling of losing independence, time, appearance, and job identity . Becoming a mother for most new mothers is not the same as what they expected and used to think about it . Having heard from the society and medias that becoming a mother is an easy and fulfilling natural experience, new mothers are usually reluctant to talk about the problems they face in this regard . In spite of the extensive surveys carried out about maternity experiences in different countries, this could be due to the lack of a safe and proper outlet through which they can express their concerns in this experience . Being aware of new mothers postnatal challenging experiences and the factors and conditions that give rise to them expands our knowledge about the ways that they react in this situation, while identifying the differences and complexities of such experiences; this recognition could not be achieved without examining various populations and societies . Not much research information around maternal adaptation and the social and psychological factors that can affect it is available in iran . This paper reports a qualitative study of the maternal experiences from the perspective of first - time mothers in the current social and cultural environment of iran . In this qualitative study, one - to - one interviews with a semi - structured in - depth interview schedule were used to shed light upon the experiences of first - time mothers in their maternal role adaptation . This research project ran from january 2011 until july 2011 in tehran (the capital of iran) and ahwaz (a south - western city of iran). The study participants were a group of 21 first - time mothers whose average age was 26 years and the average duration of their married life was 3 years . The inclusion criteria were as follows: first - time mothers over 18 years of age with a full - term pregnancy and no experience of high - risk pregnancy, speaking in farsi, having a healthy child from 0 to 1 year old, no history of depression, no serious illness before or after giving birth, and good cooperation during interviews . They were also chosen from different demographic and ethnographic backgrounds . According the sampling strategy for qualitative studies, purposeful sampling was conducted with maximum variation and interviews were continued until they reached a saturation point . All the interviews were digitally recorded with permission of the interviewees and the moral committee of tehran university of medical sciences, with the assurance that all the information would be kept as confidential . The recorded interviews which were transcribed verbatim along with the notes taken during the interviews were carefully analyzed . As this step was in progress, new ideas and questions useful to this study emerged . Locations for the performance of interviews were decided by the participants and every single interview took 40 - 80 min . The analysis started with reading of the collected information to get an overall view of it . Then the parts containing the experiences of the participants were picked out inductively and gathered as a separate text which made the core of the analysis . It was then summarized and outlined based on the meaning units which were coded and condensed . The second and the third co - writers also examined all the collected codes and notes; the experimental categories which were obtained by making frequent references to the original texts were discussed and examined to reach an agreement . Finally, the basic meanings derived from the implied analysis of these categories introduced the theme . This research project ran from january 2011 until july 2011 in tehran (the capital of iran) and ahwaz (a south - western city of iran). The study participants were a group of 21 first - time mothers whose average age was 26 years and the average duration of their married life was 3 years . The inclusion criteria were as follows: first - time mothers over 18 years of age with a full - term pregnancy and no experience of high - risk pregnancy, speaking in farsi, having a healthy child from 0 to 1 year old, no history of depression, no serious illness before or after giving birth, and good cooperation during interviews . They were also chosen from different demographic and ethnographic backgrounds . According the sampling strategy for qualitative studies, purposeful sampling was conducted with maximum variation and interviews were continued until they reached a saturation point . All the interviews were digitally recorded with permission of the interviewees and the moral committee of tehran university of medical sciences, with the assurance that all the information would be kept as confidential . The recorded interviews which were transcribed verbatim along with the notes taken during the interviews were carefully analyzed . As this step was in progress, new ideas and questions useful to this study emerged . Locations for the performance of interviews were decided by the participants and every single interview took 40 - 80 min . The analysis started with reading of the collected information to get an overall view of it . Then the parts containing the experiences of the participants were picked out inductively and gathered as a separate text which made the core of the analysis . It was then summarized and outlined based on the meaning units which were coded and condensed . The second and the third co - writers also examined all the collected codes and notes; the experimental categories which were obtained by making frequent references to the original texts were discussed and examined to reach an agreement . Finally, the basic meanings derived from the implied analysis of these categories introduced the theme . The central theme extracted from the analysis of interviews with 21 first - time mothers, named internal conflicts, consisted of four categories as follows: 1 . The scrutiny of the first - time mothers experiences in this study reveals the existence of a sense of unpreparedness in them during the first days or weeks after child birth . First - time mothers came up with a change in their feelings and emotions after giving birth . They repeatedly used phrases like emergence of strange feelings or feeling a change in deeds and attitudes, and unknown or vague sense of motherhood . Confusion, disturbance, bewilderment, as well as ambivalence caused by the new condition were repeatedly pointed out in their expressions . Mothers expectations, imaginations, and dreams about child's appearance and the way of life before and after giving birth are not the same . A 24-year - old mother, for example, referring to her 5-month - old baby said: to tell the truth, i didn't really like him at first, after 9 months bearing the burden of holding a child, i expected something more than just a little thing . I asked myself: is that all? In addition to this, mother is suddenly encountered with others new expectations for which she is not prepared . They expect her to be like a typical mother and act in a way that best suits her new role . For instance, when i joke they say: you have a child now; this is not a proper thing for you to do any more . Among mothers experiences during the first weeks after giving birth, lack of control and domination over affairs is quite conspicuous . In studying their experiences, we repeatedly encountered phrases like sometimes i find it unbearable and i m hard - pressed on every side, which denote the hardship of the new situation and mother's inconvenience with it . As for the role of child temperament in giving a sense of discontentment to them, a mother with a 1.5-month - old baby sometimes i breast feed him so much that i feel like dying . These pressures on mothers give way to physical and psychological disorders which are frequently remarked in their speeches through phrases like nerve - racking, she constantly feels obligated to bear the imposed restrictions for the sake of her child . Having to stay at home and taking care of the child, changing their routines, wearing special clothing suitable for breast feeding, and ignoring their dreams and desires are all instances of restraints that pave the way for nostalgic feelings about the past . They felt nostalgic for their past desires, their previous appearance, and the images they used to hold about themselves; they missed participation in social activities and whatever else they had lost due to being a mother . A mother with a 1.5-month - old baby said: i feel like a stay - at home - mom . Sometimes i don't find time to take bath, leave alone sleeping or making myself look pretty, i crave going to the park or shopping, but when i see that i should feed the baby or change his nappy, then i say, forget it; it's better to stay at home . Fear of losing the baby and anxiety about his / her health, which seem to be a never - ending worry for first - time mothers, and a sense of incompetence that reveals itself with diffidence and inhibition enhance this lack of control over affairs . A mother of a 7-month - old baby said: at first it was so difficult, i was inexperienced and always got flustered . In the very early days under such circumstances, more than any other time, she feels troubled by finding herself alone and having no time to care for both the baby and herself; she expects to receive informational help and practical support (especially from her mother) as well as emotional support (especially from her husband). I think being alone and having no one to help make everything difficult to me, it's really difficult . The experiences of the participants reveal the fact that mothers in the first days after child birth, in spite of what they have seen or heard about strong maternal feelings about the newborn, have no feeling toward the baby and they involve themselves more with the changes and the new condition than expressing their maternal affections to the child . As a result of these imperfect or undeveloped maternal feelings, they feel sinful and force themselves to exhibit maternal feelings and behaviors . One of the participants with a 7-month - old child said: everybody said that it feels really good to become a mother, but i didn't have a good feeling . I haven't really said this to anybody so far, but first, i didn't like him that much . Perhaps because i was very annoyed . At first i breastfed him as a duty and i didn't like to do it, if it was possible i would give him formula milk . The pressure aroused by the changes and the new condition in the first days or weeks after child birth deviates mother's concentration and gives way to a kind of instability in her relations with her husband and the society . Mother is conscious and worried about the fragility of physical and emotional relations with her husband . She is constantly faced with his complaints in this regard and consciously attempts for the betterment of their shaking marital relations . I used to care a lot for my husband, but now i can't, perhaps because i m very busy, i have less patience for my husband . He is always grumbling at me . In addition to this, detachment from normal social relations (paying visits to family and friends) brings about a sense of isolation which intensifies her conflicts . The central theme extracted from the analysis of interviews with 21 first - time mothers, named internal conflicts, consisted of four categories as follows: 1 . The scrutiny of the first - time mothers experiences in this study reveals the existence of a sense of unpreparedness in them during the first days or weeks after child birth . First - time mothers came up with a change in their feelings and emotions after giving birth . They repeatedly used phrases like emergence of strange feelings or feeling a change in deeds and attitudes, and unknown or vague sense of motherhood . Confusion, disturbance, bewilderment, as well as ambivalence caused by the new condition were repeatedly pointed out in their expressions . Mothers expectations, imaginations, and dreams about child's appearance and the way of life before and after giving birth are not the same . A 24-year - old mother, for example, referring to her 5-month - old baby said: to tell the truth, i didn't really like him at first, after 9 months bearing the burden of holding a child, i expected something more than just a little thing . I asked myself: is that all? In addition to this, mother is suddenly encountered with others new expectations for which she is not prepared . They expect her to be like a typical mother and act in a way that best suits her new role . For instance, when i joke they say: you have a child now; this is not a proper thing for you to do any more . Among mothers experiences during the first weeks after giving birth, lack of control and domination over affairs is quite conspicuous . In studying their experiences, we repeatedly encountered phrases like sometimes i find it unbearable and i m hard - pressed on every side, which denote the hardship of the new situation and mother's inconvenience with it . As for the role of child temperament in giving a sense of discontentment to them, a mother with a 1.5-month - old baby sometimes i breast feed him so much that i feel like dying . These pressures on mothers give way to physical and psychological disorders which are frequently remarked in their speeches through phrases like nerve - racking, she constantly feels obligated to bear the imposed restrictions for the sake of her child . Having to stay at home and taking care of the child, changing their routines, wearing special clothing suitable for breast feeding, and ignoring their dreams and desires are all instances of restraints that pave the way for nostalgic feelings about the past . They felt nostalgic for their past desires, their previous appearance, and the images they used to hold about themselves; they missed participation in social activities and whatever else they had lost due to being a mother . A mother with a 1.5-month - old baby said: i feel like a stay - at home - mom . Sometimes i don't find time to take bath, leave alone sleeping or making myself look pretty, i crave going to the park or shopping, but when i see that i should feed the baby or change his nappy, then i say, forget it; it's better to stay at home . Fear of losing the baby and anxiety about his / her health, which seem to be a never - ending worry for first - time mothers, and a sense of incompetence that reveals itself with diffidence and inhibition enhance this lack of control over affairs . A mother of a 7-month - old baby said: at first it was so difficult, i was inexperienced and always got flustered . In the very early days under such circumstances, more than any other time, she feels troubled by finding herself alone and having no time to care for both the baby and herself; she expects to receive informational help and practical support (especially from her mother) as well as emotional support (especially from her husband). I think being alone and having no one to help make everything difficult to me, it's really difficult . The experiences of the participants reveal the fact that mothers in the first days after child birth, in spite of what they have seen or heard about strong maternal feelings about the newborn, have no feeling toward the baby and they involve themselves more with the changes and the new condition than expressing their maternal affections to the child . As a result of these imperfect or undeveloped maternal feelings, they feel sinful and force themselves to exhibit maternal feelings and behaviors . One of the participants with a 7-month - old child said: everybody said that it feels really good to become a mother, but i didn't have a good feeling . I haven't really said this to anybody so far, but first, i didn't like him that much . Perhaps because i was very annoyed . At first i breastfed him as a duty and i didn't like to do it, if it was possible i would give him formula milk . The pressure aroused by the changes and the new condition in the first days or weeks after child birth deviates mother's concentration and gives way to a kind of instability in her relations with her husband and the society . Mother is conscious and worried about the fragility of physical and emotional relations with her husband . She is constantly faced with his complaints in this regard and consciously attempts for the betterment of their shaking marital relations . I used to care a lot for my husband, but now i can't, perhaps because i m very busy, i have less patience for my husband . He is always grumbling at me . In addition to this, detachment from normal social relations (paying visits to family and friends) brings about a sense of isolation which intensifies her conflicts . The scrutiny of the first - time mothers experiences in this study reveals the existence of a sense of unpreparedness in them during the first days or weeks after child birth . First - time mothers came up with a change in their feelings and emotions after giving birth . They repeatedly used phrases like emergence of strange feelings or feeling a change in deeds and attitudes, and unknown or vague sense of motherhood . Confusion, disturbance, bewilderment, as well as ambivalence caused by the new condition were repeatedly pointed out in their expressions . Mothers expectations, imaginations, and dreams about child's appearance and the way of life before and after giving birth are not the same . A 24-year - old mother, for example, referring to her 5-month - old baby said: to tell the truth, i didn't really like him at first, after 9 months bearing the burden of holding a child, i expected something more than just a little thing . I asked myself: is that all? In addition to this, mother is suddenly encountered with others new expectations for which she is not prepared . They expect her to be like a typical mother and act in a way that best suits her new role . For instance, when i joke they say: you have a child now; this is not a proper thing for you to do any more . Among mothers experiences during the first weeks after giving birth, lack of control and domination over affairs is quite conspicuous . In studying their experiences sometimes i find it unbearable and i m hard - pressed on every side, which denote the hardship of the new situation and mother's inconvenience with it . As for the role of child temperament in giving a sense of discontentment to them, a mother with a 1.5-month - old baby these pressures on mothers give way to physical and psychological disorders which are frequently remarked in their speeches through phrases like nerve - racking, she constantly feels obligated to bear the imposed restrictions for the sake of her child . Having to stay at home and taking care of the child, changing their routines, wearing special clothing suitable for breast feeding, and ignoring their dreams and desires are all instances of restraints that pave the way for nostalgic feelings about the past . They felt nostalgic for their past desires, their previous appearance, and the images they used to hold about themselves; they missed participation in social activities and whatever else they had lost due to being a mother . A mother with a 1.5-month - old baby sometimes i don't find time to take bath, leave alone sleeping or making myself look pretty, i crave going to the park or shopping, but when i see that i should feed the baby or change his nappy, then i say, forget it; it's better to stay at home . Fear of losing the baby and anxiety about his / her health, which seem to be a never - ending worry for first - time mothers, and a sense of incompetence that reveals itself with diffidence and inhibition enhance this lack of control over affairs . A mother of a 7-month - old baby said: at first it was so difficult, i was inexperienced and always got flustered . In the very early days under such circumstances, more than any other time, she feels troubled by finding herself alone and having no time to care for both the baby and herself; she expects to receive informational help and practical support (especially from her mother) as well as emotional support (especially from her husband). I think being alone and having no one to help make everything difficult to me, it's really difficult . The experiences of the participants reveal the fact that mothers in the first days after child birth, in spite of what they have seen or heard about strong maternal feelings about the newborn, have no feeling toward the baby and they involve themselves more with the changes and the new condition than expressing their maternal affections to the child . As a result of these imperfect or undeveloped maternal feelings, they feel sinful and force themselves to exhibit maternal feelings and behaviors . One of the participants with a 7-month - old child said: everybody said that it feels really good to become a mother, but i didn't have a good feeling . I haven't really said this to anybody so far, but first, i didn't like him that much . Perhaps because i was very annoyed . At first i breastfed him as a duty and i didn't like to do it, if it was possible i would give him formula milk . The pressure aroused by the changes and the new condition in the first days or weeks after child birth deviates mother's concentration and gives way to a kind of instability in her relations with her husband and the society . Mother is conscious and worried about the fragility of physical and emotional relations with her husband . She is constantly faced with his complaints in this regard and consciously attempts for the betterment of their shaking marital relations . I used to care a lot for my husband, but now i can't, perhaps because i m very busy, i have less patience for my husband . He is always grumbling at me . In addition to this, detachment from normal social relations (paying visits to family and friends) brings about a sense of isolation which intensifies her conflicts . As revealed in this research, the first postnatal days, weeks, and months for iranian new mothers are associated with a great deal of stress and conflicts . These conflicts, which are in turn the outcome of a collection of issues, diminish mother's capacity for adapting to her new role while introducing us with the aspects that should be discerned and attended to so as to improve and increase mothers psychological healthiness in this stage . According to the findings of this research, both mothers and others do not have the essential information and perception into the situation; neither do they have a proper mental background about mothers state in this transition . In iran, as in many other countries, the training and supervision given during pregnancy and after delivery are generally centered on physical condition of mothers rather than their psychological or mental health condition . The critical role of these training sessions during pregnancy is generally limited to unregistered training concerning nutrition, personal health, etc . Therefore, despite the promotion of the level and amount of training given to mothers in recent years, neither midwives nor mothers and families have proper information and a sound mental image about becoming a mother and the changes and problems associated with that . Lack of proper training about what really happens after childbirth, and the variety and contradiction that exists in the tips and instructions given by professional advisors and common people around that could be confusing for new mothers . Neither the family nor the society grants her proper time and opportunity to adapt to her maternal role, and they expect her to perform in a way she is not prepared for . Consequently, she finds her real postnatal experience contrary to the happy scenario she used to imagine . This state of unpreparedness is not only evident among iranian new mothers, but also reported in some other studies performed in this regard . Raynor (2006) has acknowledged the existence of a vast range of contradictory and opposing feelings and thoughts that appear after giving birth . Wilkins (2006) and miller (2011) have also revealed in their studies that mothers unfulfilled expectations during pregnancy and after delivery signify a distance between what they had expected and what they faced in reality . (2007) who reported that mothers pre - childbirth expectations were consistent with what they experienced after that . Refer to some major themes like unpreparedness, loneliness, and loss, which stand out among the themes they have encountered in motherhood process, and assert that nurses and midwives should talk to mothers about the challenges to which they are exposed in this respect . Insufficient dominance and control over affairs is one of the major challenges for mothers in the motherhood process; feeling of hardship and discontentment which results from the overwhelming new condition, child's temperament, and mother's physical and mental tensions, and a sense of loss and restriction alike are influential in creating stress and conflict . A feeling of extreme and overwhelming tiredness, and loss of physical and mental energy after delivery have been indicated in many studies . According to troy (1999), women within the whole first year after delivery feel tired and de - energized . Nystrom and ohrling have also admitted that the most outstanding theme in parents experiences in the first year after childbirth is that they live in a a new overwhelming world . It goes without saying that child's difficult temperament also plays an important part in the way parents experience it . Many studies have acknowledged a direct association between parents distresses and having a child with difficult temperament . Feeling nostalgic about their past and what they have lost, added to the restrictions and undertakings imposed by the new condition, as barkley et al . (1997) have also indicated, leave new mothers with a sense of discontentment, grief, and depression . Insufficient control over affairs (practical control) can reduce self - confidence and hinder maternal role attainment . Studies have shown that mothers self - reliance could account for the absence or existence of stress and anxiety in mothers; i.e., mothers with more self - confidence can pass through the transition state more easily while their general health is less intimidated . Fear and anxiety which have been frequently observed in iranian first - time mothers and is most likely rooted in protective culture of eastern mothers the most significant aspect of this anxiety is fear of losing the child, as it has been mentioned by cornford et al . One thing that concerns women after delivery is that they do not want to be left alone; when they do not have someone around to help, they take the difficulties much more seriously; they imagine that they could be more supported when others are around . In this research, getting support from different sources, such as practical support especially provided by new moms mothers, affectional support mainly given by their husbands, and informational or subjective support offered by midwives or other advisors, is maintained as an essential element that facilitates a successful adaptation to the maternal role . They like to be understood and totally appreciated by others, especially their husbands, about their appearance, performance, house - keeping, and motherly behaviors; otherwise, their control and dominance could be disrupted . Seeking support from others could be a sign of mother's endeavor to keep or make some time for themselves to relax and get accustomed to the new condition . The study participants revealed that they have not yet accepted the child as an integral being and impose motherly deeds and maternal feelings upon themselves as they have seen or heard from others . Therefore, there is an inverse relationship between social support and neediness, i.e., mothers sense of neediness to get more support implies the weak state of the supporting system . According to researches, they have always heard that becoming a mother must be the happiest event in a woman's life or that maternal love is endless and stable . But now, being afraid of the fact that their experience has questioned all the established cultural beliefs and that it may incite negative judgments about them, they prefer not to convey their feelings while this intensifies their internal conflicts . According to the experiences of participants in this study, the duration and the range and intensity of these mental states differ from one to another . A mother, for instance, claimed that it was only the day after delivery that she had such feelings, while some others said that it took about a week or even more than a month to pass through these states . Some have only experienced one or few of these states; yet, with respect to their social relationship, all unanimously admitted the instability of their relationships, especially matrimonial relationships . Although most couples believe that having a child can strengthen their relationship, most of the researches reveal that after childbirth, parents do not form satisfactory relationships and their mutual and positive relations diminish . Mothers spend more time on child care and house chores and less time with their husbands, family, friends, work, etc ., while this can decrease marital satisfaction and bring about social exclusion . Likewise, their desire for sexual activities dissipates, while both parents are aware of these changes . Mothers awareness of these changes and the fear that they may get worse help to foster more anxiety and intensify their internal conflict . With regard to ethnic diversity in iran findings of this research, therefore, do not reflect maternal experiences of all ethnic groups living in iran . Despite the existing restrictions, this study attempts to present a proper description of view points and experiences of today's iranian first - time mothers . According to the present research findings, experiences of iranian first - time mothers in dealing with challenges and strains of maternal role adaptation have a lot in common with counterpart studies that have been mainly conducted in western countries . The intense attachment of personal, social, and cultural factors which give rise to these challenges and crises denotes the existence of some other reasons beyond biomedical factors . At the same time, the embedded resemblance of these experiences despite social, cultural, and geographic differences admits the existence of shared factors in creating the aforementioned crisis, which calls for additional research so as to provide awareness and proper outlooks toward such experiences among different groups of people around the world . What is evident is that the discrepancies between subjective expectations and postnatal experiences take an influential part in causing these problems . The more accurate information mothers and families have about this transitory stage, the better they can get prepared to tackle with it . This specifies the significant role of midwives, midwifery educators, and healthcare policy makers and practitioners to include these concepts in training programs and protocols of healthcare and support services in due time, form, and content that is in accordance with mothers mental and psychological needs . With regard to ethnic diversity in iran, best attempt has been made to choose study participants with different ethnic backgrounds . Findings of this research, therefore, do not reflect maternal experiences of all ethnic groups living in iran . Despite the existing restrictions, this study attempts to present a proper description of view points and experiences of today's iranian first - time mothers . According to the present research findings, experiences of iranian first - time mothers in dealing with challenges and strains of maternal role adaptation have a lot in common with counterpart studies that have been mainly conducted in western countries . The intense attachment of personal, social, and cultural factors which give rise to these challenges and crises denotes the existence of some other reasons beyond biomedical factors . At the same time, the embedded resemblance of these experiences despite social, cultural, and geographic differences admits the existence of shared factors in creating the aforementioned crisis, which calls for additional research so as to provide awareness and proper outlooks toward such experiences among different groups of people around the world . What is evident is that the discrepancies between subjective expectations and postnatal experiences take an influential part in causing these problems . The more accurate information mothers and families have about this transitory stage, the better they can get prepared to tackle with it . This specifies the significant role of midwives, midwifery educators, and healthcare policy makers and practitioners to include these concepts in training programs and protocols of healthcare and support services in due time, form, and content that is in accordance with mothers mental and psychological needs.
A key determinant of a successful glaucoma filtering surgery (gfs) is the healing response of the sclera, tenon tissue, and conjunctiva . Histologically, functional blebs show low inflammatory response with scattered conjunctival epithelium cells and fibroblasts, whereas dysfunctional blebs are characterized by dense collagen tissues and corkscrew blood vessels . Agents that modify the healing process have been used to improve the success of gfs . Antiproliferative agents such as 5-fluoroacil (5-fu) and mitomycin - c (mmc) inhibit the proliferation of fibroblasts . Both intraoperative use and postoperative use of these two agents however, the use of these antiproliferative agents can lead to very thin blebs that are prone to leakage, hypotony, and higher incidence of endophthalmitis [3, 4]. Other modulating agents such as growth factors inhibitors have been investigated for their potential use in gfs . Among these, antitransforming growth factor-2 (tgf-2) has shown promising results in experimental studies but has failed to show any superiority to placebo group in clinical trial . In addition to inflammation and fibroblast proliferation, angiogenesis also plays an integral role in the wound healing process . The level of vascular endothelium growth factor (vegf) has been shown to be upregulated in the aqueous humour of human and rabbit eyes after trabeculectomy . Case series have shown that postoperative subconjunctival injection of bevacizumab (scb) decreased the bleb vascularity and iop . At the time of writing, three published prospective studies have evaluated the adjunct use of scb in trabeculectomy and phacotrabeculectomy for patients with open angle glaucoma (oag) [810]. The study by grewal and associates is a prospective noncomparative case series of twelve trabeculectomies augmented with scb (1.25 mg) immediately at the end of the surgery . Mmc and 5-fu were not used . At six months, successful outcome (iop between 616 mmhg) was achieved in eleven out of the twelve eyes . In their study, blebs increased their vascularity after 3 months . The authors hypothesized that the use of scb might decrease the incidence of cystic thin blebs, frequently noted after mmc augmented trabeculectomies . The study by nilforushan and associates is a randomized comparative study comparing the uses of scb and mmc in trabeculectomy surgery . Thirty - six eyes were randomized to receive either scb (2.5 mg) or mmc (0.02% for 3 minutes). The eyes that received scb had a statistically higher iop level compared to the eyes that received mmc (13.6 3.2 mmhg versus 9.6 2.7 mmhg). In their study, the study by sengupta and associates is a randomized study comparing the uses of mmc (0.03% for 3 minutes), direct intraoperative bevacizumab application on the scleral using a sponge and three doses of scb (1.25 mg) in patients undergoing phacotrabeculectomy . In their study, the patients randomized to the scb group received two doses of scb during the surgery and the third dose at one week . At six months, they reported a higher rate of success (iop less than 18 mmhg) in the scb group compared to those of the other two groups (90% versus 60%). We intend to examine the use of scb in the postoperative management of combined cataract and glaucoma surgery including both trabeculectomy and nonpenetrating glaucoma surgery (npgs). The study was a retrospective review of patients who had glaucoma filtering surgery (gfs) and subconjunctival bevacizumab injection (scb) as part of their postoperative management . All patients have given written informed consent to receive scb . All surgeries and postoperative followups the study subjects were identified through searching a computerized internal medical database by typing the keywords such as subconjunctival bevacizumab, a control group was selected matching the demographic data and surgical procedures of the scb group . Subjects from the control group received similar postoperative management as the scb group with the exception of subconjunctival bevacizumab injection . Demographics data, types of gfs, intraocular pressure (iop) and the number of topical iop - lowering medications used pre- and postsurgery, the frequency and timing of scb injections, other interventions used in the postoperative period, and complications were recorded . The types of gfs included trabeculectomy and npgs with or without phacoemulsification and posterior chamber intraocular lens implantation (pciol). In both gfs procedures, a conjunctival incision was made 1.5 mm posterior to the limbus, and a half thickness 3.5 3.5 mm scleral flap was dissected toward the limbus . In npgs, a second deeper flap was dissected and excised, leaving an anterior trabeculodescemet window and the anterior wall of schlemm's canal unroofed . In trabeculectomy surgery, a sclerostomy was created using a kelly punch, and a peripheral iridectomy was performed with vannas scissors . In both cases, mmc (0.2 mg / ml) was routinely applied for 2 minutes on the bare sclera followed by thorough irrigation of conjunctival tissues with balanced salt solution, the scleral flap was closed with interrupted 10.0 nylon sutures, and the conjunctiva was closed using 10.0 biosorb using the modified wise technique . In combined gfs and cataract extraction, the anterior chamber was entered under the scleral flap with a 2.75 mm keratome, and phacoemulsification was performed using a direct chop technique followed by cortical clean - up and implantation of a foldable intraocular lens . At the end of surgery, all patients received topical moxifloxacin four times per day for one week, nonsteroidal anti - inflammatory four times per day for one week, and topical steroid tapering off for over two months . During the postoperative follow - up period, all patients could have the following interventions at the surgeon's discretion: subconjunctival injection of 5-fluorouracil (5-fu), laser suture lysis, bleb needling with a 30-gauge needle with or without 5-fu, laser goniopuncture, and subconjunctival injection of betamethasone or triamcinolone acetonide . Needling was performed using a 30-gauge needle tip to the flap area and then beneath the flap to puncture episcleral fibrous tissue . The needle tip was passed several times beneath the flap until bleb elevation was observed . Ml) was occasionally used during the same session depending on the clinical appearance of the bleb . Scleral flap sutures were lysed using an argon green laser or red laser (0.1 s, 400420 mw, 50 um spot size). Goniopunctures with nd: yag laser (4 mj) were performed using a latina gonioscopy contact lens . In addition to the previously mentioned interventions, all patients in the scb group also received at least one dose of scb postoperatively . After topical anesthesia, 1.25 mg (0.05 ml) of bevacizumab was injected in the subconjunctival space superior to the bleb using a 30-gauge needle . Main outcome measurements included iop and topical medication use recorded on the last clinical visit . The database retrieved 30 eyes of 28 patients who had scb injection following either trabeculectomy or npgs in postoperative period . Matching for age, diagnosis, and surgical procedures, we selected 28 control patients who did not received scb as part of their postoperative management . Demographic data and postoperative management for each group age, preoperative iop, and medications used were not significantly different in each group . The mean follow - up time was 19.6 (11.5) and 16.8 (8.2) months for the control and scb groups, respectively . The majority of patients had combined npgs with phaco - pciol (scb group: 24/30 eyes; control group: 20/30 eyes); few patients had phacotrabeculectomy, npgs, and trabeculectomy alone (table 1). In the control group, eight eyes from the control group did not undergo any intervention during their postoperative period . The rest of the control group (22/30 eyes) underwent one or a combination of the following treatments: 5-fu injection (12/30 eyes), suture lysis (9/30 eyes), goniopuncture (11/30 eyes), needling (2/30 eyes), and subconjunctival injection of triamcinolone acedonide (1/30 eyes). Patients from the scb group received similar pattern of postoperative management, but more patients underwent needling compared to those of the control group: 5-fu injection (10/30 eyes), suture lysis (7/30 eyes), goniopuncture (8/30 eyes), needling (5/30 eyes), and subconjunctival injection of triamacinolone (2/30 eyes) and betamethasone (1/30 eyes). Sixteen eyes of the scb group received only bevacizumab as their postoperative management with no other intervention required . Compared to the control groups, the scb group had fewer patients who required different types of interventions: 14/30 eyes in the scb group versus 22/30 eyes in the control group (table 2). Iop was reduced from 21.9 (9.8) mmhg preoperatively to 11.9 (4.7) mmhg on the last follow - up visit in the control group . In the scb group, the average iop was reduced from 19.6 (8.9) mmhg preoperatively to 14.0 (4.7) mmhg on the last visit . The number of iop - lowering medications was reduced from 3.1 (1.0) to 0.4 (0.8) in the control group and from 3.0 (1.1) to 0.7 (1.1) in the scb group . The reductions in number of medications and iop were statistically significant in both groups (p <0.001). The mean final iop was slightly higher in the scb group (14.0 4.7 mmhg) compared to that of the control group (11.9 4.7 mmhg); however the difference was not statistically significant (p = 0.36). In fact, there were no statistical differences in final iop, the number of medications or reductions in iop between the scb and control groups (tables 3 and 4). The complications observed in the control group included one case of blebitis requiring topical antibiotic treatment, two cases of iris incarceration, and one case of bleb leak requiring medical treatment . In the scb group, three patients developed branch retinal vein occlusion (brvo) during their follow - up periods, two patients had choroidal detachments, and one patient had a bleb leak that required surgical repair . Two of the three patients were not known for any systemic risk factors such as hypertension, diabetes, or atherosclerosis, whereas the third patient with brvo had had central retinal vein occlusion in the past (table 5). Studies have shown that anti - vegf agents could modulate wound healing through antifibroblast effect in addition to its antiangiogenic role . Have also demonstrated that the application of bevacizumab inhibited tenon fibroblasts proliferation, reduced collagen deposition, and induced apoptosis of fibroblasts [6, 13]. In vivo animal studies in rabbits undergoing trabeculectomy have demonstrated the efficacy of bevacizumab in prolonging bleb survival compared to 5-fu injection . One study has also shown that the bleb survival rate was the highest in the rabbits receiving a combination of subconjunctival 5-fu and bevacizumab injections (100% survival rate) compared to the rabbits receiving only one of the two injections (5-fu: 25% survival rate or bevacizumab: 50% survival rate), suggesting that a combination of antimetabolite and anti - vegf might be superior to the use of either agent alone . Such synergetic effect of antimetabolite and anti - vegf has been reported in clinical trials involving patients treated for colorectal cancer where delayed wound healing was observed in the group treated with systemic bevacizumab and 5-fu compared to the group treated with 5-fu - based chemotherapy alone . Clinical studies on the combined use of anti - vegf and antifibrotic agents are limited to the use of ranibizumab . In a prospective study, kahook compared the effect of combined intravitreal injection of ranibizumab and topical mmc to mmc alone in patients undergoing trabeculectomy . His study found that patients receiving a combination of mmc and anti - vegf demonstrated more diffuse bleb, but both groups had the same final iop . Our comparative series attempted to illustrate the combined use of scb in addition to other postoperative interventions including the use of antimetabolites such as mmc and 5-fu . We also did not find significant advantage in terms of the final iop value or the reduction in medications . Unfortunately, the difference in bleb morphology between the scb and control groups could not be compared due to lack of documentation; whether the addition of bevacizumab would influence the bleb morphology could not be determined in this series . The optimal timing and delivery method of bevacizumab in gfs are yet to be determined . Pharmacokinetics studies in rabbits have shown that both subconjunctival and intravitreal administrations of bevacizumab reached adequate intraocular concentration, whereas the topical route did not . Topical use of anti - vegf is, however, sufficient in regressing corneal neovascularization . It is debatable if the topical administration of anti - vegf will be sufficient in glaucoma surgery as the target place is the subconjunctival space . Topical administration has the advantage of less systemic absorption but requires frequent dosing, whereas a single intravitreal or subconjunctival injection may be sufficient as the duration of bevacizumab can be detected at least up to two weeks in aqueous humour . The duration of injection will cover the peak inflammatory and healing phase that occurs for about one week after filtering surgeries . In the prospective studies on anti - vegf use in gfs, subconjunctival injection is the most commonly used route except in one study that used intravitreal injection [810, 17]. Most of studies also used a single injection intraoperatively or postoperatively . Only the study by sengupta and there is no study comparing a single use of bevacizumab to repeated injections . In our study, scb was given postoperatively when the bleb appeared to be hypervascular . This approach is reasonable as newly formed vessels depend on vegf to survive, and the inhibition of vegf will result in regression of neovascularization . Clinically, decreased bleb vascularity has been observed after one single injection of bevacizumab into the bleb in some case reports as well as in our series of patients (figure 1). In our opinion, intravitreal or subconjunctival injection may not differ much in the management of gfs, as both methods achieve adequate aqueous concentration and have similar pharmacokinetics . Intravitreal injection has the additional ocular risks of inflammation, retinal detachment, and endophthalmitis . One ongoing study is investigating the use of topical ranibizumab four times a day for one month in postoperative period for trabeculectomy; its results may illustrate if topical treatment will be also an option in the management of glaucoma surgery . We observed three cases of branch retinal vein occlusion (brvo) in the group of 28 patients receiving bevacizumab . This high - incidence brvo is probably coincidental since the onset of brvo was seven to eight weeks after the injection of bevacizumab . Other series on glaucoma filtering surgery did not report any vascular complication related to the use of anti - vegf agents [8, 9, 17]. However, brvo is a serious ocular complication, and we should still be cautious about the possible vascular side effects of anti - vegf agents . Systemic use of bevacizumab is associated with various vascular events such as hypertension, hemorrhage, and thromboembolism . Regarding the ocular use of bevacizumab, rare events such as retinal ischemia, central retinal artery occlusion, subretinal hemorrhages, and systemic complications such as elevated blood pressure, stroke, and myocardial infarctions have also been reported in surveys and retrospective studies [22, 23]. Pooled results from clinical trials have suggested a higher incidence of stroke in patients receiving a higher dose of ranibizumab (0.5 mg) compared to that in those receiving a lower dose (0.3 mg). Some studies have also shown that oag patients had elevated vegf levels in their aqueous humour and plasma compared to normal controls [25, 26]. The presence of vegf is likely important for normal function and repair of vascular endothelium, and the blockage of vegf may lead to endothelial dysfunction [19, 25]. Although there is no definitive evidence suggesting that glaucoma patients are prone to side effect of anti - vegf agents, as their use may be more frequent in glaucoma patients, the safety profile of anti - vegf agents should be followed . In conclusion, our comparative series did not found that subconjunctival injection of bevacizumab had additional benefit in term of iop reduction . The bleb morphology could not be assessed in this series due to the retrospective nature of the study and lack of bleb description in every patient . Whether bevacizumab will influence the bleb morphology and whether this influence will be clinically relevant need to be addressed by prospective studies with longer follow - up time . In our opinion, subconjunctival and intravitreal injections of anti - vegf would have the same efficacy as the pharmacokinetics of either delivery route will cover the inflammatory phase after filtering surgeries . Of significance, there were three cases of brvo observed in the bevacizumab group . Clinicians should monitor potential side effects of anti - vegf agents as their safety profile in glaucoma patients may not be the same as that in amd patients.
Currarino syndrome (cs) is a hereditary pathology that is characterized by a triad of malformations: sacrococcygeal bone defect, presacral mass, and anorectal malformation . Radiologic imaging modalities such as ultrasonography (us), computed tomography (ct), and magnetic resonance imaging (mri) play a vital part in diagnosis and follow - up of this pathology . In this report, we aim to present the magnetic resonance imaging (mri) findings of a case with complete cs recognized in adulthood . A 20-year - old male patient who underwent anal atresia operation in childhood was referred to our department for evaluation of anal sphincter status with transrectal us . Calibration of one - third of the inferior rectum was observed to be very thin, while one - third of the superior and middle segments of the rectum was seen to be dilated . The left half of s2 and s3 vertebrae, more distal sacral vertebrae, and coccyx were not seen . We also detected anterior meningocele related to spinal canal that originated from the neural foramen of s2 and s3 vertebrae . The lesion was isointense with cerebrospinal fluid in all of the sequences and its size was 4.5 3.5 cm . 20-year - old man with anal atresia operation history in childhood diagnosed with currarino syndrome . (a) t2-weighted sagittal magnetic resonance image shows calibre of one - third of inferior rectum is very thin (thick white arrows), while superior and middle segments of the rectum are dilated (arrowheads). Also, the images demonstrate anterior meningocele (asterisk) at the posterior of the rectum related to spinal canal that originated from the neural foramen of s2 and s3 vertebrae . (b) fat - saturated t2-weighted axial magnetic resonance image shows partial cleft at l5 vertebra corpus (thin white arrow) and dilated rectum in front of it . On (c) t1-weighted and (d) fat - saturated t2-weighted axial magnetic resonance images, left half of the sacrum is not seen . In this part, spinal canal relationship of anterior meningocele (asterisk) and its indentation to the adjacent rectum is also observed . (e) t2-weighted coronal magnetic resonance images demonstrate the contiguity of the sacral defect and anterior meningocele (asterisk) more clearly (r = rectum, b = bladder). Currarino syndrome is a rare inherited disorder that is characterized by sacrococcygeal bone defect, presacral mass, and anorectal malformation . The complete form of this syndrome shows all three abnormalities and is rarer than the incomplete form which includes one or two of the disease components mentioned above . Although the patients with this syndrome may present with complex conditions like recurrent urinary system infections or meningitis, chronic constipation is the most common finding . Some of the cases are asymptomatic and can be diagnosed during adulthood only on x - rays and us examinations that are performed for different reasons . In addition to rectal digital examination, x - rays, us, ct, and mri are very helpful in the diagnosis of the syndrome . Sacrococcygeal bone defect is a prerequisite for the diagnosis of cs and its spectrum changes from mild lateralization of the coccyx to multilevel hypoplasia of the sacral segments (scimitar sacrum). In our case, there was partial cleft at l5 vertebra corpus and s1 vertebra was partially deformed . Also, the left half of s2 and s3 vertebrae, more distal sacral vertebrae, and coccyx were absent . Rectal and/or anal stenosis which may be fistulized to ascending spinal canal is the most common anorectal malformation which is seen in cs . Anal atresia or ectopy, imperforated anus, rectal duplication, and rectourethral, rectovaginal, and rectovesical fistulas may be also associated with the syndrome . In our patient, there was heterogeneity of the cutaneous and subcutaneous soft tissue around the anus secondary to earlier anal atresia operation . Enteric cyst, lipoma, hamartoma, dermoid and epidermoid cysts may be seen as well . Us, ct, and mri are frequently used for diagnosing the mass lesion and determining its nature . Mri is an invaluable technique to recognize accompanying additional pathologies like tethered cord, syringomyelia, and intradural lipoma . In our patient, we detected a 4.5 3.5 cm size anterior meningocele related to the spinal canal . Cloacal malformation with anal atresia, double vagina, rectovesical fistula, urogenital sinus, neurogenic bladder, vesicoureteral reflux, and multicystic kidney are other related pathologies of cs . Mri is a very useful imaging modality to diagnose cs and accurately detect all the three components . Besides, it can show the different aspects of other cloacal malformations including the uterus, vagina, maldeveloped puborectalis, and external anal sphincter . Although these anomalies are not surgically correctable, early detection almost always provides better prognosis and helps in patient management . Another major advantage of mri over other imaging modalities is the ability to detect associated anomalies of anorectal malformations, especially of the spinal cord, spine, and urogenital system . Kassir et al ., also reported that mri is a specific and sensitive non - invasive diagnostic tool in patients with anorectal malformations . Mri provides excellent information about the postoperative anatomy of anorectal malformations . Due to its superior soft tissue characterization, multiplanar imaging ability, and lack of ionizing radiation exposure these unique features make mri a very appropriate modality in follow - up of patients treated for anorectal abnormalities . The treatment strategy changes based on the type of pathology . The management of the anorectal anomaly and presacral mass may be performed with single - stage surgical procedure . If the mass is a meningocele, a staged operation must be preferred due to the risk of meningitis . Cs should be obviously considered in patients with sacrococcygeal bone defect, presacral mass, and anorectal malformation . Mri may be used safely in diagnosis of the syndrome, in detecting the accompanying mass lesions and other pathologies, and in the follow - up investigations after treatment in patients with cs.
Dyslipidemia may cause complications such as stroke, kidney failure, coronary heart disease, and atherosclerosis . High concentrations of triglyceride - rich lipoproteins, and apolipoprotein b in infants with a low gestational age; and increase of apo c-1 rich in high - density lipoprotein (hdl) with low birth weight are potential risk factors for cardiovascular disease in the future . It is suggested that the decreasing activity of the lipoprotein lipase, hepatic lipase and lecithin - cholesterol acyltransferase enzymes in preterm infants might lead to high concentrations of lipoproteins . A study in spain reported that the cord blood hdl - cholesterol (hdl - c) level was lower in premature than in term newborns, but very low - density lipoprotein (vldl) levels were higher in preterm than in term newborns . In a study in japan, cord blood samples of preterm newborns had higher concentrations of vldl, ldl - cholesterol (ldl - c), and hdl - c subclasses compared with their term counterparts . Reported that the concentrations of total cholesterol, ldl - c, hdl - c, and apolipoprotein - b were higher in preterm- than in full - term newborns . A previous study in iran showed that the total cholesterol and hdl - c of female newborns were higher than in male newborns . Limited experience exists on the difference of the cord blood concentrations according to the gestational age . The aim of this investigation is to compare the cord blood lipid concentration in term, late preterm and preterm newborns in a sample of iranian neonates . This cross - sectional study was conducted in 2013 among 100 neonates in isfahan, iran . Neonates with a gestational age of 37 weeks were considered as term, those with gestational age of <34 weeks as preterm, and those with 34 gestational age <37 weeks, as late preterm neonates . Newborns were selected by simple sampling from the maternity ward of shaheed beheshti teaching hospital, affiliated to isfahan university of medical sciences in isfahan, the second large city in iran . Immediately after delivery, 2 ml of cord blood was collected from the fetal part of the umbilical cord . Cord blood was allowed clot and then stored at 20c until analysis in <1 month . Lipid profile and apolipoproteins a and b were analyzed in milad laboratory by autoanalyzer (hitachi 911, japan). Chicago, il, usa) was used for statistical analysis using one - way analysis of variance . Cord blood concentrations of triglycerides, total cholesterol and ldl - c were significantly different in term, late preterm, and preterm newborns [table 1]. Term infants had the highest level of triglycerides (47.03 mg / dl), whereas preterm infants had its lowest concentration (61.69 mg / dl). Our analysis showed that in each three groups under study, the difference of total cholesterol level was significant . The lowest amount of cholesterol content was documented in term infants (72.51 mg / dl). No significant difference existed in the mean level of hdl - c, apolipoprotein a and apolipoprotein b levels in the three studied groups (p> 0.05). The present study demonstrated that the term infants had the highest concentration of cord blood triglycerides . The highest amount of cholesterol in cord blood was documented in late preterm infants . In the total study group, no significant difference existed in the mean of hdl, apolipoprotein a and apolipoprotein b levels . Lipid profile is a marker of an underlying cardiovascular status, and direct correlation exists between the abnormalities in lipid profile and incidence of many chronic diseases . Among various factors theorized in the development of atherosclerosis, increased plasma levels of cholesterol and or triglycerides are considered to be of most important factors . Atherosclerosis begins early in life, and the studies conducted on cord blood lipid profile had inconsistent findings . Some findings of the current study are not consistent with a previous study on cord blood cholesterol, which found higher cholesterol in preterm than in term newborns . In another study, preterm infants had a high level of cholesterol concentrations . In this investigation, we did not document significant difference in the mean cord blood level of hdl - c, apolipoprotein a and apolipoprotein b levels in the total study group . Consistent with another investigation, the current study indicated that differences in the gestational age do not affect cord blood hdl - c level . A previous study showed that ldl - c concentrations decline from 30 - 33 to 42 weeks of gestation . Our findings also demonstrated that term newborns had lower cord blood ldl - c concentration than preterm and late preterm infants, however the differences were not significant . Haridas and acharya in their study concluded that preterm infant have higher triglycerides and total cholesterol levels, but statically significant difference was found only for total cholesterol . Mathur et al . Concluded that in preterm neonates, total cholesterol was significantly high . Doneg et al ., reported that total cholesterol, ldl - c, and hdl - c levels of cord blood were significantly higher in preterm infants; also the triglycerides content was lower in preterm neonates ., found that total cholesterl, ldl - c, and hdl - c of cord blood were higher in preterm neonates compared with term neonates, with statically significant difference in total cholesterol and ldl - c levels, but the corresponding figure was not significant for hdl - c . It is suggested that decrease of lipoprotein lipase, hepatic lipase, and lecithin cholesterol acyltransferase enzymes activity in preterm newborns might increase lipoprotein concentrations in preterm than in term infants . In addition to genetic factors that are of major determinants of lipid levels, fetal growth retardation might also establish a lifelong irreversible atherogenic profile, and the history of low birth weight, or preterm birth are associated with various risk factors including increase in apolipoprotein b levels . In our study, higher levels of triglycerides, total - cholesterol, and ldl - c in preterm and late preterm infants could be explained by the fact that preterm newborns lack both hepatic carbohydrate and subcutaneous adipose stores . Increase in cord blood cholesterol level may reflect the metabolic adaptation to provide adequate energy, especially to vital organs like brain . Given the early life origins of adult diseases, primordial and primary prevention should be emphasized . Future longitudinal studies with long - term follow - up are necessary to verify the clinical implications of the current findings . Future longitudinal studies with long - term follow - up are necessary to verify the clinical implications of the current findings . The present study assumes that cord blood lipid profile differs according to the gestational age . All authors contributed in the study design and conduct, as well as in drafting and revising the manuscript . All authors approved the final version of the paper, and take the responsibility for its content.
The specific causes of tle in the elderly have not been clearly disclosed . With advancing age underlying coexisting factors are more likely to be identified, and the proportion of people classified as idiopathic is less when compared to the younger age groups . Nonetheless, up to half of elderly patients with epilepsy go without an identified cause . In the under 65-year age group, head trauma, brain tumors, and cns infections idiopathic tle has been reported in adults with familiar history of epilepsy (mean age of onset: 25.5; range: 1145 years). There are however case reports of mesial temporal lobe sclerosis (mts) in elderly persons with new onset seizures . These accounts highlight the challenging differential diagnosis overlap with primary cognitive disorders and nonparaneoplastic limbic encephalitis . The etiology of hippocampal damage is discussed in a retrospective study of 38 patients with adult onset tle . The median age at onset was 37.8 years (range: 21.0 to 78.7 years). A total of seven patients, 60 years and older, were included in this report . In all cases, the common features encompass frequent cpss (range <1 to 600 seizures per month) developing over one year (defined as the median time from the initial onset of seizures to the time of assessment). Based on mri evidence of hippocampal atrophy, history of causative events, concomitant disease, cerebrospinal fluid results, and autoantibodies patients were classified in one of four etiologic categories; secondary hippocampal sclerosis (hs); idiopathic hs; definite limbic encephalitis (le) and mri defined possible le . Two patients had experienced a severe head trauma; two, old infarct; one a porencephalic cyst and one neurofibromatosis type i. only two patients in this subgroup were 60 years and older . The latter reported 500 seizures per month . In the idiopathic hs group, all seven subjects had unilateral hippocampal abnormalities . Definite le included nine patients with 5 (56%) showing bilateral hippocampal abnormalities on mri . Six of the nine patients had sequential mris with initial hippocampal swelling, followed by normalization and ultimately atrophy and high - intensity signal . Four patients had positive voltage gated potassium channel antibodies (vgkc abs) and one anit - hu antibodies . The remaining four patients were diagnosed with rectal, small cell lung cancer (sclc), testicular tumor with ma2 antibodies, and a uterine leiomyosarcoma, respectively . In this subgroup of definite le, three patients were 60 years and older . Vgkc abs, rectal cancer, and small cell lung cancer were documented in one patient each . Finally, 11 patients were classified as mri defined possible le based on repeated mris with initial hippocampal swelling, normalization and atrophy, and absence of malignancy or antibodies (one exception with atypical anti - hu antibodies). Two patients, 60 years and older, were included, both with unilateral hippocampal involvement . This report delineates idiopathic and secondary hs as well as paraneoplastic and non - paraneoplastic le as causative factors of late onset tle emphasizing bilateral hs in the latter two subgroups, however, not infrequent with secondary hs . Memory impairment was more common with bilateral hs and in all cases frequent cpss developed over a relatively short period of time . The few elderly subjects included in this series appear to follow these general premises . A potential etiologic factor of tle in the elderly is represented in generalized convulsive status epilepticus (gcse) recognized to occur more often in the elderly with an estimated incidence of 86 per 100000 [12, 13]. A complication of gcse is subsequent damage to the hippocampus [14, 15]. Mortality after gcse is significant . Survivors would be at risk of subsequent partial seizures originating after hippocampal damage . The clinical features of various partial seizure types have been recently reported comparing patients 55 years of age and older (mean 65.2 8.53) to a group between 18 and 45 years of age (mean 33.6 6.75); each group encompassed 55 consecutive subjects . Partial seizures with loss of awareness and lack of prominent automatisms were classified as dialeptic seizures and in the older group accounted for 60% of all focal seizures and 52% of the younger patients . Partial seizures with prominent mouth and/or hand automatisms happened in 18% of the older patients while in 14% of the younger group . The authors did not specifically report on the localization of the origin of these seizures . In this series not surprisingly, the older group had a later age at seizure onset (53.6 20.23 yrs versus 23.4 12.12 yrs), seizure - free period greater than 1 year (20% versus 8%), and cerebral vascular disease as a risk factor (20% versus 6%). The younger group reported more than one seizure per month (54% versus 28%) and a history of febrile seizures (20% versus 6%). For the older and younger age groups, risk factors included trauma 30% in each group, cns infection in 6% and 4% and cns tumor in 8% and 6% . Among the 28 patients that had no recollection of their seizures, 17 (61%) were from the older age group . In total, 9 subjects (18%), all of the older age group, reported subtle perception of transient confusion (p = 0.002). The total number of subjects with aura (defined as partial seizures without loss of awareness) was less common in the older age group (54% versus 76% p = 0.03). No significant differences could be demonstrated in the comparison of symptoms consisting of auras (psychic, autonomic, abdominal, visual, somatosensory or gustatory, auditory, olfactory, and vertigo) or generalized tonic clonic seizures (gtcss) occurrence . Approximately one - third of patients in each group had a normal eeg, while 26% of the older group and 20% of the younger group had nonspecific finding . Focal epileptiform discharges were shown in 42% of the older and 23% of the younger subjects . Video - eeg monitoring was limited to a total of 9 patients yielding an additional 10% of older and 8% of younger patients with focal epileptiform and/or focal seizures . In patients with epilepsy, transient epileptic amnesia is an emerging concept described in middle - aged and older people with an evident response to antiepileptic drug treatment (aed). A characteristic feature is prolonged periods of ante- and retrograde memory impairment with a mean duration between 30 and 60 minutes and subsequent amnesia that occurs upon awakening . Transient amnesia may be the sole clinical manifestation in approximately one - third of patients . Repetitive questioning is present in up to half of patients requiring differentiation from transient global amnesia . Interictal epileptiform abnormalities may be demonstrated in one - third of patients and ictal electrophysiological correlates have been recorded uni o bilaterally in the temporal lobes . Brain imaging is usually clear, or lesions involve the temporal lobe [18, 19]. The diagnosis of tle in the elderly poses a challenge (see table 1). Memory lapses, brief gaps in the flow of conversation, and blank stare and confusion may be the only clinical manifestations . Clinical features resemble tia, delirium, congestive heart failure, cardiac arrhythmia, orthostatic hypotension, vasodepressor episodes, metabolic dysfunction with hypoglycemia, hyponatremia, sepsis, or drug toxicity . A small number of randomized controlled trials (rcts) have been conducted in elderly patients testing the efficacy of aeds . Studies do not specifically address tle but serve to evaluate efficacy and safety issues of aeds in the setting of epilepsy in the elderly . Recently, a group of 77 people with a mean age of 68 years and partial onset seizures were randomized in a pilot study to topiramate (tpm) 50 mg or 200 mg doses as add - on or monotherapy . Seizure freedom was similar with the lower and higher doses (52% and 58%, resp .) As well as seizure frequency (0.26/month and 0.33/month, resp . ). More than 60% in both study groups complained of adverse side effects with somnolence, dizziness, and headache being the most common . One randomized controlled trial (rct) tested carbamazepine (cbz) versus lamotrigine (lmt) in a total of 64 subjects (mean age: 67 years) with partial seizures (simple or complex) with or without secondary generalization presenting on average between 8 and 12 months after stroke . Stroke was classified as cortical in 64% of patients allocated to lmt and 66.7% of the cbz group . Subcortical strokes were diagnosed in 36% and 33.3%, respectively, in these study groups . Subjects allocated to cbz reported significantly more adverse effects than lmt leading to study withdrawal (31% versus 3%, p = 0.02). On completion of the first year, 72% of patients on lmt and 44% on cbz were seizure - free (p = 0.05). A total of 590 elderly patients (mean age: 72 years) with newly diagnosed epilepsy with any seizure type were randomly allocated to gabapentin (gbp), lmt, or cbz . In total, the etiology was similar among groups with an approximate one - third due to cerebral infarction . The localization of the origin of cpss was not accounted for . A mild cognitive impairment or memory problems were present in 35% and 25% of patients, respectively . At 12 months lmt was significantly best retained than cbz (p <0.0001) or gbp (p = 0.015). At one year, seizure freedom was not significantly different between groups (lmt 51.4%, gbp 47.4%, and cbz 64.3%). The time to first, second, fifth, and tenth seizure in the first year was also similar and no significant statistical differences were demonstrated between groups . Of the seven patients requiring hospital admission due to hypersensitivity reactions, one was due to lmt and six to cbz . A total of 150 elderly people (mean age: 77 years) with any seizure type resulting from idiopathic, symptomatic, or cryptogenic epilepsies were included and randomized in a 2: 1 ratio to either lmt or cbz . Cerebral infarction was disclosed in 30% of the lmt group and 38% in the cbz group . In 79% the daily lmt median dose was 100 mg (range: 75300 mg), and in 82% the daily cbz median dose was 400 mg (range: 200800 mg). Seizure freedom during the 16 weeks of the study period was significantly different favoring lmt over cbz (39% versus 21%, p = 0.027). Seizure outcome in 16 patients 50 years and older (mean 55.5 yrs, range 5072) was compared to 184 younger patients (mean: 32.9 yrs, range: 1649) undergoing anterior temporal lobectomy (atl). All patients had pathologically confirmed unilateral hs and mri lacked evidence of any other pathology . Following atl older patients were less often seizure - free than the younger patients (56% versus 79%, p = 0.041). Postsurgical complications were more frequent in the older patients (25% versus 4.4%, p = 0.009). Boling et al . The mean age at surgery was 54 years and followed for up to 64 months . Seizure freedom was reached by 61%, and 72% were able to reduce or stop their antiepileptic drugs . This older set of patients underwent multiple comparisons with subjects grouped in decades from ages 10 to 49 years . In the four resulting groups sirven et al . Reviewed a total of 30 patients aged 50 years and older (mean 54.2 4.7) and compared them to 340 subjects younger than 50 years (mean 32.8 7.7). These groups were followed for 4.0 2.7 years and 5.0 2.9 years, respectively . Seizure freedom was 52% in the older group and 75% in the younger group (p = 0.008). A discriminant function analysis disclosed that age at surgery and duration of epilepsy explained the largest fraction of variance . The authors consider that results could have been impacted by the fact that surgery in the older group was performed in their early 50's with only 5 patients 59 years and older . In this retrospective analysis, as population grows, an increment is expected in the number of subjects 65 years and older identified with tle . Countries with a longer life expectancy are going to be impacted to a greater extent . More often than not a coexisting significant medical condition may be revealed as well as acute or chronic brain involvement . Clinical diagnosis is elusive with subtle presenting features such as recurrent memory lapses or periods of confusion . Nevertheless, the typical signs of cpss are likely . The undisputable advantage of video - eeg monitoring recognized in the investigation of people with epilepsy is clearly applicable to elderly populations [2022, 38, 39].
Heterozygous hnf4a mutations cause a beta cell phenotype of neonatal hyperinsulinism with macrosomia and young onset diabetes . Autosomal dominant idiopathic fanconi syndrome (a renal proximal tubulopathy) is described but no genetic cause has been defined . We report six patients heterozygous for the p.r76w hnf4a mutation who have fanconi syndrome and nephrocalcinosis in addition to neonatal hyperinsulinism and macrosomia . All six displayed a novel phenotype of proximal tubulopathy, characterised by generalised aminoaciduria, low molecular weight proteinuria, glycosuria, hyperphosphaturia and hypouricaemia, and additional features not seen in fanconi syndrome: nephrocalcinosis, renal impairment, hypercalciuria with relative hypocalcaemia, and hypermagnesaemia . This was mutation specific, with the renal phenotype not being seen in patients with other hnf4a mutations . In silico modelling shows the r76 residue is directly involved in dna binding and the r76w mutation reduces dna binding affinity . The target(s) selectively affected by altered dna binding of r76w that results in fanconi syndrome is not known . The hnf4a r76w mutation is an unusual example of a mutation specific phenotype, with autosomal dominant atypical fanconi syndrome in addition to the established beta cell phenotype.
Ureteritis cystica (uc) is usually suspected when defects of filling are seen in the ureter in contrasted images of the urinary tract . Although is the condition is usually an incidental finding during the evaluation of the urinary tract, it presents occasionally with acute flank pain . A urinalysis showed a ph of 6, was positive for haemoglobin and leukocyte esterase (+ +). Bilateral renal morphology and function were normal including a normal left ureter . In the distal right ureter cytology of the ureteral washings was also negative for malignant cells . To further refine the diagnosis computerized axial tomography (ct) and urological magnetic resonance (mru) both studies showed that the right ureter was occupied from the crossing of the iliac vessels to the bladder by material with characteristics soft tissue . There was no evidence of retroperitoneal, iliac or inguinal lymphadenopathy, or hepatic lesions (figs . 2 and 3). In view of these findings a biopsy was negative for cancer . With the presumptive diagnosis of ureteritis cystica, we opted for observation with annual follow - up . The patient remains asymptomatic and free of urinary tract infections, haematuria or renal pain . Ureteritis cystica is defined as the cystic transformation of the epithelial nest of brunn, with the appearance of numerous cysts containing clear fluid with sizes between 1 and 10 mm with flattened epithelial walls . It is considered of the result of irritation in nonspecific chronic inflammations.1 other etiological factors that have been postulated include bilharziasis, vitamin a excess and increased immunoglobulin a. none of these factors have been proven to play a specific role.2 ureteritis cystica is an infrequent condition which is predominantly found in adults females,3 but also reported to occur in men and children . Although usually unilateral, bilateral cases have been described.4 the location of the cysts is predominantly in the proximal ureter, but they can be found at any level of the urothelium . The lesions are benign with low potential for degeneration, although occasionally it has been associated with bladder carcinoma or renal carcinoma.4 the clinical presentation is variable . Ureteritis cystica is usually detected during the evaluation of urinary tract infections (82%), lithiasis (53%) or haematuria (52%).57 the most commonly used imaging techniques are excretory urography and retrograde pyelography . In these imaging studies one can observe numerous defects of filling with well - defined, rounded smooth contours often with a scalloping appearance.7 in cases where the diagnosis is uncertain, as was the case in our patient, ct scan and mru can be used to better define the nature of the lesions, their extent, the presence of other abnormalities . Other lesion that can have a similar appearance include, (radiolucent stones, clots, veins, air bubbles, tuberculous ureteritis).8 when the imaging studies are not conclusive, ureteroscopy plays a fundamental roll in the ability to directly visualize the cystic formations and to take biopsies in order to realize an anatomo - pathological analysis.7 in fact, many authors consider endoscopic diagnosis as the most appropriate in these cases.6 the treatment consists of eliminating the process which is causing the inflammation (infection, lithiasis), although in those cases in which obstruction other measures may be appropriate . In table 1 imaging studies should be complemented with ureteroscopy and biopsy when indicated when tumour is suspected.
Gastroschisis is a right - sided, small, and full - thickness paraumbilical defect of the abdominal wall that occurs in 1 of 4000 births . Unlike an omphalocele, theories concerning the etiology of gastroschisis are usually considered to be the result of a vascular insult . Cardiac and genitourinary abnormalities have been associated with gastroschisis, but presence of extra - gastrointestinal anomalies warrants search for alternative diagnosis . In the presented case, gastroschisis associated with spinal and lower limb anomalies in early age group mother (primiparous) has been presented . A 22-years - old full - term primiparous patient underwent an ultrasound examination, we found large paraumbilical defect with herniation of stomach, small and large bowel loops, liver, gallbladder, and right kidney . Herniated stomach and bowel loops were rest over internal os [figure 1]. Amount of liquor was very less and fetus looked flexed and twisted in breech presentation . Spinal anomalies were observed block cervical vertebrae with spina bifida of cervical and lumber spines . In lower limbs anomalies, mildly short, thin left femur and left tibia with very thin fiber - like left fibula were seen . Both feet were rudimentary, especially left foot, with no defined toe while right foot was with four abnormal toes . Usg image of fetal abdomen showing herniated bowel loop, right kidney, and fetal liver resting on maternal internal os usg image of fetus showing two vessel cords after delivery, we took photographs of dead abnormal newborn . Stomach, intestinal loops, liver, gallbladder, and right kidney were herniated through the large abdominal defect [figure 3]. Both lower limbs were mildly short; left leg being thinner with deformed feet and small three buds of toes while right foot presented with four abnormal toes [figure 4]. Left side hip was small with kypo - scoliotic curvature of vertebral column photograph of newborn showing herniated bowel loops and liver with intact umbilical cord photograph of newborn showing small and thin left leg with deformed both feet ct scan and radiograph of newborn were also obtained [figures 5 - 7] and the above said findings were corroborated by the images . Ct vrt image of newborn showing blocked cervical vertebrae with cervical and lumbosacral spina bifida ct vrt image of newborn showing short, thin left femur and tibia with thin fiber - like fibula and rudimentary left iliac bone anterioposterior radiograph image of newborn showing blocked cervical vertebrae with cervical and lumbosacral spina bifida, short and thin left femur, thin tibia and rudimentary left iliac bone gastroschisis is a congenital anterior abdominal wall defect, adjacent and usually to the right of the umbilical cord insertion . This differentiates it from an omphalocele, which usually is covered by a membranous sac and more frequently is associated with other structural and chromosomal anomalies . In addition, however, gastroschisis may be associated with gastrointestinal anomalies such as intestinal atresia, stenosis, and malrotation . By the study of stoll c et al . Of omphalocele and gastroschisis and associated malformations, they assessed these associated malformations ascertained between 1979 and 2003 in 334 262 consecutive births . These included patients with chromosomal abnormalities (25, 29.0%) and non - chromosomal syndromes . Malformations of the musculoskeletal system (31, 23.5%), urogenital system (27, 20.4%), cardiovascular system (20, 15.1%), and central nervous system (12, 9.1%) were the most common, other congenital malformations in patients with omphalocele and non - syndromic multiple congenital anomalies (mca). Performed an international study to identify malformation patterns and to evaluate the role of maternal age in non - isolated cases of gastroschisis and associated defects . Case - by - case information from 24 registries, all members of the international clearinghouse for birth defects surveillance and research (icbdsr) were evaluated . Their results showed that of 3 322 total cases, 469 non - isolated cases were registered (14.1%): 41 chromosomal syndromes, 24 other syndromes, and 404 mca . Among mca, four groups of anomalies were most frequent: cns (4.5%), cardiovascular (2.5%), limb (2.2%), and kidney anomalies (1.9%). No similar patterns emerged except two patterns resembling limb - body wall complex and omphalocele - exstrophy - imperforate anus - spinal defects (oeis). In both of them, the gastroschisis could be misclassified . Chromosomal trisomies and possibly non - syndromic mca are associated with an older maternal age more than isolated cases . On consideration of their data and the most valid studies published in the literature, the best estimate of the proportion of gastroschisis associated with major unrelated defects is about 10%, with a few cases associated to recognizable syndromes . Recognized syndromes with gastroschisis seem to be so exceptional that the well documented and validated cases are worth being published as interesting case report . An appropriate case definition in etiological studies should include only isolated gastroschisis after an appropriate definition of isolated and non - isolated cases and a thorough case - by - case review . Reported a rare case of a newborn baby with an abdominal wall defect, together with multiple congenital abnormalities and diagnosed as gastroschisis . There were multiple defects seen as spinal deformity, imperforate anus, esophageal fistula, and lower limb deformity (congenital talipes equinovarus) along with the webbing of neck . There were also ischemic changes present over the left upper limb in the form of cyanosis . The diagnosis made was gastroschisis and omphalocele along with spinal deformity . In the presented case, head, face, neck, thorax, and both upper limbs were normal . Although gastroschisis has rare associated malformations, but if present, pentalogy of cantrell, limb - body wall complex, etc . Other birth defects are associated with gastroschisis, most commonly, abnormalities of the cardiac and genitourinary . The present case was not completely considered within any known alternative diagnosis of gastroschisis - associated complex, non - syndromic and syndromic anomalies . The presented case of gastroschisis, therefore, highlight the associations of both spinal and lower limbs anomalies in primiparous, which was proven to be a rare case.
Pediatric ms (pms) is defined by an initiation prior to the age of 16 years (sadaka et al ., 2012; renoux et al ., 2007). Although a variety of features characteristic for pms have been described recently (e.g., hummel et al ., 2013; bigi and banwell, 2012; sadaka et al ., 2012; vargas - lowy et al ., 2012; polman et al ., 2011), it is currently unclear whether pms denotes a pathoetiologically homogeneous or heterogeneous clinical phenotype . In particular, recent studies found evidence for differences in the gender distribution between patients with early onset pms (eopms) as compared to later onset pms (lopms; banwell et al ., 2007). Another study evaluated the validity of the revised mcdonald criteria 2010 for diagnosing pms and found that these criteria are well suited for children with onset at the age of 11 or older, but considerably less well for the subgroup of children with earlier onset (sadaka et al ., 2012). Comparing characteristics of hyperintense lesions in t2-weighted (t2w) brain mr images between eopms and lopms patients, chabas and colleagues found that eopms lesions were less well - defined . Consistently, these authors claimed that mri - based diagnostic criteria for prepubertal ms might have to be revised (chabas et al ., 2008). In recent years, the application of computer - based classification algorithms for the analysis of mri data of neurological (e.g., kloeppel et al ., 2008) as well as psychiatric disorders (e.g., weygandt et al ., 2012) has become more and more frequent . In ms research, a variety of neuroimaging studies used this approach to identify lesion - related and non - lesion - related mri biomarkers for adult ms extracted from brain images acquired with clinical routine mri acquisition protocols . For example, we applied this approach to diagnose relapsing remitting ms (rrms) by evaluating voxel intensity patterns (weygandt et al ., 2011), and wavelet representations of these patterns (hackmack et al ., 2012a) extracted from brain areas containing normal - appearing brain tissue measured with t2w mri sequences . In a further study, we used tissue intensity patterns to predict symptom severity in rrms (hackmack et al ., 2012b). Finally, another group used the approach to differentiate between subgroups of ms (i.e. Patients with short vs. long disease duration; high vs. low white matter (wm) lesion load; benign vs. non - benign ms) based on gray matter (gm) probability maps (bendfeldt et al ., 2012). In the present study, we assessed whether classification techniques can also be applied in pediatric neuroimaging by searching for mri - based diagnostic biomarkers specific for eopms . In particular, we conducted three main diagnostic classification analyses to identify brain regions containing the most relevant diagnostic information for eopms . In analysis 1, we investigated differential diagnostic separability of eopms vs. lopms patients using logistic regression . This was done independently for areas located in gm and for areas located in wm based on local tissue probability parameters . In analysis 2, we searched for diagnostic information for separation of eopms patients vs. healthy control (hc) subjects separately for gm and wm areas . Finally, in analysis 3 we tested local diagnostic separability of lopms vs. hc based on local gm and wm tissue probability parameters importantly, lopms and hc were matched with eopms with regard to gender, the lopms and eopms group were additionally matched in terms of disease duration and lesion load . In three supplementary analyses, the same comparisons were additionally computed for pms subgroups matched for age, gender and lesion load . Participants were included in the study in a three - step procedure . In the first step, 79 ms patients with a disease onset prior to the age of 16 years and 20 hc subjects scanned with a standardized mr protocol (see brain imaging) were included . All pediatric ms patients were diagnosed according to the revised mcdonald diagnostic criteria 2010 (polman et al ., 2011). Each patient was followed - up at the german national center for multiple sclerosis in childhood and adolescence, university medical center, gttingen, germany . Compatible with the heuristic for age - based patient subdivision presented in ruggieri et al . (2004), patients with disease onset prior to the age of twelve were assigned to the eopms group, and patients with onset at 12 years or older were assigned to the lopms group . In the second step, we (mw) conducted an mri quality assessment procedure leading to exclusion of the data of eight eopms patients, 20 lopms patients, and five healthy participants (primarily due to slight motion artifacts). Finally, the six eopms patients with the longest and the 14 lopms patients with the shortest disease duration were excluded in the third step in order to match the two groups in terms of disease duration . Following these steps, 15 patients with eopms, 16 patients with lopms, and 15 hc subjects were finally included . For the supplementary analyses, two alternative pms groups were composed to match these groups in terms of participant age . Specifically, the five youngest eopms patients and the 13 oldest lopms patients were excluded . Thus, 16 patients with eopms, 17 patients with lopms, and 15 hc subjects were included in these supplementary analyses . Consent was obtained according to the declaration of helsinki, and the local research ethics committee of the medical faculty, georg - august university, gttingen approved the study . Whole - brain high in - plane resolution t2w images with 32 or 35 contiguous axial slices (tr = 4500 ms, te = 103 ms, flip angle = 130, voxel resolution = 0.6 0.6 4 mm, no gap, matrix size = 384 384) were acquired with a turbo spin echo sequence on a 3 tesla whole - body mri (magnetom trio, siemens, erlangen, germany) using a clinical routine 12-channel head coil . In particular, in the three main analyses evaluating pms groups matched for disease duration (in the three supplementary analyses evaluating pms groups matched for participant age) images of 2 (2) eopms patients had 32 axial slices, images of 13 (14) had 35 slices . For lopms patients, 9 (4) had 32 and 7 (13) had 35 slices, 3 (3) hc subjects had 32, 12 (12) hc subjects 35 slices . A -test for stochastic independence showed that the number of slices acquired was partly balanced across groups i.e. Eopms vs. lopms: = 6.23, p = 0.013 (= 0.67, p = 0.412), eopms vs. hc: = 0.24, p = 0.624 (= 0.32, p = 0.570), and lopms vs. hc = 4.29, p = 0.038 (= 0.06, p = 0.810). Preprocessing included a manual lesion mapping procedure, two sequential segmentation runs, resampling and within - subject standardization of modulated tissue probability maps . In particular, we (hh) started preprocessing by performing a manual lesion mapping based on high in - plane resolution t2w - images (voxel size = 0.6 0.6 4 mm) of individual subjects using the osirix software toolbox (osirix foundation, geneva, switzerland). Subsequently, images were segmented into gm, wm and csf in a two - run segmentation procedure using the unified segmentation approach in spm8 (wellcome trust centre for neuroimaging, institute of neurology, ucl, london, uk http://www.fil.ion.ucl.ac.uk/spm). The two - run procedure was applied to minimize age - dependent deformation effects (e.g., wilke et al ., 2002) and tissue misclassification (e.g., wilke et al ., 2003) that might result in a typical one - run procedure from using adult reference data during normalization / segmentation of pediatric brain images . In the first segmentation run, unmodulated tissue probability maps and spatially normalized lesion masks were generated as output files for each subject . These output files were then used to compute customized tissue probability templates for the second segmentation run separately for each pair of groups (eopms & lopms, eopms & hc and lopms & hc) and for both matching procedures . In particular, customized tissue probability templates were determined by computing the voxel - wise averages across not modulated tissue probability maps for gm, wm and csf of each subject included in the given pair of groups . To avoid lesion - induced artifacts, subjects having a lesion at a given voxel coordinate (as defined by the normalized lesion masks) were excluded from the averaging procedure . In the second segmentation run, these customized tissue probability templates for gm, wm, and csf were used (please note that the images of the eopms and lopms patients were segmented twice within each matching framework: once for the comparison with the hc subjects, once for the comparison with the other pms group). Unmodulated tissue probability maps and spatially normalized lesion maps were generated as output files for each subject . After segmentation, the modulated probability maps were resampled to a voxel resolution of 6 6 6 mm using trilinear interpolation to increase sensitivity of the diagnostic classification analyses and to decrease the substantial computational load imposed by the voxel - wise permutation procedure used for statistical inference (please see section mri - based classification analyses). In the next step, unmodulated probability maps were used to generate binary masks for gm, wm, and csf . This was done for each pair of groups and matching framework separately to facilitate a tissue - specific search for diagnostic biomarkers in the classification analyses . Specifically, we first computed the voxel - wise average for each of the three tissue types across unmodulated tissue probability maps (voxel resolution = 2 2 2 mm) of all subjects included in a given pair of groups that did not have a lesion at a given coordinate . Whether a lesion was present for a given coordinate or not was determined based on the normalized lesion masks determined in the second segmentation run . Then, we resampled these averaged maps to a voxel resolution of 6 6 6 mm . Finally, a voxel coordinate was assigned to that tissue class for which the maximal averaged probability score among the three tissues was computed . Following this procedure, the gm mask comprised 4498 (supplementary analysis using age - matched samples: 4641) voxels for the contrast eopms vs. lopms, the wm mask for this comparison comprised 2379 (2513) voxels . For eopms vs. hc, the gm mask consisted of 5015 (4951) voxels, the mask for wm consisted of 2186 (2301) voxels . Finally, for lopms vs. hc the gm mask consisted of 4632 (4727) voxels, the mask for wm consisted of 2519 (2510) voxels . In a final step, we computed a within - subject z - transformation of the masked, resampled, and modulated gm and wm tissue probability maps for each subject to account for potential between group differences of overall brain volume . The resulting z - transformed, masked, resampled and modulated tissue probability maps then entered the classification analyses (see below). We conducted three separate main analyses to discover diagnostic information that potentially differentiates eopms, lopms and hc . In particular, we searched for diagnostic information for separation of eopms vs. lopms patients contained in modulated tissue probability parameters extracted from gm (wm) areas in classification analysis 1a (1b). In classification analysis 2a (2b), we searched for diagnostic information for eopms patients vs. hc subjects in gm (wm) areas . Finally, in analysis 3a (3b), we searched for diagnostic information for separation of lopms patients vs. hc subjects based on gm (wm) tissue parameters . In these main analyses, pms groups were matched with regard to disease duration, lesion load and age . In three supplementary analyses (s1s3) the same comparisons were computed for pms subgroups matched for gender, age and lesion load . The processing steps did not differ between analyses, except for group- and tissue - specific brain masks and the tissue - specific modulated probability maps used for the analyses (see preprocessing). For each voxel, we determined the diagnostic classification accuracy in a leave - one - out cross - validation (cv) framework . In particular, we started the analysis by extracting the modulated tissue probability score (from either the modulated gm or wm probability map) of each subject included in the analysis (ntotal) for a given voxel (e.g., for the comparison eopms vs. lopms in the disease duration matching framework: ntotal = 15[neopms] + 16[nlopms] = 31). Then, the data of all but one subject (ntotal 1) were used as training data to compute a logistic regression model using the glmfit function included in matlab 7.6 (the mathworks inc ., in the next step, this model was used to compute the predicted class membership probability of the left out or test subject respectively based on its modulated tissue probability score . This procedure was repeated ntotal times i.e. Until each subject included in the analysis was once excluded from building the regression model / once treated as test subject . In the next step, we computed the root mean square error (rmse) between true class labels (eopms vs. lopms: eopms = 1, lopms = 0; eopms vs. hc: eopms = 1, hc = 0; lopms vs. hc: lopms = 1, hc = 0) and predicted class membership probabilities as primary diagnostic accuracy measure for each voxel coordinate . As secondary / illustrative accuracy measure, we computed the mean of sensitivity and specificity (mss). For that measure, a correctly classified subject belonging to the group named first in each comparison (e.g., an eopms patient in the comparison eopms vs. lopms) was considered as true positive, a correctly classified subject belonging to the second group (lopms in the example) was considered as true negative, etc . The diagnostic classification procedure was repeated for each voxel coordinate included in the respective analysis and yielded a diagnostic accuracy map for that analysis which denoted the rmse (mss) score for each voxel . To test for significance of diagnostic accuracy obtained for each voxel, we used permutation testing to compute the probability of the observed rmse - score under the null hypothesis . The false discovery rate (fdr; benjamini and hochberg, 1995) criterion was used to correct for multiple comparisons . Following the fdr criterion, the number of permutations necessary to assess the significance of accuracy in each voxel equals the number of tests (i.e. Voxels) included in each analyses divided by the false positive rate for a single test (single test = 0.05). Thus, for example in analysis 1a we permuted the class labels of each voxel 4498 (number of voxels in gm mask for eopms vs. lopms) / 0.05 (single test) = 89,960 times . Then, we used the resulting permutation distribution of rmse - scores for each voxel to assess the significance of the observed rmse - score under the null hypothesis (good et al ., 2005). At this point, we would like to mention that due to the direct link between number of voxels in an analysis on one hand and the number of tests per analysis and the number of permutations necessary per test on the other permutation testing might easily involve a massive computational burden if the number of voxels is very high . For that reason, we decided to resample the voxel resolution during image preprocessing in order to decrease the computational load . In the results section, we report coordinates for which rmse is significant on a threshold corrected for multiple comparisons following the fdr criterion (fdr = 0.05) and also voxel coordinates significant according to an uncorrected threshold (uncorrected = 5 10). Results reported correspond to coordinates in the anatomical standard space of the mni - brain template (tzourio - mazoyer et al ., 2002). Since the classification analyses were conducted in customized template space, it was necessary to map the coordinates identified in the analyses to mni - space first . Lopms patients and hc subjects did not differ from eopms patients with regard to gender (eopms vs. lopms: = 1.70, p = 0.193; eopms vs. hc: = 1.43, p = 0.232). Following from the disease duration matching procedure, the average age in the lopms group was significantly higher than in the eopms group (eopms vs. lopms: t = 5.9, p = 2.3 10). In addition, the average age in the lopms group was also higher as in the hc group (lopms vs. hc: t = 5.0, p = 2.7 10). A t - test for independent samples revealed that both pms subgroups were comparable in terms of t2w lesion volume (t = 0.14, p = 0.886). . Please see table s1 for participant characteristics of pms subgroups matched by age and gender . In this analysis, we searched for differential diagnostic information for separation of eopms vs. lopms patients contained in modulated tissue probability parameters extracted from gm (wm) areas in classification analysis 1a (1b). Consistently, wm analysis 1b revealed diagnostic information in wm areas located in the vicinity of frontal gyri please see table s2 for results for this group comparison based on pms subgroups matched for age, gender and lesion load . In classification analysis 2a (2b), we searched for diagnostic features separating between eopms patients and hc subjects contained in modulated tissue probability parameters extracted from gm (wm) areas . In analysis 2a, however, consistent with analysis 1, also a coordinate in superior frontal gyrus was found . Analysis 2b revealed wm coordinates close to the striatum, cerebellum but also postcentral gyrus and calcarine fissure . . Please see table s3 for results regarding this group comparison based on eopms patients that were matched to lopms patients in terms of age, gender and lesion load . Finally, in analysis 3a (3b) we searched for gm (wm) areas containing diagnostic information for the separation of lopms patients and hc subjects . Consistent with the difference in the presence of disease and the difference in mean participant age between the groups, analyses 3a and b revealed a comparatively large number of coordinates with significant diagnostic information . Notably, contrary to analysis 1 and 2, no frontal or orbitofrontal gyri or wm coordinates in the vicinity of these areas were identified . Please see table s4 for results regarding this group comparison based on lopms patients that were matched to eopms patients in terms of age, gender and lesion load . In this study, we identified diagnostic features in conventional mri images separating between eopms patients, lopms patients and hc subjects by applying computer - based classification techniques to locally specific brain tissue probability information . Three main analyses were conducted that investigated diagnostic separability within the three pairs of groups (eopms vs. lopms, eopms vs. hc, and lopms vs. hc) based on gray as well as white matter voxel tissue probability information . In these analyses, the pms groups were matched by disease duration, lesion load and gender whereas the same comparisons were computed in three supplementary analyses (s1 s3) based on pms groups matched for age, lesion load and gender ., 2013; polman et al ., 2011; morgen et al ., 2006; prinster et al ., 2006; filippi and rocca, 2005), a set of widely distributed brain areas was identified across analyses . Among these, areas were detected that are discussed as key markers for ms such as wm areas in vicinity of e.g. Caudate nucleus and thalamus (i.e. Periventricular wm areas, cf . Polman et al ., 2011) and for these areas, very high accuracy has been obtained . Specifically, for the separation of eopms and hc (based on pms groups matched for age, gender and lesion load, analysis s2), an accuracy of 87.1% has been reached for a wm coordinate close to caudate nucleus (mni: 18, 18, 30). However, besides these well established markers temporal and frontal gm areas have also been identified, which are less well known to be diagnostically relevant, although their general involvement in ms has repeatedly been reported (e.g., morgen et al ., 2006; prinster et al ., for example, 87.1% accuracy has been reached for a gm locus in the middle temporal pole (mni: 16, 10, 38) for the separation of lopms and hc (based on pms groups matched for disease duration, gender and lesion load, analysis 3). Unfortunately, identification of biomarkers for the separation of eopms patients from hc subjects or even for eopms vs. lopms patients alone does not necessarily imply that the markers are also specific for eopms . To the contrary, many of the markers found for eopms vs. hc markers found for eopms vs. lopms are not necessarily specific as pms subgroups differed in terms of age in analysis 1 (or disease duration in analysis s1). These differences cannot be easily avoided by e.g. Another matching procedure because they follow from the conjunction of a) the mutual dependence of the factors disease onset, disease duration and age (e.g., age is the sum of disease onset and duration) and b) the definition of eopms and lopms in terms of their different disease onset, disease duration and age (e.g., age is the sum of disease onset and duration) and b) the definition of eopms and lopms in terms of their different disease onset . Thus, within this constellation, balancing one of the factors disease duration or age automatically results in inducing a bias in the other . Consequently, balancing alone cannot be sufficient to guarantee specificity of biomarkers identified in the comparison eopms vs. lopms . Instead, an additional group comparison must be taken into account that is comparably characterized by a difference in age or disease duration . Specifically, given that certain findings are made only in the eopms vs. lopms comparison but not for a pair of groups that is similarly characterized by one of these confounding factors, it is unlikely that underlying differences observed in eopms vs. lopms are induced by the confound since its effect should be comparable across analyses . A comparison for which this condition was fulfilled was lopms vs. hc (analysis 3). In particular, the average age in the lopms group was higher than in the eopms group and (comparably) higher than in the hc group (see table 1). Consequently, biomarkers identified in the eopms vs. lopms but not in the lopms vs. hc comparison should not depend on differences in participant age (and disease duration) and should thus be specific for eopms . This specific result pattern was found for frontal gyri and wm areas located in the vicinity to frontal gm gyri . In particular, analysis 1 identified a coordinate in middle ([mni: 24, 32, 18], mss = 80.6%) and inferior frontal gyri ([mni: 36, 30, 20], 77.3% mss) with diagnostic information . In addition, a wm area in vicinity to superior frontal gyrus ([mni: 18, 54, 12), mss = 74.2%) also contained diagnostic information . On the contrary, thus, taken together the integration of findings made across analyses conducted in the study suggest that disease - related alterations of frontal gyri starting early after disease onset is a diagnostically relevant feature specific for eopms . Besides identifying brain mri - based biomarkers specific for eopms, the study also demonstrates that computer - based classification approaches are able to utilize subtle tissue variations for diagnosis, extracted from gm but also wm that are less characterized by pronounced intensity alterations than hyperintense wm lesions in t2w - images . This was revealed by the gm analyses conducted as most of the coordinates with diagnostic information did not contain lesions (see tables 24 and s1s3). Non - lesion gm based separation of eopms and hc is in line with findings of studies showing gm loss in adult ms patients compared to hc (weygandt et al ., 2011; ceccarelli et al ., 2009; morgen et al ., 2006; prinster et al ., 2006; bakshi et al ., 2002). Within these studies (i.e. Ceccarelli et al ., 2009; bakshi et al ., 2002), it was assumed that elevated iron deposition might lead to tissue loss in deep gray matter nuclei which in turn induces signal hypointensity in t2w - images . Speculatively, this mechanism might also underly gm - based separability of pms groups and hc . Interestingly, utilization of non - lesion related mri signals for the diagnosis of eopms vs. hc by the regression approach in addition to utilization of lesion - related signals was also supported by white matter analyses . In particular, although high diagnostic accuracy was frequently linked to high lesion occurrence in these analyses what is in line with the diagnostic guidelines (polman et al ., 2011) implying that lesions are the only mri features indicative of ms high lesion occurrence alone was frequently not sufficient to explain accuracy obtained by the regression approach . This can be seen e.g. In analysis 2b for a wm coordinate (mni: 24, 34, 40) located in the vicinity of postcentral gyrus . Although a substantial proportion of eopms patients had a lesion at this coordinate (i.e. 4 out of 15 or 26.7%, see table 3) accuracy expectable by this lesion occurrence was lower than accuracy obtained by the logistic regression approach . Specifically, when following the diagnostic guidelines (polman et al ., 2011) suggesting that lesions are the only mri alterations indicative of ms, and when additionally assuming that lesions do only occur in patients but not controls, this lesion occurrence should go along with a diagnostic accuracy of 63.4% . In particular, given that four out of 15 patients, who should present with lesions, had a lesion at this coordinate (sensitivity = 26.7%), and none of 15 controls, who should not have lesions, had a lesion (specificity = 100%), a mean of sensitivity and specificity of 63.4% follows . Contrary to these 63.4% however, the logistic regression approach evaluating continuous tissue probability parameters (instead of the dichotomously scaled presence of a lesion marker evaluated by human raters) obtained 80.0% mean of sensitivity and specificity . Consequently, the discrepancy between these accuracies suggests that the regression approach utilizes further mri signals in addition to lesion - induced signal alterations . Speculatively, wm characterized by only slight signal hyperintensities being too weak to be classified as lesion by a human rater (i.e. So - called dirty - appearing white matter; ge et al . . This interpretation would be compatible with our recent findings (weygandt et al ., 2011). At first glance, the moderate sample sizes of groups investigated might be considered a drawback of the study . However, within the specific framework of the present study several aspects have to be considered when evaluating the sample size . First, we were able to identify diagnostic information in periventricular wm areas containing lesions . Periventricular wm lesions are considered (one of) the key diagnostic marker(s) for ms (e.g., polman et al ., 2011). Thus, the sample size was sufficient to reliably identify this major mri marker for ms frequently reported in the literature . Second, from a pragmatic perspective the small prevalence of pediatric ms has to be considered . (2004), the prevalence of ms with an onset prior to the age of 16 years is approximately between 0.003% and 0.006% in the overall population . For ms with an onset prior to the age of 10 years it is even less approximately between 0.0002% and 0.0008% . Thus, eopms and even lopms are rare clinically cases . Finally, we used non - parametric permutation testing to estimate the probability of diagnostic accuracies obtained on the voxel level . Compared with parametric procedures, permutation tests rely much less on the fulfillment of distributional assumptions for a given test statistic (good, 2005) a problem that might be more prominent in moderately sized data sets as compared to larger data sets . Finally, the small deviations in the number of slices acquired for t2w - images across groups for pms groups matched with regard to disease duration, lesion load and gender might be considered as limitation of the study . However, the impact of this imbalance does not appear to be very strong since results obtained for analyses 13 are compatible with those obtained in analyses s1s3 . And for the latter analyses the number of slices was balanced across groups . In this study, we identified diagnostic features in mri acquired during clinical routine that differentiated between eopms patients, lopms patients and hc subjects by applying computer - based classification techniques to locally specific brain tissue probability information . In particular, we were able to replicate existing knowledge on the pivotal role of hyperintense wm lesions for ms diagnosis by using this approach to the separate between pms patients and hc subjects . By using the classification approach for differential diagnosis or separation of pms subgroups respectively, we found diagnostic information specific for eopms in frontal gyri and also wm areas in the vicinity of frontal gyri . These results suggest that conventional mri contains a richer set of diagnostically informative features than assumed so far . Taken together, by integrating results from diagnostic classification analyses investigating groups matched for disease duration, lesion load, participant age, and gender, we identified biomarkers specific for ms patients with very early disease onset.

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